IB Biology Course Book: 2014 Edition: Oxford IB Diploma Program [2014 ed.] 0198392117, 9780198392118

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OXFORD IB DIplOm a pROgRam m e

2 0 1 4 ED I TI O N

BIOLO GY C O U R S E C O M PA N I O N

Andrew Allott David Mindorf

3 Great Clarendon Street, Oxford, OX2 6DP, United Kingdom Oxford University Press is a department of the University of Oxford. It furthers the Universitys objective of excellence in research, scholarship, and education by publishing worldwide. Oxford is a registered trade mark of Oxford University Press in the UK and in certain other countries  Oxford University Press 2014 The moral rights of the authors have been asserted First published in 2014 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, by licence or under terms agreed with the appropriate reprographics rights organization. Enquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above. You must not circulate this work in any other form and you must impose this same condition on any acquirer British Library Cataloguing in Publication Data Data available 978-0-19-839211-8 1 3 5 7 9 10 8 6 4 2 Paper used in the production of this book is a natural, recyclable product made from wood grown in sustainable forests. The manufacturing process conforms to the environmental regulations of the country of origin. Printed in Malaysia Acknowledgements The publishers would like to thank the following for permissions to use their photographs: Cover image:  Paul Souders/Corbis; p1: Sulston & Horvitz; p2: DR YORGOS NIKAS/SCIENCE PHOTO LIBRARY; p3a: DR.JEREMY BURGESS/SCIENCE PHOTO LIBRARY; p3b: Shutterstock; p6: Ferran Garcia-Pichel, Max Planck Institute of Marine Biology, Bermen Germany; p7a: Prof. P.Motta & T. Naguro/ SPL; p7b: Andrew Allot; p7c: Andrew Allot; p7d: MICHAEL ABBEY/ SCIENCE PHOTO LIBRARY; p8a: Carolina Biological Supply Co/Visuals Unlimited, Inc.; p8b: ASTRID & HANNS-FRIEDER MICHLER/SCIENCE PHOTO LIBRARY; p9: MICHAEL ABBEY/SCIENCE PHOTO LIBRARY; p10a: DR. PETER SIVER, VISUALS UNLIMITED /SCIENCE PHOTO LIBRARY; p10b: Sulston & Horvitz; p12: JAMES CAVALLINI/SCIENCE PHOTO LIBRARY; p14a: CHRIS BARRY/VISUALS UNLIMITED, INC. /SCIENCE PHOTO LIBRARY; p14b: SIMON FRASER/DEPARTMENT OF HAEMATOLOGY, RVI, NEWCASTLE/SCIENCE PHOTO LIBRARY; p16a: TEK IMAGE/SCIENCE PHOTO LIBRARY; p17: LAWRENCE BERKELEY NATIONAL LABORATORY/ SCIENCE PHOTO LIBRARY; p19: A B Dowsett/SPL; p20a: Eye of Science/SPL; p20b: CNRI/SCIENCE PHOTO LIBRARY; p21a: BIOPHOTO ASSOCIATES/SCIENCE PHOTO LIBRARY; p21b: MICROSCAPE/SCIENCE PHOTO LIBRARY; p22a: BIOPHOTO ASSOCIATES/ SCIENCE PHOTO LIBRARY; p22b: DR GOPAL MURTI/SCIENCE PHOTO LIBRARY; p22c: DR GOPAL MURTI/SCIENCE PHOTO LIBRARY; p22d: MICROSCAPE/SCIENCE PHOTO LIBRARY; p22e: DR KARI LOUNATMAA/ SCIENCE PHOTO LIBRARY; p22f: MICROSCAPE/SCIENCE PHOTO LIBRARY; p23a:  DON W. FAWCETT/SCIENCE PHOTO LIBRARY; p23b: DR. GOPAL MURTI/SCIENCE PHOTO LIBRARY; p23c: Andrew Allot; p24a: STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY; p24b: DR.JEREMY BURGESS/ SCIENCE PHOTO LIBRARY; p25a: STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY; p25b: DAVID M. PHILLIPS/SCIENCE PHOTO LIBRARY; p25c: STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY; p27: Author Image; p28: NIBSC/ SCIENCE PHOTO LIBRARY; p29: Author Image; p32: Janaka Dharmasena/ Shutterstock; p43a: OUP; p43b: Andrew Allot; p44: Herve Conge/SPL; p45: David Mayer, Consultant and CSL Liver Surgery, Queen Elizabeth Hospital, Birmingham; p46a: THOMAS DEERINCK, NCMIR/SCIENCE PHOTO LIBRARY; p46b: The VRoma Project (www.vroma.org); p48: GEORGETTE DOUWMA/ SCIENCE PHOTO LIBRARY; p49: DAVID MCCARTHY/SCIENCE PHOTO LIBRARY; p51: M.I. Walker/SPL; p53a,b,c,d: STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY; p54a,b: STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY; p55a: Dharam M Ramnani; p55b: MANFRED KAGE/SCIENCE PHOTO LIBRARY; p55c: MANFRED KAGE/SCIENCE PHOTO LIBRARY; p57: MOREDUN ANIMAL HEALTH LTD/SCIENCE PHOTO LIBRARY; p58: OUP; p54: Andrew Allot; p60: J Herve Conge, ISM/ SPL; p61: OUP; p62: Vasiliy Koval/Shutterstock; p66: LAGUNA DESIGN/SCIENCE PHOTO LIBRARY; p69a-p69b: OUP; p70: CLAIRE PAXTON & JACQUI FARROW/SCIENCE PHOTO LIBRARY; p71: DR KEITH WHEELER/SCIENCE PHOTO LIBRARY; p72: OUP; p73a: Dr. Elena Kiseleva/SPL; p73b: Dr. Gopal Murti/SPL; p73c: Dr. Elena Kiseleva/SPL; p75a: LAGUNA DESIGN/SCIENCE PHOTO LIBRARY; p75b: LAGUNA DESIGN/ SCIENCE PHOTO LIBRARY; p75c: LAGUNA DESIGN/SCIENCE PHOTO LIBRARY; p79: OUP; p80a: Andrew Allot; p80b-81: OUP; p83a: OUP; p83b: Giles Bell; p90a: OUP; p90b: www.rcsb.org; p91: www.rcsb.org; p92a: Yikrazuul/Wikipedia; p92b: OUP; p95: JAMES KING-HOLMES/SCIENCE

PHOTO LIBRARY; p101-102: OUP; p110: SPL; p116: Author Image; p122:  Tony Rusecki / Alamy; p123a: OUP; p123b: Glenn Tattersall; p124a: MATTHEW OLDFIELD/SCIENCE PHOTO LIBRARY; p124b: Author Image; p152: OUP; p126a: OUP; p126b: Petrov Andrey/Shutterstock; p130a: OUP; p130b: OUP; p130c: Andrew Allott; p131c: Andrew Allott; p132a: OUP; p133: William Allott; p134: OUP; p141: OUP; p143a: Jax.org; p143b: Jax.org; p143c: Jax.org; p144: www.ncbi.nlm.nih.gov/pubmed; p146a: Eye of Science/SPL; p146b: Eye of Science/SPL; p148: MAURO FERMARIELLO/SCIENCE PHOTO LIBRARY; p150a: M .Wurtz/Biozentrum/University o fBasel/SPL; p150b: Kwangshin Kim/SPL; p151: www.ncbi.nlm.nih.gov; p152: Dr. Oscar Lee Miller, Jr of the University of Virginia; p155a: OUP; p155b: Andrew Allot; p156: OUP; p158a: DEPT. OF CLINICAL CYTOGENETICS, ADDENBROOKES HOSPITAL/SCIENCE PHOTO LIBRARY; p158b: Tomasz Markowski/ Dreamstime; p159: L. WILLATT, EAST ANGLIAN REGIONAL GENETICS SERVICE/SCIENCE PHOTO LIBRARY; p160-161b: OUP; p162a: Andrew Allot; p164a,b,c,d: Andrew Allot; p165a,b,c,d: Andrew Allot; p166a: OUP; p166b: OUP; p166c: OUP; p169: OUP; p171a: OUP; p171b: OUP; p172: William Allott; p176: Enrico Coen; p177-184a: OUP; p184b: OUP; p186: RIA NOVOSTI/ SCIENCE PHOTO LIBRARY; p188: VOLKER STEGER/SCIENCE PHOTO LIBRARY; p189: OUP; p190a: Andrew Allot; p190b: DAVID PARKER/SCIENCE PHOTO LIBRARY; p190c-196c: OUP; p197: WALLY EBERHART, VISUALS UNLIMITED /SCIENCE PHOTO LIBRARY; p198a: GERARD PEAUCELLIER, ISM /SCIENCE PHOTO LIBRARY; p198b: GERARD PEAUCELLIER, ISM /SCIENCE PHOTO LIBRARY; p198c: Author Image; p199: PHILIPPE PLAILLY/SCIENCE PHOTO LIBRARY; p201: OUP; p202: Parinya Hirunthitima/Shutterstock; p203a: OUP; p203b: OUP; p203c: ERIC GRAVE/SCIENCE PHOTO LIBRARY; p203d: OUP; p204a,b,c,d: Andrew Allot; p205a: Author Image; p205b: CreativeNature.nl/Shutterstock; p205c: Author Image; p206: OUP; p207: OUP; p207b: Author Image; p209: Author Image; p210: OUP; p211: OUP; p212a: OUP; p212b: Andrew Allott; p214: Andrew Allott; p215a: OUP; p215b: Andrew Allott; p215c: Andrew Allott; p215d: Rich Lindie/Shutterstock; p215e: OUP; p217a: OUP; p217b: Andrew Allott; p217d: OUP; p221: Giorgiogp2/Wikipedia; p223a: Andrew Allott; p223b: Andrew Allott; p224: OUP; p225a: OUP; p225b: Andrew Allott; p225c: Andrew Allott; p228-242b: OUP; p243: Erik Lam/Shutterstock; p244: Sinclair Stammers/SPL; p246a: Wikipedia; p246b: Daiju AZUMA; p246c: Wikipedia; p246d: Shutterstock; p248a: Andrew Allott; p248b Andrew Allott; p250a: OUP; p250b: OUP; p251a: OUP; p251b: OUP; p251c: OUP; p251d: OUP; p251e: PETER CHADWICK/SCIENCE PHOTO LIBRARY; p253: OUP; p259: Author Image; p261: OUP; p262a: OUP; p262b: OUP; p264: Andrew Allot; p265: Kipling Brock/Shutterstock; p270a: Author Image; p270b: Author Image; p272: OUP; p276a: OUP; p276b: BOB GIBBONS/SCIENCE PHOTO LIBRARY; p279: BSIP VEM/SCIENCE PHOTO LIBRARY; p281: Dennis Kunkel/Photolibrary; p282: Author Image; p283a: Andrew Allot; p283b: OUP; p286: Author Image; p290: Public Domain/Wikipedia; p292a: OUP; p292b: OUP; p294a: OUP; p294b: BIOPHOTO ASSOCIATES/SCIENCE PHOTO LIBRARY; p298: Andrew Allot; p299: OUP; p302: OUP; p303a: OUP; p303b: Andrew Allot; p304a: OUP; p304b: OUP; p305: JAMES CAVALLINI/SCIENCE PHOTO LIBRARY; p306: ST MARYS HOSPITAL MEDICAL SCHOOL/SCIENCE PHOTO LIBRARY; p307: OUP; p308: Wikipedia; p309: OUP; p315: OUP; p317: DU CANE MEDICAL IMAGING LTD/SCIENCE PHOTO LIBRARY; p318: OUP; p320a: OUP; p320b: THOMAS DEERINCK, NCMIR/SCIENCE PHOTO LIBRARY; p323: OUP; p325: BSIP VEM/SCIENCE PHOTO LIBRARY; p327: OUP; p328a: SCIENCE VU, VISUALS UNLIMITED /SCIENCE PHOTO LIBRARY; p328b: OUP; p330:  J. ZBAEREN/EURELIOS/SCIENCE PHOTO LIBRARY; p331: OUP; p332: OAK RIDGE NATIONAL LABORATORY/US DEPARTMENT OF ENERGY/SCIENCE PHOTO LIBRARY; p333: OUP; p334: POWER AND SYRED/SCIENCE PHOTO LIBRARY; p339: CHASSENET/BSIP/SCIENCE PHOTO LIBRARY; p340: Author Image; p343: SIMON FRASER/SCIENCE PHOTO LIBRARY; p344:  LEE D. SIMON/SCIENCE PHOTO LIBRARY; p346: SPL; p348: Image of PDB ID 1aoi (K. Luger, A.W. Mader, R.K. Richmond, D.F. Sargent, T.J. Richmond (1997) structure of the core particle at 2.8 A resolution Nature 389: 251-260) created with Chimera (UCSF Chimera--a visualization system for exploratory research and analysis. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. J Comput Chem. 2004 Oct;25(13):160512. ); p349: Public Domain/Wikipedia; p351: SCIENCE PHOTO LIBRARY; p352: Andrew Allot; p353: Charvosi/Wikipedia; p357: Axel Bueckert/ Shutterstock; p358: PNAS.Org; p359: DR ELENA KISELEVA/SCIENCE PHOTO LIBRARY; p363a: Jmol; p363b: RCSB.org; p367:  1970 American Association for the Advancement of Science. Miller, O. L. et al. Visualization of bacterial genes in action. Science 169,392395 (1970). All rights reserved; p368a: Nobelprize.org; p368b: POWER AND SYRED/SCIENCE PHOTO LIBRARY; p368c: SINCLAIR STAMMERS/SCIENCE PHOTO LIBRARY; p370a: Andrew Allot; p373: Shutterstock; p375: RAMON ANDRADE 3DCIENCIA/SCIENCE PHOTO LIBRARY; p387a: CNRI/SCIENCE PHOTO LIBRARY; p387b: Petrov Andrey/Shutterstock; p387c: Prof. Kenneth R Miller/ SPL; p387d: Andrew Allot; p387e: Andrew Allot; p388: Dr. Carmen Manella, Wadsworth Center,New York State Department of Health; p390: Prof. Kenneth R Miller/ SPL; p392: Andrew Allot; p398: Andrew Allot; p399: Barrie Juniper; p403: POWER AND SYRED/SCIENCE PHOTO LIBRARY; p404: SINCLAIR STAMMERS/ SCIENCE PHOTO LIBRARY; p405a: Smugmug.Com; p405b: SCIENCE PHOTO LIBRARY; p406a: POWER AND SYRED/SCIENCE PHOTO LIBRARY; p406b: DR KEITH WHEELER/SCIENCE PHOTO LIBRARY; p410: SIDNEY MOULDS/ SCIENCE PHOTO LIBRARY; p411: DR KEITH WHEELER/SCIENCE PHOTO Continued on back page.

Contents 1 Cell Biology Introduction to cells Ultrastructure o cells Membrane structure Membrane transport The origin o cells C ell division

7 Nucleic acids (AHL) 1 16 25 33 45 51

2 Molecular Biology Molecules to metabolism 61 Water 68 C arbohydrates and lipids 73 Proteins 87 E nzymes 96 S tructure o D NA and RNA 1 05 D NA replication, transcription and translation 111 C ell respiration 1 22 Photosynthesis 1 29

3 Genetics Genes C hromosomes Meiosis Inheritance Genetic modication and biotechnology

1 41 1 49 1 59 1 68 1 87

4 Ecology S pecies, communities and ecosystems E nergy fow C arbon cycling C limate change

2 01 21 3 220 229

2 41 2 49 258 2 63

6 Human physiology D igestion and absorption The blood system D eence against inectious diseases Gas exchange Neurones and synapses Hormones, homeostasis and reproduction

3 43 355 3 62

8 Metabolism, cell respiration and photosynthesis (AHL) Metabolism C ell respiration Photosynthesis

3 73 3 80 3 89

9 Plant biology (AHL) Transport in the xylem o plants Transport in the phloem o plants Growth in plants Reproduction in plants

403 41 2 42 2 42 9

10 Genetics and evolution (AHL) Meiosis Inheritance Gene pool and speciation

2 79 2 89 3 02 31 0 31 9 329

Antibody production and vaccination Movement The kidney and osmoregulation S exual reproduction

5 75 5 82 5 91

C Ecology and conservation Species and communities C ommunities and ecosystems Impacts o humans on ecosystems C onservation o biodiversity Population ecology The nitrogen and phosphorous cycles

603 61 3 62 5 63 5 642 649

D Human physiology

43 9 445 45 5

11 Animal physiology (AHL)

5 Evolution and biodiversity E vidence or evolution Natural selection C lassication and biodiversity C ladistics

D NA structure and replication Transcription and gene expression Translation

Environmental protection Medicine B ioormatics

Human nutrition D igestion Functions o the liver The heart Hormones and metabolism Transport o respiratory gases

65 9 671 678 684 694 699

Internal Assessment (with thanks to Mark Headlee for his assistance with this chapter) 708

Index

71 3

465 476 485 499

A Neurobiology and behaviour Neural development The human brain Perception o stimuli Innate and learned behaviour Neuropharmacology Ethology

513 518 526 533 5 41 5 48

B Biotechnology and bioinformatics Microbiology: organisms in industry 557 B iotechnology in agriculture 5 65

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Course book defnition

The IB Learner Profle

The IB D iploma Programme course books are resource materials designed to support students throughout their two- year D iploma Programme course o study in a particular subj ect. They will help students gain an understanding o what is expected rom the study o an IB D iploma Programme subj ect while presenting content in a way that illustrates the purpose and aims o the IB . They refect the philosophy and approach o the IB and encourage a deep understanding o each subj ect by making connections to wider issues and providing opportunities or critical thinking.

The aim o all IB programmes to develop internationally minded people who work to create a better and more peaceul world. The aim o the programme is to develop this person through ten learner attributes, as described below.

The books mirror the IB philosophy o viewing the curriculum in terms o a whole- course approach; the use o a wide range o resources, international mindedness, the IB learner prole and the IB D iploma Programme core requirements, theory o knowledge, the extended essay, and creativity, action, service ( C AS ) . E ach book can be used in conj unction with other materials and indeed, students o the IB are required and encouraged to draw conclusions rom a variety o resources. Suggestions or additional and urther reading are given in each book and suggestions or how to extend research are provided. In addition, the course companions provide advice and guidance on the specic course assessment requirements and on academic honesty protocol. They are distinctive and authoritative without being prescriptive.

IB mission statement The International B accalaureate aims to develop inquiring, knowledgeable and caring young people who help to create a better and more peaceul world through intercultural understanding and respect. To this end the organization works with schools, governments and international organizations to develop challenging programmes o international education and rigorous assessment. These programmes encourage students across the world to become active, compassionate and lielong learners who understand that other people, with their dierences, can also be right.

iv

Inquirers: They develop their natural curiosity. They acquire the skills necessary to conduct inquiry and research and snow independence in learning. They actively enj oy learning and this love o learning will be sustained throughout their lives. Knowledgeable: They explore concepts, ideas, and issues that have local and global signicance. In so doing, they acquire in-depth knowledge and develop understanding across a broad and balanced range o disciplines. Thinkers: They exercise initiative in applying thinking skills critically and creatively to recognize and approach complex problems, and make reasoned, ethical decisions. C ommunicators: They understand and express ideas and inormation condently and creatively in more than one language and in a variety o modes o communication. They work eectively and willingly in collaboration with others. Princip led: They act with integrity and honesty, with a strong sense o airness, j ustice and respect or the dignity o the individual, groups and communities. They take responsibility or their own action and the consequences that accompany them. O p en-minded: They understand and appreciate their own cultures and personal histories, and are open to the perspectives, values and traditions o other individuals and communities. They are accustomed to seeking and evaluating a range o points o view, and are willing to grow rom the experience. C aring: They show empathy, compassion and respect towards the needs and eelings o others. They have a personal commitment to service, and to act to make a positive dierence to the lives o others and to the environment. Risk-takers: They approach unamiliar situations and uncertainty with courage and orethought, and have the independence o spirit to explore new roles, ideas, and strategies. They are brave and articulate in deending their belies.

B alanced: They understand the importance o intellectual, physical and emotional ballance to achieve personal well- being or themselves and others. Refective: They give thoughtul consideration to their own learning and experience. They are able to assess and understand their strengths and limitations in order to support their learning and personal development.

What constitutes malpractice? Malpractice is behaviour that results in, or may result in, you or any student gaining an unair advantage in one or more assessment component. Malpractice includes plagiarism and collusion. Plagiarism is defned as the representation o the ideas or work o another person as your own. The ollowing are some o the ways to avoid plagiarism: 

words and ideas o another person to support ones arguments must be acknowledged



passages that are quoted verbatim must be enclosed within quotation marks and acknowledged



C D -Roms, email messages, web sites on the Internet and any other electronic media must be treated in the same way as books and j ournals



the sources o all photographs, maps, illustrations, computer programs, data, graphs, audio- visual and similar material must be acknowledged i they are not your own work



works o art, whether music, flm dance, theatre arts or visual arts and where the creative use o a part o a work takes place, the original artist must be acknowledged.

A note on academic honesty It is o vital importance to acknowledge and appropriately credit the owners o inormation when that inormation is used in your work. Ater all, owners o ideas ( intellectual property) have property rights. To have an authentic piece o work, it must be based on your individual and original ideas with the work o others ully acknowledged. Thereore, all assignments, written or oral, completed or assessment must use your own language and expression. Where sources are used or reerred to, whether in the orm o direct quotation or paraphrase, such sources must be appropriately acknowledged.

How do I acknowledge the work of others? The way that you acknowledge that you have used the ideas o other people is through the use o ootnotes and bibliographies. Footnotes ( placed at the bottom o a page) or endnotes ( placed at the end o a document) are to be provided when you quote or paraphrase rom another document, or closely summarize the inormation provided in another document. You do not need to provide a ootnote or inormation that is part o a body o knowledge. That is, defnitions do not need to be ootnoted as they are part o the assumed knowledge. B ibliograp hies should include a ormal list o the resources that you used in your work. Formal means that you should use one o the several accepted orms o presentation. This usually involves separating the resources that you use into dierent categories ( e.g. books, magazines, newspaper articles, internet-based resources, C ds and works o art) and providing ull inormation as to how a reader or viewer o your work can fnd the same inormation. A bibliography is compulsory in the E xtended Essay.

C ollusion is defned as supporting malpractice by another student. This includes: 

allowing your work to be copied or submitted or assessment by another student



duplicating work or dierent assessment components and/or diploma requirements.

O ther orms o malp ractice include any action that gives you an unair advantage or aects the results o another student. Examples include, taking unauthorized material into an examination room, misconduct during an examination and alsiying a C AS record.

v

Using your IB Biology Online Resources What is Kerboodle? Kerboodle is an online learning platorm. I your school has a subscription to IB B iology Kerboodle O nline Resources you will be able to access a huge bank o resources, assessments, and presentations to guide you through this course.

What is in your Kerboodle Online Resources? There are three main areas or students on the IB B iology Kerboodle: planning, resources, and assessment.

Resources There a hundreds o extra resources available on the IB B iology Kerboodle O nline. You can use these at home or in the classroom to develop your skills and knowledge as you progress through the course. Watch videos and animations o experiments, difcult concepts, and science in action. Hundreds o worksheets  read articles, perorm experiments and simulations, practice your skills, or use your knowledge to answer questions. Look at galleries o images rom the book and see their details close up. Find out more by looking at recommended sites on the Internet, answer questions, or do more research.

Planning B e prepared or the practical work and your internal assessment with extra resources on the IB B iology Kerboodle online. Learn about the dierent skills that you need to perorm an investigation. Plan and prepare experiments o your own. Learn how to analyse data and draw conclusions successully and accurately.

One of hundreds of worksheets.

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Practical skills presentation.

Assessment C lick on the assessment tab to check your knowledge or revise or your examinations. Here you will fnd lots o interactive quizzes and examstyle practice questions. Formative tests: use these to check your comprehension, theres one auto-marked quiz or every sub-topic. E valuate how confdent you eel about a sub-topic, then complete the test. You will have two attempts at each question and get eedback ater every question. The marks are automatically reported in the markbook, so you can see how you progress throughout the year. Summative tests: use these to practice or your exams or as revision, theres one auto- marked quiz or every topic. Work through the test as i it were an examination  go back and change any questions you arent sure about until you are happy, then submit the test or a fnal mark. The marks are automatically reported in the markbook, so you can see where you may need more practice. Assessment practice: use these to practice answering the longer written questions you will come across when you are examined. These worksheets can be printed out and perormed as a timed test.

Don't forget! You can also fnd extra resources on our ree website www.oxfordsecondary.co.uk/ib-biology Here you can fnd all o the answers and even more practice questions. vii

Introduction This book is a companion or students o B iology in the International B accalaureate D iploma Programme. B iology is the most popular choice o science subj ect as part o the IB diploma. The study o biology should lead students to appreciate the interconnectedness o lie within the biosphere. With a ocus on understanding the nature o science, IB B iology will allow you to develop a level o scientifc literacy that will better prepare you to act on issues o local and global concern, with a ull understanding o the scientifc point o view. The structure o this book is closely based on the biology programme in the S ubj ect Guide. S ubheadings restate the specifc assessment statements. Topics 1  6 explain in detail the C ore material that is common to both S L and HL courses. Topics 7  1 1 explain the AHL ( additional higher level material) . Topics A, B , C and D cover the content o the options. All topics include the ollowing elements:

Understanding The specifcs o the content requirements or each sub- topic are covered in detail. C oncepts are presented in ways that will promote enduring understanding.

Applications These sections help you to develop your understanding by studying a specifc illustrative example or learning about a signifcant experiment in the history o biology.

Skills topics These sections encourage you to apply your understanding through practical activities and analysis o results rom classic biological research. In some cases this involves instructions or handling data rom experiments and also use o IC T. Some o the skills sections involve experiments with known outcomes, aimed at promoting understanding through doing and seeing. O thers involve ideas or experimental work with unknown outcomes, where you can defne the problem and the methods. These are a valuable opportunities to build the skills that are assessed in IA ( see page 708) .

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Nature of science Here you can explore the methods o science and some o the knowledge issues that are associated with scientifc endeavour. This is done using careully selected examples, including biological research that led to paradigm shits in our understanding o the natural world.

Theory of Knowledge These short sections have headings that are equivocal ` knowledge questions. The text that follows often details one possible answer to the knowledge question. We encourage you draw on these examples of knowledge issues in your TOK essays. Of course, much of the material elsewhere in the book, particularly in the nature of science sections, can be used to prompt TOK discussions.

activity A variety of short topics are included under this heading with the focus in all cases on active learning. We encourage you research these topics yourself, using information available in textbooks or on the Internet. The aim is to promote an independent approach to learning. We believe that the optimal approach to learning is to be active  the more that you do for yourself, guided by your teacher, the better you will learn.

Data-based questions These questions involve studying and analysing data from biological research  this type of question appears in both Paper 2 and Paper 3 for SL and HL IB Biology. Answers to these questions can be found at www.oxfordsecondary.co.uk/ib-biology

End -of-Topic Questions At the end o each topic you will fnd a range o questions, including both past IB B iology exam questions and new questions. Answers can be ound at www.oxordsecondary. co.uk/ib- biology

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CE LL B I O LO GY

Introduction There is an unbroken chain o lie rom the rst cells on Earth to all cells ound in organisms alive today. Eukaryotes have a much more complex cell structure than prokaryotes. The evolution o multicellular organisms allowed cell specialization and cell replacement. C ell division is essential but is carried out dierently

in prokaryotes and eukaryotes. While evolution has resulted in a biological world o enormous diversity, the study o cells shows us that there are also universal eatures. For example, the fuid and dynamic structure o biological membranes allows them to control the composition o cells.

1.1 Introduction to cells Understanding  According to the cell theory, living organisms   

  

are composed o cells. Organisms consisting o only one cell carry out all unctions o lie in that cell. Surace area to volume ratio is important in the limitation o cell size. Multicellular organisms have properties that emerge rom the interaction o their cellular components. Specialized tissues can develop by cell dierentiation in multicellular organisms. Dierentiation involves the expression o some genes and not others in a cells genome. The capacity o stem cells to divide and dierentiate along dierent pathways is necessary in embryonic development. It also makes stem cells suitable or therapeutic uses.

Nature of science

Applications  Questioning the cell theory using atypical

examples, including striated muscle, giant algae and aseptate ungal hyphae.  Investigation o unctions o lie in Paramecium and one named photosynthetic unicellular organism.  Use o stem cells to treat Stargardts disease and one other named condition.  Ethics o the therapeutic use o stem cells rom specially created embryos, rom the umbilical cord blood o a new-born baby and rom an adults own tissues.

Skills

 Looking or trends and discrepancies: although

 Use o a light microscope to investigate the

most organisms conorm to cell theory, there are exceptions.  Ethical implications o research: research involving stem cells is growing in importance and raises ethical issues.

structure o cells and tissues.  Drawing cell structures as seen with the light microscope.  Calculation o the magnifcation o drawings and the actual size o structures shown in drawings or micrographs. 1

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C E LL B I O LO G Y

The cell theory Living organisms are composed of cells. The internal structure of living organisms is very intricate and is built up from very small individual parts. O rgans such as the kidney and the eye are easily visible. If they are dissected we can see that large organs are made of a number of different tissues, but until microscopes were invented little or nothing was discovered about the structure of tissues. From the 1 7th century onwards biologists examined tissues from both plants and animals using microscopes. Although there was much variation, certain features were seen again and again. A theory was developed to explain the basic features of structure  the cell theory. This states that cells are the fundamental building blocks of all living organisms. The smallest organisms are unicellular  they consist of j ust one cell. Larger organisms are multicellular  they are composed of many cells. C ells vary considerably in size and shape but they share certain common features: 

Every living cell is surrounded by a membrane, which separates the cell contents from everything else outside.



C ells contain genetic material which stores all of the instructions needed for the cells activities.



Many of these activities are chemical reactions, catalysed by enzymes produced inside the cell.



C ells have their own energy release system that powers all of the cells activities.

S o, cells can be thought of as the smallest living structures  nothing smaller can survive.

 Figure 1 Coloured scanning electron micrograph (SEM)

2

of a human embryo on the tip of a pin

1 .1 I n tro d u ctI o n to ce lls

Exceptions to the cell theory Looking for trends and discrepancies: although most organisms conform to cell theory, there are exceptions. An early stage in scientifc investigation is to look or trends  things that appear to be ound generally rather than j ust in specifc cases. These trends can lead to the development o a theory. A scientifc theory is a way o interpreting the natural world. Theories allow us to make predictions. S ometimes exceptions to a general trend are ound. These are called discrepancies. S cientists have to j udge whether the discrepancies are common or serious enough to make predictions too unreliable to be useul. The theory is then discarded. The cell theory is an example o where scientists have looked or trends and discrepancies. Robert Hooke was the frst to use the word cell or structures in living organisms. He did this in 1 665 ater examining cork and other parts o plants. Ater describing cells in cork he wrote this:

 Figure 2

Robert Hookes drawing of cork cells

Aiviy

Nor is this kind of texture peculiar to cork only, for upon examination with my microscope I have found that the pith of the Elder or almost any other tree, the inner pith of the Cany hollow stems of several other vegetables: as of Fennel, Carrets, Daucus, Bur-docks, Teasels, Fearn, some kind of Reeds etc. have much such a kind of Schematisme, as I have lately shown that of cork. S o Hooke wasnt content with looking at j ust one type o plant tissue  he looked at many and discovered a general trend. S ince Hookes day biologists have looked at tissues rom a huge variety o living organisms. Many o these tissues have been ound to consist o cells, so the cell theory has not been discarded. However, some discrepancies have been discovered  organisms or parts o organisms that do not consist o typical cells. More discrepancies may be discovered, but it is extremely unlikely that the cell theory will ever be discarded, because so many tissues do consist o cells.

 Figure 3

What is the unit of life: the boy or his cells?

These two answers represent the holistic and the reductionist approach in biology.

image viewed here

Using light microscopes

eyepiece lens

Use of a light microscope to investigate the structure of cells and tissues. Try to improve your skill at using microscopes as much as you can. 

Learn the names o parts o the microscope.



Understand how to ocus the microscope to get the best possible image.



Look ater your microscope so it stays in perect working order.



Know how to troubleshoot problems.

turret

coarse-focusing knob ne-focusing knob

objective lens specimen stage light from mirror or light bulb



Figure 4 Compound light microscope

3

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C E LL B I O LO G Y

Focusing 

Put the slide on the stage, with the most promising region exactly in the middle o the hole in the stage that the light comes through.



Always ocus at low power rst even i eventually you need high power magnication.



Focus with the larger coarse- ocusing knobs rst, then when you have nearly got the image in ocus make it really sharp using the smaller ne- ocusing knobs.



I you want to increase the magnication, move the slide so the most promising region is exactly in the middle o the eld o view and then change to a higher magnication lens.

Looking after your microscope 

Always ocus by moving the lens and the specimen urther apart, never closer to each other.



Make sure that the slide is clean and dry beore putting it on the stage.



Never touch the suraces o the lenses with your ngers or anything else.



C arry the microscope careully with a hand under it to support its weight securely.

Troubleshooting Problem: Nothing is visible when I try to ocus. Solution: Make sure the specimen is actually under the lens, by careully positioning the slide. It is easier to nd the specimen i you ocus at low power rst.

Problem: A circle with a thick black rim is visible. Solution: There is an air bubble on the slide. Ignore it and try to improve your technique or making slides so that there are no air bubbles.

Problem: There are blurred parts o the image

Types of slide The slides that we examine with a microscope can be permanent or temporary. Making permanent slides is very skilled and takes a long time, so these slides are normally made by experts. Permanent slides o tissues are made using very thin slices o tissue. Making temporary slides is quicker and easier so we can do this or ourselves.

Examining and drawing plant and animal cells Almost all cells are too small to be seen with the naked eye, so a microscope is needed to study them. It is usually easy to see whether a cell is rom a plant or an animal, even though there are many dierent cell types in both the plant and animal kingdoms. 

Place the cells on the slide in a layer not more than one cell thick.



Add a drop o water or stain.



C areully lower a cover slip onto the drop. Try to avoid trapping any air bubbles.



Remove excess fuid or stain by putting the slide inside a olded piece o paper towel and pressing lightly on the cover slip.

It is best to examine the slide rst using low power. Move the slide to get the most promising areas in the middle o the eld o view and then move up to high power. D raw a ew cells, so you remember their structure. cover slip cells

carefully lower the cover slip stain or water

even when I ocus it as well as I can. gently squeeze to remove exces uid

Solution: Either the lenses or the slide have dirt on them. Ask your teacher to clean it.

Problem: The image is very dark. Solution: Increase the amount o light passing through the specimen by adj usting the diaphragm.

Problem: The image looks rather bleached. Solution: D ecrease the amount o light passing through the specimen by adj usting the diaphragm.

4

cover slip folded a er towel

slide

 Figure 5 Making a

temporary mount

1 .1 I n tro d u ctI o n to ce lls

1 Moss leaf

2 B anana fruit cell 10 m

3 Mammalian liver cell 5 m

20 m

Use a moss plant with very thin leaves. Mount a single lea in a drop o water or methylene blue stain.

Scrape a small amount o the sot tissue rom a banana and place on a slide. Mount in a drop o iodine solution.

S crape cells rom a reshly cut surace o liver ( not previously rozen) . S mear onto a slide and add methylene blue to stain.

4 Leaf lower epidermis

5 Human cheek cell

6 White blood cell

20 m

2 m

10 m

Peel the lower epidermis o a lea. The cell drawn here was rom Valeriana. Mount in water or in methylene blue.  Figure 6 Plant and animal cell drawings

A thin layer o mammalian blood can be smeared over a slide and stained with Leishmans stain.

S crape cells rom the inside o your cheek with a cotton bud. S mear them on a slide and add methylene blue to stain.

Drawing cells Drawing cell structures as seen with the light microscope. C areul drawings are a useul way o recording the structure o cells or other biological structures. Usually the lines on the drawing represent the edges o structures. D o not show unnecessary detail and only use aint shading. D rawings o structures seen using a microscope will be larger than the structures actually are  the drawing shows them magnifed. O n page 6 the method or calculating the magnifcation o a drawing is explained. E verything on a drawing should be shown to the same magnifcation. a) Use a sharp pencil with a hard lead to draw single sharp lines.

b) Join up lines careully to orm continuous structures such as cells

c) D raw lines reehand, but use a ruler or labelling lines. cell

bad 

good

bad

good

bad

cell

good

Figure 7 Examples of drawing styles

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C E LL B I O LO G Y

Calculation o magnifcation and actual size Calculation o the magnifcation o drawings and the actual size o structures shown in drawings or micrographs. When we look down a microscope the structures that we see appear larger than they actually are. The microscope is magniying them. Most microscopes allow us to magniy specimens by two or three dierent actors. This is done by rotating the turret to switch rom one obj ective lens to another. A typical school microscope has three levels o magnifcation: 

 40 ( low power)



 1 00 ( medium power)



 400 ( high power)

I we take a photo down a microscope, we can magniy the image even more. A photo taken down a microscope is called a micrograph. There are many micrographs in this book, including electron micrographs taken using an electron microscope.

It is very important when using this ormula to make sure that the units or the size o the image and actual size o the specimen are the same. They could both be millimetres ( mm) or micrometres ( m) but they must not be dierent or the calculation will be wrong. Millimetres can be converted to micrometres by multiplying by one thousand. Micrometres can be converted to millimetres by dividing by one thousand. S cale bars are sometimes put on micrographs or drawings, or j ust alongside them. These are straight lines, with the actual size that the scale bar represents. For example, i there was a 1 0 mm long scale bar on a micrograph with a magnifcation o  1 0, 000 the scale bar would have a label o 1 m.

EXAMPLE:

When we draw a specimen, we can make the drawing larger or smaller, so the magnifcation o the drawing isnt necessarily the same as the magnifcation o the microscope.

The length o an image is 3 0 mm. It represents a structure that has an actual size o 3 m. D etermine the magnifcation o the image.

To fnd the magnifcation o a micrograph or a drawing we need to know two things: the size o the image ( in the drawing or the micrograph) and the actual size o the specimen. This ormula is used or the calculation:

3 0 mm = 3 0  1 0 - 3 m 3 m = 3  1 0 - 6 m

Either:

size o image magnifcation = ___ actual size o specimen

30  1 0 - 3 Magnifcation = _ 3  1 0-6 = 1 0, 000 

Or: 3 0 mm = 3 0, 000 m

I we know the size o the image and the magnifcation, we can calculate the actual size o a specimen.

3 0, 000 Magnifcation = _ 3 = 1 0, 000 

Data-based questions 1

a)

D etermine the magnifcation o the string o Thiomargarita cells in fgure 8, i the scale bar represents 0.2 mm [3 ]

b) Determine the width o the string o cells.

[2]  Figure 8

6

Thiomargarita

1 .1 I n tro d u ctI o n to ce lls

2

b) D etermine the length o the cheek cell.

In fgure 9 the actual length o the mitochondrion is 8 m. a) D etermine the magnifcation o this electron micrograph.

[2 ]

[2 ]

b) C alculate how long a 5 m scale bar would be on this electron micrograph. [2 ] c) Determine the width o the mitochondrion.

[1 ]

 Figure 10

4

a)

Human cheek cell

Using the width o the hens egg as a guide, estimate the actual length o the ostrich egg ( fgure 1 1 ) . [2 ]

b) E stimate the magnifcation o the image.  Figure 9

3

[2 ]

Mitochondrion

The magnifcation o the human cheek cell rom a compound microscope ( fgure 1 0) is 2 , 000  . a) C alculate how long a 2 0 m scale bar would be on the image.

[2 ]  Figure 11

Ostrich egg

Testing the cell theory Questioning the cell theory using atypical examples, including striated muscle, giant algae and aseptate fungal hyphae. To test the cell theory you should look at the structure o as many living organisms as you can, using a microscope. Instructions or microscope use are given on page 4. In each case you should ask the question, D oes the organism or tissue ft the trend stated in the cell theory by consisting o one or more cells?

In humans they have an average length o about 3 0 mm, whereas other human cells are mostly less than 0.03 mm in length. Instead o having one nucleus they have many, sometimes as many as several hundred.

Three atypical examples are worth considering: 

Striated muscle is the type o tissue that we use to change the position o our body. The building blocks o this tissue are muscle fbres, which are similar in some ways to cells. They are surrounded by a membrane and are ormed by division o pre-existing cells. They have their own genetic material and their own energy release system. However muscle fbres are ar rom typical. They are much larger than most animal cells.

 Figure 12

Striated muscle fbres

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C E LL B I O LO G Y





Fungi consist o narrow thread-like structures called hyphae. These hyphae are usually white in colour and have a fuy appearance. They have a cell membrane and, outside it, a cell wall. In some types o ungi the hyphae are divided up into small cell-like sections by cross walls called septa. However, in aseptate ungi there are no septa. Each hypha is an uninterrupted tube-like structure with many nuclei spread along it. Algae are organisms that eed themselves by photosynthesis and store their genes inside nuclei, but they are simpler in their structure and organization than plants. Many algae consist o one microscopic cell. There are vast numbers o these unicellular algae in the oceans and they orm the basis o most marine ood chains. Less common are some algae that grow to a much larger size, yet they still seem to be single cells. They are known as giant algae. Acetabularia is one example. It can grow to a length o as much as 1 00 mm, despite only having one nucleus. I a new organism with a length o 1 00 mm was discovered, we would certainly expect it to consist o many cells, not just one.

 Figure 13

Aseptate hypha

 Figure 14 Giant alga

Unicellular organisms Organisms consisting of only one cell carry out all functions of life in that cell. The unctions o lie are things that all organisms must do to stay alive. S ome organisms consist o only one cell. This cell thereore has to carry out all the unctions o lie. B ecause o this the structure o unicellular organisms is more complex than most cells in multicellular organisms. Unicellular organisms carry out at least seven unctions o lie: 

Nutrition  obtaining ood, to provide energy and the materials needed or growth.



Metabolism  chemical reactions inside the cell, including cell respiration to release energy.



Growth  an irreversible increase in size.



Response  the ability to react to changes in the environment.



Excretion  getting rid o the waste products o metabolism.



Homeostasis  keeping conditions inside the organism within tolerable limits.



Reproduction  producing ospring either sexually or asexually.

Many unicellular organisms also have a method o movement, but some remain in a xed position or merely drit in water or air currents.

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1 .1 I n tro d u ctI o n to ce lls

Limitations on cell size Surface area to volume ratio is important in the limitation of cell size. In the cytoplasm of cells, large numbers of chemical reactions take place. These reactions are known collectively as the metabolism of the cell. The rate of these reactions ( the metabolic rate of the cell) is proportional to the volume of the cell. For metabolism to continue, substances used in the reactions must be absorbed by the cell and waste products must be removed. S ubstances move into and out of cells through the plasma membrane at the surface of the cell. The rate at which substances cross this membrane depends on its surface area. The surface area to volume ratio of a cell is therefore very important. If the ratio is too small then substances will not enter the cell as quickly as they are required and waste products will accumulate because they are produced more rapidly than they can be excreted. Surface area to volume ratio is also important in relation to heat production and loss. If the ratio is too small then cells may overheat because the metabolism produces heat faster than it is lost over the cells surface.

same cube unfolded

 Figure 15 Volume and

surace area

o a cube

Functions of life in unicellular organisms Investigation of functions of life in Paramecium and one named photosynthetic unicellular organism. Paramecium is a unicellular organism that can be cultured quite easily in the laboratory. Alternatively collect some pond water and use a centrifuge to concentrate the organisms in it to see if Paramecium is present. Place a drop of culture solution containing Paramecium on a microscope slide. Add a cover slip and examine the slide with a microscope. The nucleus o the cell can divide to produce the extra nuclei that are needed when the cell reproduces. Oten the reproduction is asexual with the parent cell dividing to orm two daughter cells. Food vacuoles contain smaller organisms that the Paramecium has consumed. These are gradually digested and the nutrients are absorbed into the cytoplasm where they provide energy and materials needed or growth. The cell membrane controls what chemicals enter and leave. It allows the entry o oxygen or respiration. Excretion happens simply by waste products difusing out through the membrane.

The contractile vacuoles at each end o the cell ll up with water and then expel it through the plasma membrane o the cell, to keep the cells water content within tolerable limits. Metabolic reactions take place in the cytoplasm, including the reactions that release energy by respiration. Enzymes in the cytoplasm are the catalysts that cause these reactions to happen. Beating o the cilia moves the Paramecium through the water and this can be controlled by the cell so that it moves in a particular direction in response to changes in the environment.

 Figure 16 Paramecium

9

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C E LL B I O LO G Y

Chlamydomonas is a unicellular alga that lives in soil and freshwater habitats. It has been used widely for research into cell and molecular biology. Although it is green in colour and carries out photosynthesis it is not a true plant and its cell wall is not made of cellulose. The nucleus o the cell can divide to produce genetically identical nuclei or asexual reproduction. Nuclei can also use and divide to carry out a sexual orm o reproduction. In this image, the nucleus is concealed by chloroplasts.

The contractile vacuoles at the base o the fagella ll up with water and then expel it through the plasma membrane o the cell, to keep the cells water content within tolerable limits. Photosynthesis occurs inside chloroplasts in the cytoplasm. Carbon dioxide can be converted into the compounds needed or growth here, but in the dark carbon compounds rom other organisms are sometimes absorbed through the cell membrane i they are available.

Metabolic reactions take place in the cytoplasm, with enzymes present to speed them up.

The cell wall is reely permeable and it is the membrane inside it that controls what chemicals enter and leave. Oxygen is a waste product o photosynthesis and is excreted by diusing out through the membrane.  Figure 17

Beating o the two fagella moves the Chlamydomonas through the water. A lightsensitive eyespot allows the cell to sense where the brightest light is and respond by swimming towards it.

Chlamydomonas

Multicellular organisms Multicellular organisms have properties that emerge from the interaction of their cellular components. S ome unicellular organisms live together in colonies, for example a type of alga called Volvox aureus. E ach colony consists of a ball made of a protein gel, with 5 00 or more identical cells attached to its surface. Figure 1 8 shows two colonies, with daughter colonies forming inside them. Although the cells are cooperating, they are not fused to form a single cell mass and so are not a single organism.  Figure 18 Volvox colonies

O rganisms consisting of a single mass of cells, fused together, are multicellular. O ne of the most intensively researched multicellular organisms is a worm called Caenorhabditis elegans. The adult body is about one millimetre long and it is made up of exactly 95 9 cells. This might seem like a large number, but most multicellular organisms have far more cells. There are about ten million million cells in an adult human body and even more in organisms such as oak trees or whales. Although very well known to biologists, Caenorhabditis elegans has no common name and lives unseen in decomposing organic matter. It feeds on the bacteria that cause decomposition. C. elegans has a mouth, pharynx, intestine and anus. It is hermaphrodite so has both male and female reproductive organs. Almost a third of the cells are neurons, or

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1 .1 I n tro d u ctI o n to ce lls

nerve cells. Most o these neurons are located at the ront end o the worm in a structure that can be regarded as the animals brain. Although the brain in C. elegans coordinates responses to the worms environment, it does not control how individual cells develop. The cells in this and other multicellular organisms can be regarded as cooperative groups, without any cells in the group acting as a leader or supervisor. It is remarkable how individual cells in a group can organize themselves and interact with each other to orm a living organism with distinctive overall properties. The characteristics o the whole organism, including the act that it is alive, are known as emergent properties. E mergent properties arise rom the interaction o the component parts o a complex structure. We sometimes sum this up with the phrase: the whole is greater than the sum o its parts. A simple example o an emergent property was described in a C hinese philosophical text written more than 2 , 5 00 years ago: Pots are fashioned from clay. But its the hollow that makes the pot work.  S o, in biology we can carry out research by studying component parts, but we must remember that some bigger things result rom interactions between these components.

Cell diferentiation in multicellular organisms Specialized tissues can develop by cell dierentiation in multicellular organisms. In multicellular organisms dierent cells perorm dierent unctions. This is sometimes called division o labour. In simple terms, a unction is a job or a role. For example the unction o a red blood cell is to carry oxygen, and the unction o a rod cell in the retina o the eye is to absorb light and then transmit impulses to the brain. Oten a group o cells specialize in the same way to perorm the same unction. They are called a tissue. B y becoming specialized, the cells in a tissue can carry out their role more efciently than i they had many dierent roles. They can develop the ideal structure, with the enzymes needed to carry out all o the chemical reactions associated with the unction. The development o cells in dierent ways to carry out specifc unctions is called dierentiation. In humans, 2 2 0 distinctively dierent highly specialized cell types have been recognized, all o which develop by dierentiation.

toK Hw a w i wh  m i b ha ah? An emergent property o a system is not a property o any one component o the system, but it is a property o the system as a whole. Emergence reers to how complex systems and patterns arise rom many small and relatively simple interactions. We cannot thereore necessarily predict emergent properties by studying each part o a system separately (an approach known as reductionism) . Molecular biology is an example o the success that a reductionist approach can have. Many processes occurring in living organisms have been explained at a molecular level. However, many argue that reductionism is less useul in the study o emergent properties including intelligence, consciousness and other aspects o psychology. The interconnectivity o the components in cases like these is at least as important as the unctioning o each individual component. One approach that has been used to study interconnectivity and emergent properties is computer modelling. In both animal behaviour and ecology, a programme known as the Game o Lie has been used. It was devised by John Conway and is available on the Internet. Test the Game o Lie by creating initial confgurations o cells and seeing how they evolve. Research ways in which the model has been applied.

Gene expression and cell diferentiation Dierentiation involves the expression o some genes and not others in a cells genome. There are many dierent cell types in a multicellular organism but they all have the same set o genes. The 2 2 0 cell types in the human body have the same set o genes, despite large dierences in their structure and activities. To take an example, rod cells in the retina o the eye produce a pigment that absorbs light. Without it, the rod cell would not be able to do its j ob o sensing light. A lens cell in the eye produces no pigments and is transparent. I it did contain pigments, less light would

11

1

C E LL B I O LO G Y pass through the lens and our vision would be worse. While they are developing, both cell types contain the genes for making the pigment, but these genes are only used in the rod cell. This is the usual situation  cells do not j ust have genes with the instructions that they need, they have genes needed to specialize in every possible way. There are approximately 2 5 , 000 genes in the human genome, and these genes are all present in a body cell. However, in most cell types less than half of the genes will ever be needed or used. When a gene is being used in a cell, we say that the gene is being expressed. In simple terms, the gene is switched on and the information in it is used to make a protein or other gene product. The development of a cell involves switching on particular genes and expressing them, but not others. C ell differentiation happens because a different sequence of genes is expressed in different cell types. The control of gene expression is therefore the key to development. An extreme example of differentiation involves a large family of genes in humans that carry the information for making receptors for odorants  smells. These genes are only expressed in cells in the skin inside the nose, called olfactory receptor cells. Each of these cells expresses j ust one of the genes and so makes one type of receptor to detect one type of odorant. This is how we can distinguish between so many different smells. Richard Axel and Linda B uck were given the Nobel Prize in 2 004 for their work on this system.

Stem cells The capacity o stem cells to divide and diferentiate along diferent pathways is necessary in embryonic development. It also makes stem cells suitable or therapeutic uses. A new animal life starts when a sperm fertilizes an egg cell to produce a zygote. An embryo is formed when the zygote divides to give two cells. This two- cell embryo divides again to produce a four- cell embryo, then eight, sixteen and so on. At these early stages in embryonic development the cells are capable of dividing many times to produce large amounts of tissue. They are also extremely versatile and can differentiate along different pathways into any of the cell types found in that particular animal. In the 1 9th century, the name stem cell was given to the zygote and the cells of the early embryo, meaning that all the tissues of the adult stem from them. S tem cells have two key properties that have made them one of the most active areas of research in biology and medicine today.

 Figure 19

12

Embryonic stem cells



S tem cells can divide again and again to produce copious quantities of new cells. They are therefore useful for the growth of tissues or the replacement of cells that have been lost or damaged.



S tem cells are not fully differentiated. They can differentiate in different ways, to produce different cell types.

1 .1 I n tro d u ctI o n to ce lls

Embryonic stem cells are thereore potentially very useul. They could be used to produce regenerated tissue, such as skin or people who have suered burns. They could provide a means o healing diseases such as type 1 diabetes where a particular cell type has been lost or is malunctioning. They might even be used in the uture to grow whole replacement organs  hearts or kidneys, or example. These types o use are called therapeutic, because they provide therapies or diseases or other health problems. There are also non-therapeutic uses or embryonic stem cells. One possibility is to use them to produce large quantities o striated muscle fbres, or meat, or human consumption. The bee burgers o the uture may thereore be produced rom stem cells, without the need to rear and slaughter cattle. It is the early stage embryonic stem cells that are the most versatile. Gradually during embryo development the cells commit themselves to a pattern o dierentiation. This involves a series o points at which a cell decides whether to develop along one pathway or another. Eventually each cell becomes committed to develop into one specifc cell type. Once committed, a cell may still be able to divide, but all o these cells will dierentiate in the same way and they are no longer stem cells. Small numbers o cells remain as stem cells, however, and they are still present in the adult body. They are present in many human tissues, including bone marrow, skin and liver. They give some human tissues considerable powers o regeneration and repair. The stem cells in other tissues only allow limited repair  brain, kidney and heart or example.

Therapeutic uses of stem cells Use of stem cells to treat Stargardts disease and one other named condition. There are a ew current uses o stem cells to treat diseases, and a huge range o possible uture uses, many o which are being actively researched. Two examples are given here: one involving embryonic stem cells and one using adult stem cells.

Stargardts disease The ull name o this disease is S targardts macular dystrophy. It is a genetic disease that develops in children between the ages o six and twelve. Most cases are due to a recessive mutation o a gene called AB C A4. This causes a membrane protein used or active transport in retina cells to malunction. As a consequence, photoreceptive cells in the retina degenerate. These are the cells that detect light, so vision becomes progressively worse. The loss o vision can be severe enough or the person to be registered as blind.

Researchers have developed methods or making embryonic stem cells develop into retina cells. This was done initially with mouse cells, which were then injected into the eyes o mice that had a condition similar to Stargardts disease. The injected cells were not rejected, did not develop into tumours or cause any other problems. The cells moved to the retina where they attached themselves and remained. Very encouragingly, they caused an improvement in the vision o the mice. In November 2 01 0, researchers in the United S tates got approval or trials in humans. A woman in her 5 0s with S targardts disease was treated by having 5 0, 000 retina cells derived rom embryonic stem cells inj ected into her eyes. Again the cells attached to the retina and remained there during the our- month trial. There was an improvement in her vision, and no harmul side eects.

13

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C E LL B I O LO G Y

Further trials with larger numbers o patients are needed, but ater these initial trials at least, we can be optimistic about the development o treatments or S targardts disease using embryonic stem cells.

can be done by treating the patient with chemicals that kill dividing cells. The procedure is known as chemotherapy. However, to remain healthy in the long term the patient must be able to produce the white blood cells needed to ght disease. S tem cells that can produce blood cells must be present, but they are killed by chemotherapy. The ollowing procedure is thereore used: 

A large needle is inserted into a large bone, usually the pelvis, and fuid is removed rom the bone marrow.



S tem cells are extracted rom this fuid and are stored by reezing them. They are adult stem cells and only have the potential or producing blood cells.



A high dose o chemotherapy drugs is given to the patient, to kill all the cancer cells in the bone marrow. The bone marrow loses its ability to produce blood cells.



The stem cells are then returned to the patients body. They re- establish themselves in the bone marrow, multiply and start to produce red and white blood cells.

 Figure 20 Stargardts disease

leukemia This disease is a type o cancer. All cancers start when mutations occur in genes that control cell division. For a cancer to develop, several specic mutations must occur in these genes in one cell. This is very unlikely to happen, but as there are huge numbers o cells in the body, the overall chance becomes much larger. More than a quarter o a million cases o leukemia are diagnosed each year globally and there are over 2 00, 000 deaths rom the disease.

In many cases this procedure cures the leukemia completely.

Once the cancer-inducing mutations have occurred in a cell, it grows and divides repeatedly, producing more and more cells. Leukemia involves the production o abnormally large numbers o white blood cells. In most cancers, the cancer cells orm a lump or tumour but this does not happen with leukemia. White blood cells are produced in the bone marrow, a sot tissue in the hollow centre o large bones such as the emur. They are then released into the blood, both in normal conditions and when excessive numbers are produced with leukemia. A normal adult white blood cell count is between 4, 000 and 1 1 ,000 per mm 3 o blood. In a person with leukemia this number rises higher and higher. C ounts above 30,000 per mm 3 suggest that a person may have leukemia. I there are more than 1 00, 000 per mm 3 it is likely that the person has acute leukemia. To cure leukemia, the cancer cells in the bone marrow that are producing excessive numbers o white blood cells must be destroyed. This

14

 Figure 21

Removal of stem cells from bone marrow

1 .1 I n tro d u ctI o n to ce lls

The ethics of stem cell research Ethical implications o research: research involving stem cells is growing in importance and raises ethical issues. S tem cell research has been very controversial. Many ethical obj ections have been raised. S cientists should always consider the ethical implications o their research beore doing it. S ome o the research that was carried out in the past would not be considered ethically acceptable today, such as medical research carried out on patients without their inormed consent.

D ecisions about whether research is ethically acceptable must be based on a clear understanding o the science involved. S ome people dismiss all stem cell research as unethical, but this shows a misunderstanding o the dierent possible sources o the stem cells being used. In the next section, three possible sources o stem cells and the ethics o research involving them are discussed.

Sources of stem cells and the ethics of using them Ethics o the therapeutic use o stem cells rom specially created embryos, rom the umbilical cord blood o a new-born baby and rom an adults own tissues. Stem cells can be obtained rom a variety o sources. E mbryos can be deliberately created by ertilizing egg cells with sperm and allowing the resulting zygote to develop or a ew days until it has between our and sixteen cells. All o the cells are embryonic stem cells.



B lood can be extracted rom the umbilical cord o a new- born baby and stem cells obtained rom it. The cells can be rozen



embyi m   









Almost unlimited growth potential. Can dierentiate into any type in the body. More risk o becoming tumour cells than with adult stem cells, including teratomas that contain dierent tissue types. Less chance o genetic damage due to the accumulation o mutations than with adult stem cells. Likely to be genetically dierent rom an adult patient receiving the tissue. Removal o cells rom the embryo kills it, unless only one or two cells are taken.

and stored or possible use later in the babys lie. 

S tem cells can be obtained rom some adult tissues such as bone marrow.

These types o stem cell vary in their properties and thereore in their potential or therapeutic use. The table below gives some properties o the three types, to give the scientifc basis or an ethical assessment.

c b m  

Easily obtained and stored.



Commercial collection and storage services already available.





Fully compatible with the tissues o the adult that grows rom the baby, so no rejection problems occur. Limited capacity to dierentiate into dierent cell types  only naturally develop into blood cells, but research may lead to production o other types.



Limited quantities o stem cells rom one babys cord.



The umbilical cord is discarded whether or not stem cells are taken rom it.

A m  

Difcult to obtain as there are very ew o them and they are buried deep in tissues.



Less growth potential than embryonic stem cells.



Less chance o malignant tumours developing than rom embryonic stem cells.



Limited capacity to dierentiate into dierent cell types.



Fully compatible with the adults tissues, so rejection problems do not occur.



Removal o stem cells does not kill the adult rom which the cells are taken.

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C E LL B I O LO G Y

Stem cell research has been very controversial. Many ethical obj ections have been raised. There are most obj ections to the use of embryonic stem cells, because current techniques usually involve the death of the embryo when the stem cells are taken. The main question is whether an early stage embryo is as much a human individual as a new- born baby, in which case killing the embryo is undoubtedly unethical. When does a human life begin? There are different views on this. Some consider that when the sperm fertilizes the egg, a human life has begun. Others say that early stage embryos have not yet developed human characteristics and cannot suffer pain, so they should be thought of simply as groups of stem cells. Some suggest that a human life truly begins when there is a heartbeat, or bone tissue or brain activity. These stages take place after a few weeks of development. Another view is that it is only when the embryo has developed into a fetus that is capable of surviving outside the uterus. Some scientists argue that if embryos are specially created by in vitro fertilization (IVF) in order to obtain stem cells, no human that would otherwise

have lived has been denied its chance of living. However, a counterargument is that it is unethical to create human lives solely for the purpose of obtaining stem cells. Also, IVF involves hormone treatment of women, with some associated risk, as well as an invasive surgical procedure for removal of eggs from the ovary. If women are paid for supplying eggs for IVF this could lead to the exploitation of vulnerable groups such as college students. We mu st no t fo rge t e thical argume nts in favo ur o f the u se o f e mb ryo nic ste m ce lls. The y have the p o te ntial to allo w me tho ds o f tre atme nt fo r dise ase s and disab ilitie s that are cu rre ntly incu rab le , so the y co u ld gre atly re du ce the su ffe ring o f so me individuals.

 Figure 22

Harvesting umbilical cord blood

1.2 ultrastrctre of cells Understanding  Prokaryotes have a simple cell structure

without compartments.  Eukaryotes have a compartmentalized cell structure.  Prokaryotes divide by binary fssion.  Electron microscopes have a much higher resolution than light microscopes.

Nature of science

16

Applications  The structure and unction o organelles within

exocrine gland cells o the pancreas.  The structure and unction o organelles within palisade mesophyll cells o the lea.

Skills

 Developments in scientifc research ollow

 Drawing the ultrastructure o prokaryotic cells

improvements in apparatus: the invention o electron microscopes led to greater understanding o cell structure.

based on electron micrographs.  Drawing the ultrastructure o eukaryotic cells based on electron micrographs.  Interpretation o electron micrographs to identiy organelles and deduce the unction o specialized cells.

1 . 2 u lt r A s t r u c t u r e o f c e l l s

th invnin  h n mip Developments in scientifc research ollow improvements in apparatus: the invention o electron microscopes led to greater understanding o cell structure. Much o the progress in biology over the last 1 50 years has ollowed improvements in the design o microscopes. In the second hal o the 1 9th century improved light microscopes allowed the discovery o bacteria and other unicellular organisms. C hromosomes were seen or the rst time and the processes o mitosis, meiosis and gamete ormation were discovered. The basis o sexual reproduction, which had previously eluded William Harvey and many other biologists, was seen to be the usion o gametes and subsequent development o embryos. The complexity o organs such as the kidney was revealed and mitochondria, chloroplasts and other structures were discovered within cells. There was a limit to the discoveries that could be made though. For technical reasons that are explained later in this sub-topic, light microscopes cannot produce clear images o structures smaller than 0.2 micrometres (m) . (A micrometre is a thousandth o a millimetre.) Many biological structures are smaller than this. For example, membranes in cells are about 0.01 m thick. Progress was hampered until a dierent type o microscope was invented  the electron microscope. Electron microscopes were developed in Germany during the 1 930s and came into use in research laboratories in the 1 940s and 5 0s. They allowed

images to be produced o things as small as 0.001 m  2 00 times smaller than with light microscopes. The structure o eukaryotic cells was ound to be ar more intricate than most biologists had expected and many previous ideas were shown to be wrong. For example, in the 1 890s the light microscope had revealed darker green areas in the chloroplast. They were called grana and interpreted as droplets o chlorophyll. The electron microscope showed that grana are in act stacks o fattened membrane sacs, with the chlorophyll located in the membranes. Whereas mitochondria appear as tiny structureless rods or spheres under the light microscope, the electron microscope revealed them to have an intricate internal membrane structure. The electron microscopes revealed what is now called the ultrastructure o cells, including previously unknown eatures. Ribosomes, lysosomes and the endoplasmic reticulum were all discovered and named in the 1 95 0s, or example. It is unlikely that there are structures as signicant as these still to be discovered, but improvements in the design o electron microscopes continue and each improvement allows new discoveries to be made. A recent example, described in subtopic 8.2 , is electron tomography  a method o producing 3 - D images by electron microscopy.

The resolution of electron microscopes Electron microscopes have a much higher resolution than light microscopes. I we look at a tree with unaided eyes we can see its individual leaves, but we cannot see the cells within its leaves. The unaided eye can see things with a size o 0.1 mm as separate objects, but no smaller. To see the cells within the lea we need to use a light microscope. This allows us to see things with a size o down to about 0.2 m as separate objects, so cells can become individually visible  they can be distinguished. Making the separate parts o an obj ect distinguishable by eye is called resolution. The maximum resolution o a light microscope is 0. 2 m, which is 2 00 nanometres ( nm) . However powerul the lenses o a light microscope are, the resolution cannot be higher than this because it is limited by the wavelength o light ( 400700 nm) . I we try to resolve smaller obj ects by

 Figure 1

An electron microscope

in use

17

1

C E LL B I O LO G Y making lenses with greater magnifcation, we fnd that it is impossible to ocus them properly and get a blurred image. This is why the maximum magnifcation with light microscopes is usually  400. Beams o electrons have a much shorter wavelength, so electron microscopes have a much higher resolution. The resolution o modern electron microscopes is 0.001 m or 1 nm. Electron microscopes thereore have a resolution that is 200 times greater than light microscopes. This is why light microscopes reveal the structure o cells, but electron microscopes reveal the ultrastructure. It explains why light microscopes were needed to see bacteria with a size o 1 micrometre, but viruses with a diameter o 0.1 micrometres could not be seen until electron microscopes had been invented.

resolutio

Unaided eyes Light microscopes

Ativity commee ad siee While still a young student in Berlin in the late 1920s Ernst Ruska developed magnetic coils that could ocus beams o electrons. He worked on the idea o using these lenses to obtain an image as in a light microscope, but with electron beams instead o light. During the 1930s he developed and refned this technology. By 1939 Ruska had designed the frst commercial electron microscope. In 1986 he was awarded the Nobel Prize in Physics or this pioneering work. Ruska worked with the German frm Siemens. Other companies in Britain, Canada and the United States also developed and manuactured electron microscopes. 

18

Scientists in dierent countries usually cooperate with each other but commercial companies do not. What are the reasons or this dierence?

Electron microscopes

Millimetes (mm)

Miometes (m)

naometes (m)

0.1

100

100,000

0.0002

0.2

200

0.000001

0.001

1

Prokaryotic cell structure Prokaryotes have a simple cell structure without compartments . All organisms can be divided into two groups according to their cell structure. Eukaryotes have a compartment within the cell that contains the chromosomes. It is called the nucleus and is bounded by a nuclear envelope consisting o a double layer o membrane. Prokaryotes do not have a nucleus. Prokaryotes were the frst organisms to evolve on Earth and they still have the simplest cell structure. They are mostly small in size and are ound almost everywhere  in soil, in water, on our skin, in our intestines and even in pools o hot water in volcanic areas. All cells have a cell membrane, but some cells, including prokaryotes, also have a cell wall outside the cell membrane. This is a much thicker and stronger structure than the membrane. It protects the cell, maintains its shape and prevents it rom bursting. In prokaryotes the cell wall contains peptidoglycan. It is oten reerred to as being extracellular. As no nucleus is present in a prokaryotic cell its interior is entirely flled with cytoplasm. The cytoplasm is not divided into compartments by membranes  it is one uninterrupted chamber. The structure is thereore simpler than in eukaryotic cells, though we must remember that it is still very complex in terms o the biochemicals that are present, including many enzymes. O rganelles are present in the cytoplasm o eukaryotic cells that are analogous to the organs o multi- cellular organisms in that they are distinct structures with specialized unctions. Prokaryotes do not have cytoplasmic organelles apart rom ribosomes. Their size, measured in S vedberg units ( S ) is 70S, which is smaller than those o eukaryotes.

1 . 2 u lt r A s t r u c t u r e o f c e l l s Part o the cytoplasm appears lighter than the rest in many electron micrographs. This region contains the DNA o the cell, usually in the orm o one circular DNA molecule. The DNA is not associated with proteins, which explains the lighter appearance compared with other parts o the cytoplasm that contain enzymes and ribosomes. This lighter area o the cell is called the nucleoid  meaning nucleus-like as it contains DNA but is not a true nucleus.

Cell division in prokaryotes Prokaryotes divide by binary fssion. All living organisms need to produce new cells. They can only do this by division o pre- existing cells. C ell division in prokaryotic cells is called binary fssion and it is used or asexual reproduction. The single circular chromosome is replicated and the two copies o the chromosome move to opposite ends o the cell. D ivision o the cytoplasm o the cell quickly ollows. E ach o the daughter cells contains one copy o the chromosome so they are genetically identical.

dawing pkayi  Draw the ultrastructure o prokaryotic cells based on electron micrographs. B ecause prokaryotes are mostly very small, their internal structure cannot be seen using a light microscope. It is only with much higher magnifcation in electron micrographs that we can see the details o the structure, called the ultrastructure. D rawings o the ultrastructure o prokaryotes are thereore based on electron micrographs. Shown below and on the next page are two electron micrographs o E. coli, a bacterium ound in our intestines. One o them is a thin section and shows the internal structure. The other has been prepared by a dierent technique and shows the external structure. A drawing o each is also shown. B y comparing the drawings with the electron micrographs you can learn how to identiy structures within prokaryotic cells. E lectron micrograp h of Escherichia coli (1 2 m in length)

D rawing to help interp ret the electron micrograp h ribosomes

cell wall

plasma membrane

cytoplasm

nucleoid (region containing naked DNA)

Aiviy oh nam  pkay Biologists sometimes use the term bacteria instead o prokaryote. This may not always be appropriate because the term prokaryote encompasses a larger group o organisms than true bacteria (Eubacteria) . It also includes organisms in another group called the Archaea. There is a group o photosynthetic organisms that used to be called blue-green algae, but their cell structure is prokaryotic and algae are eukaryotic. This problem has been solved by renaming them as Cyanobacteria. 

What problems are caused by scientists using dierent words or things than nonscientists?

19

1

C E LL B I O LO G Y

Electron micrograph of Escherichia coli showing surface features pili

agellum

Shown below is another micrograph o a prokaryote. You can use it to practice your skill at drawing the ultrastructure o prokaryotic cells. You can also fnd other electron micrographs o prokaryotic cells on the internet and try drawing these. There is no need to spend a long time drawing many copies o a particular structure, such as the ribosomes. You can indicate their appearance in one small representative part o the cytoplasm and annotate your drawing to say that they are ound elsewhere.

Activity Garlic cells and compartmentalization Garlic cells store a harmless sulphur-containing compound called alliin in their vacuoles. They store an enzyme called alliinase in other parts o the cell. Alliinase converts alliin into a compound called allicin, which has a very strong smell and favour and is toxic to some herbivores. This reaction occurs when herbivores bite into garlic and damage cells, mixing the enzyme and its substrate. Perhaps surprisingly, many humans like the favour, but to get it garlic must be crushed or cut, not used whole. 

20

You can test this by smelling a whole garlic bulb, then cutting or crushing it and smelling it again.

 Figure 2

Brucella abortus (Bangs bacillus) , 2 m in length

Eukaryotic cell structure Eukaryotes have a compartmentalized cell structure. Eukaryotic cells have a much more complicated internal structure than prokaryotic cells. Whereas the cytoplasm o a prokaryotic cell is one undivided space, eukaryotic cells are compartmentalized. This means that they are divided up by partitions into compartments. The partitions are single or double membranes. The most important o these compartments is the nucleus. It contains the cells chromosomes. The compartments in the cytoplasm are known as organelles. Just as each organ in an animals body is specialized

1 . 2 u lt r A s t r u c t u r e o f c e l l s to perform a particular role, each organelle in a eukaryotic cell has a distinctive structure and function. There are several advantages in being compartmentalized: 

Enzymes and substrates for a particular process can be much more concentrated than if they were spread throughout the cytoplasm.



S ubstances that could cause damage to the cell can be kept inside the membrane of an organelle. For example, the digestive enzymes of a lysosome could digest and kill a cell, if they were not safely stored inside the lysosome membrane.



C onditions such as pH can be maintained at an ideal level for a particular process, which may be different to the levels needed for other processes in a cell.



O rganelles with their contents can be moved around within the cell.

dawig kayi  Draw the ultrastructure o eukaryotic cells based on electron micrographs. The ultrastructure of eukaryotic cells is very complex and it is often best to draw only part of a cell. Your drawing is an interpretation of the structure, so you need to understand the structure of the organelles that might be present.

n double nuclear membrane nuclear pores

dense chromatin

chromatin

rgh pami im ribosomes

The table below contains an electron micrograph of each of the commonly occurring organelles, with a drawing of the structure. B rief notes on recognition features and the function of each organelle are included.

The nuclear membrane is double and has pores through it. The nucleus contains the chromosomes, consisting o DNA associated with histone proteins. Uncoiled chromosomes are spread through the nucleus and are called chromatin. There are oten densely staining areas o chromatin around the edge o the nucleus. The nucleus is where DNA is replicated and transcribed to orm mRNA, which is exported via the nuclear pores to the cytoplasm.

The rER consists o fattened membrane sacs, called cisternae. Attached to the outside o these cisternae are ribosomes. They are larger than in prokaryotes and are classied as 80S. The main unction o the rER is to synthesize protein or secretion rom the cell. Protein synthesized by the ribosomes o the rER passes into its cisternae and is then carried by vesicles, which bud o and are moved to the Golgi apparatus.

cisterna

21

1

C E LL B I O LO G Y

Gogi apparatus cisterna

vesicles

lysosome digestive enzymes

This organelle consists o fattened membrane sacs called cisternae, like rER. However the cisternae are not as long, are oten curved, do not have attached ribosomes and have many vesicles nearby. The Golgi apparatus processes proteins brought in vesicles rom the rER. Most o these proteins are then carried in vesicles to the plasma membrane or secretion. These are approximately spherical with a single membrane. They are ormed rom Golgi vesicles. They contain high concentrations o protein, which makes them densely staining in electron micrographs. They contain digestive enzymes, which can be used to break down ingested ood in vesicles or break down organelles in the cell or even the whole cell.

lysosome membrane

Mitohondrion inner membrane

outer membrane

crista

matrix

free ribosomes

These appear as dark granules in the cytoplasm and are not surrounded by a membrane. They have the same size as ribosomes attached to the rER  about 20nm in diameter, and known as 80S. Free ribosomes synthesize protein, releasing it to work in the cytoplasm, as enzymes or in other ways. Ribosomes are constructed in a region o the nucleus called the nucleolus.

choropast

A double membrane surrounds the chloroplast. Inside are stacks o thylakoids, which are fattened sacs o membrane. The shape o chloroplasts is variable but is usually spherical or ovoid. They produce glucose and a wide variety o other organic compounds by photosynthesis. Starch grains may be present inside chloroplasts i they have been photosynthesizing rapidly.

starch grain stroma double membrane thylakoid

Vauoes and vesies vacuole containing food

vesicles

22

A double membrane surrounds mitochondria, with the inner o these membranes invaginated to orm structures called cristae. The fuid inside is called the matrix. The shape o mitochondria is variable but is usually spherical or ovoid. They produce ATP or the cell by aerobic cell respiration. Fat is digested here i it is being used as an energy source in the cell.

large vacuole

These are organelles that consist simply o a single membrane with fuid inside. Many plant cells have large vacuoles that occupy more than hal o the cell volume. Some animals absorb oods rom outside and digest them inside vacuoles. Some unicellular organisms use vacuoles to expel excess water. Vesicles are very small vacuoles used to transport materials inside the cell.

1 . 2 u lt r A s t r u c t u r e o  c e l l s

In the cytoplasm o cells there are small cylindrical bres called microtubules that have a variety o roles, including moving chromosomes during cell division. Animal cells have structures called centrioles, which consist o two groups o nine triple microtubules. Centrioles orm an anchor point or microtubules during cell division and also or microtubules inside cilia and fagella.

Mib and ni triple microtubules

These are whip-like structures projecting rom the cell surace. They contain a ring o nine double microtubules plus two central ones. Flagella are larger and usually only one is present, as in a sperm. Cilia are smaller and many are present. Cilia and fagella can be used or locomotion. Cilia can be also be used to create a current in the fuid next to the cell.

ciia and faga

double plasma microtubule membrane

The electron micrograph below shows a liver cell with labels to identify some of the organelles that are present. mitochondrion



Using your understanding of these organelles, draw the whole cell to show its ultrastructure.

nucleus

free ribosomes

FPO

rough endoplasmic reticulum  Figure 3

Golgi a aratus

lysosome

Electron micrograph of part of a liver cell

23

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C E LL B I O LO G Y

Exocrine gland cells of the pancreas The structure and function of organelles within exocrine gland cells of the pancreas. Gland cells secrete substances  they release them through their plasma membrane. There are two types of gland cells in the pancreas. E ndocrine cells secrete hormones into the bloodstream. E xocrine gland cells in the pancreas secrete digestive enzymes into a duct that carries them to the small intestine where they digest foods.

FPO

Enzymes are proteins, so the exocrine gland cells have organelles needed to synthesize proteins in large quantities, process them to make them ready for secretion, transport them to the plasma membrane and then release them. The electron micrograph on the right shows these organelles: plasma membrane mitochondrion nucleus rough ER

Golgi apparatus vesicles lysosomes  Figure 4 Electron

micrograph of pancreas cell

Palisade mesophyll cells The structure and function of organelles within palisade mesophyll cells of the leaf. The function of the leaf is photosynthesis  producing organic compounds from carbon dioxide and other simple inorganic compounds, using light energy. The cell type that carries out most photosynthesis in the leaf is palisade mesophyll. The shape of these cells is roughly cylindrical. Like all living plant cells the cell is surrounded by a cell wall, with a plasma membrane inside it. The electron micrograph on the right shows the organelles that a palisade mesophyll cell contains: cell wall plasma membrane chloroplasts mitochondrion vacuole nucleus

24

 Figure 5 Electron

micrograph of palisade mesophyll cell

1 . 3 M e M b rAn e s tru ctu r e

Ipig h  of kayoi ll Interpret electron micrographs to identiy organelles and deduce the unction o specialized cells. I the organelles in a eukaryotic cell can be identifed and their unction is known, it is oten possible to deduce the overall unction o the cell. 

S tudy the electron micrographs in fgures 6, 7 and 8. Identiy the organelles that are present and try to deduce the unction o each cell.

 Figure 7

 Figure 6

 Figure 8

1.3 Mma  Understanding  Phospholipids orm bilayers in water due to the

amphipathic properties o phospholipid molecules.  Membrane proteins are diverse in terms o structure, position in the membrane and unction.  Cholesterol is a component o animal cell membranes.

Nature of science

Applications  Cholesterol in mammalian membranes reduces

membrane fuidity and permeability to some solutes.

Skills

 Using models as representations o the

 Drawing the fuid mosaic model.

real world: there are alternative models o membrane structure.  Falsication o theories with one theory being superseded by another: evidence alsied the DavsonDanielli model.

 Analysis o evidence rom electron microscopy that

led to the proposal o the DavsonDanielli model.  Analysis o the alsication o the DavsonDanielli model that led to the SingerNicolson model

25

1

C E LL B I O LO G Y OH hydrophilic phosphate head

P O O H C H H C O C O O

H H H H H H H

H H H H H H H H

H C O H C O

C H C H C H

H

C H C H C H

H H

C H C H C H

C H C H C H C H

H H

C H C H

H H

C C C H C H C H

H H

C H C H C H C H

C H C H H

H H

H H H

H H H

Phospholipid bilayers Phospholipids form bilayers in water due to the amphipathic properties of phospholipid molecules. Some substances are attracted to water  they are hydrop hilic. O ther substances are not attracted to water  they are hydrop hobic.

C H C H C H

Phospholipids are unusual because part o a phospholipid molecule is hydrophilic and part is hydrophobic. Substances with this property are described as amp hip athic.

hydrophobic hydrocarbon tails

C H C H C H C H

The hydrophilic part o a phospholipid is the phosphate group. The hydrophobic part consists o two hydrocarbon chains. The chemical structure o phospholipids is shown in fgure 1 . The structure can be represented simply using a circle or the phosphate group and two lines or the hydrocarbon chains.



C H H

 Figure 1 The molecular structure

o a phospholipid. The phosphate oten has other hydrophilic groups attached to it, but these are not shown in this diagram

Figure 2 Simplifed diagram o a phospholipid molecule

The two parts o the molecule are oten called phosphate heads and hydrocarbon tails. When phospholipids are mixed with water the phosphate heads are attracted to the water but the hydrocarbon tails are attracted to each other, but not to water. B ecause o this the phospholipids become arranged into double layers, with the hydrophobic hydrocarbon tails acing inwards towards each other and the hydrophilic heads acing the water on either side. These double layers are called phospholipid bilayers. They are stable structures and they orm the basis o all cell membranes. hydrophilic phosphate head hydrophobic hydrocarbon tails

phospholipid bilayer

 Figure 3

Simplifed diagram o a phospholipid bilayer

Models of membrane structure Using models as representations of the real world: there are alternative models of membrane structure. In the 1 9 2 0 s, Gorter and Grendel extracted phospholipids rom the plasma membrane o red blood cells and calculated that the area that the phospholipids occupied when

26

arranged in a monolayer was twice as large as the area o plasma membrane. They deduced that the membrane contained a bilayer o phospholipids. There were several errors in

1 . 3 M e M b rAn e s tru ctu r e

their methods but luckily these cancelled each other out and there is now very strong evidence or cell membranes being based on phospholipid bilayers.

band between. Proteins appear dark in electron micrographs and phospholipids appear light, so this appearance tted the D avson- D anielli model.

Membranes also contain protein and Gorter and Grendels model did not explain where this is located. In the 1 9 3 0s D avson and D anielli proposed layers o protein adj acent to the phospholipid bilayer, on both sides o the membrane. They proposed this sandwich model because they thought it would explain how membranes, despite being very thin, are a very eective barrier to the movement o some substances. High magnication electron micrographs o membranes were made in the 1 9 5 0s, which showed a railroad track appearance  two dark lines with a lighter

Another model o membrane structure was proposed in 1 966 by Singer and Nicolson. In this model the proteins occupy a variety o positions in the membrane. Peripheral proteins are attached to the inner or outer surace. Integral proteins are embedded in the phospholipid bilayer, in some cases with parts protruding out rom the bilayer on one or both sides. The proteins are likened to the tiles in a mosaic. B ecause the phospholipid molecules are ree to move in each o the two layers o the bilayer, the proteins are also able to move. This gives the model its name  the fuid mosaic model.

Polm wih h davodailli mol Falsifcation o theories with one theory being superseded by another: evidence alsifed the DavsonDanielli model. The D avsonD anielli model o membrane structure was accepted by most cell biologists or about 3 0 years. Results o many experiments tted the model including X- ray diraction studies and electron microscopy. In the 1 95 0s and 60s some experimental evidence accumulated that did not t with the D avson D anielli model: 

Freeze-etched electron micrograp hs. This technique involves rapid reezing o cells and then racturing them. The racture occurs along lines o weakness, including the centre o membranes. Globular structures scattered through reeze- etched images o the centre o membranes were interpreted as transmembrane proteins.



S tructure of membrane p roteins. Improvements in biochemical techniques allowed proteins to be extracted rom membranes. They were ound to be very varied in size and globular in shape so were unlike the type o structural protein that would orm continuous layers on the

 Figure 4 Freeze-etched electron micrograph of nuclear

membranes, with nuclear pores visible and vesicles in the surrounding cytoplasm. The diagram on page 28 shows the line of fracture through the centre of the inner and outer nuclear membranes. Transmembrane proteins are visible in both of the membranes

27

1

C E LL B I O LO G Y

periphery o the membrane. Also the proteins were hydrophobic on at least part o their surace so they would be attracted to the hydrocarbon tails o the phospholipids in the centre o the membrane. 

Fluorescent antibody tagging. Red or green fuorescent markers were attached to antibodies that bind to membrane proteins. The membrane proteins o some cells were tagged with red markers and other cells with green markers. The cells were used together. Within 40 minutes the red and green markers were mixed throughout the membrane o the used cell. This showed that membrane proteins are ree to move within the membrane rather than being xed in a peripheral layer.

Taken together, this experimental evidence alsied the D avsonD anielli model. A

replacement was needed that tted the evidence and the model that became widely accepted was the S ingerNicolson fuid mosaic model. It has been the leading model or over ty years but it would be unwise to assume that it will never be superseded. There are already some suggested modications o the model. An important maxim or scientists is Think it possible that you might be mistaken. Advances in science happen because scientists rej ect dogma and instead search continually or better understanding. cytoplasm

nucleus

inner membrane outer membrane

Evidence for and against the DavsonDanielli model of membrane structure Analysis of evidence from electron microscopy that led to the proposal of the DavsonDanielli model. Figure 5 shows the plasma membrane o a red blood cell and some o the cytoplasm near the edge o the cell. 1 . D escribe the appearance o the plasma membrane.

[2 ]

2 . Explain how this appearance suggested that the membrane had a central region o phospholipid with layers o protein on either side. [2 ] 3 . Suggest reasons or the dark grainy appearance o the cytoplasm o the red blood cell. [2 ] 4. C alculate the magnication o the electron micrograph assuming that the thickness o the membrane is 1 0 nanometres. [3 ] The two sets o data- based questions that ollow are based on the types o data that were used to alsiy the D avsonD anielli model o membrane structure.

28

 Figure 5 TEM

of plasma membrane of a red blood cell

1 . 3 M e M b rAn e s tru ctu r e

daa-a qio: Membranes in

Difusion o proteins in membranes

reeze-etched electron micrographs

Frye and Edidin used an elegant technique to obtain evidence or the fuid nature o membranes. They attached fuorescent markers to membrane proteins  green markers to mouse cells and red markers to human cells. In both cases, spherical cells growing in tissue culture were used. The marked mouse and human cells were then used together. At rst, the used cells had one green hemisphere and one red one, but over the minutes ollowing usion, the red and green markers gradually merged, until they were completely mixed throughout the whole o the cell membrane. B locking o ATP production did not prevent this mixing ( ATP supplies energy or active processes in the cell) .

Figure 6 shows a reeze- etched electron micrograph image o part o a cell. It was prepared by Proessor Horst Robenek o Mnster University.

tim af cll wih mak flly mix/% fio / rl rl rl rl Ma mi 1 2 3 4

 Figure 6

1

In all o the ractured membranes in the micrograph small granules are visible.

4

0





10

3

0





25

40

54





40

87

88

93

100

120

100







C alculate the mean percentage o cells with markers ully mixed or each time ater usion. [4]

2

Plot a graph o the results, including range bars or times where there was variation in the results. To do this you plot the highest and lowest results with a small bar and j oin these bars with a ruled line. You should also plot the mean result with a cross. This will lie on the range bar. [4]

3

D escribe the trend shown by the graph.

[1 ]

Identiy three mitochondria visible in the micrograph, either using labels or by describing their positions. [2 ]

4

Explain whether the results t the D avsonD anielli model or the S ingerNicolson model more closely.

[2 ]

Explain the evidence rom the micrograph that this cell was processing proteins in its cytoplasm. [2 ]

5

Explain the benet o plotting range bars on graphs. [2 ]

6

During this experiment the cells were incubated at 37 C . Suggest a reason or the researchers choosing this temperature. [1 ]

b) E xplain the signicance o these granules in the investigation o membrane structure.

3

0

1

a) S tate what these granules are.

2

5

[2 ]

[3 ]

O ne o the membranes that surround the nucleus is visible on the let o the micrograph. D educe whether it is the inner or outer nuclear membrane. ( Always give your reasons when asked to deduce something.) [2 ]

Extension questions on this topic can be ound at www.oxordsecondary. co.uk/ib- biology

29

1

7

8

9

The experiment was repeated at dierent temperatures. Figure 7 shows the results. Explain the trends shown in the graph or temperatures between 1 5 and 3 5 C . [2 ] Explain the trends shown in the graph or temperatures below 1 5 C . [2 ] When ATP synthesis was blocked in the cells, the mixing o the red and green markers still occurred. E xplain what conclusion can be drawn rom this. [1 ]

1 0 Predict, with reasons, the results o the experiment i it was repeated using cells rom arctic fsh rather than rom mice or humans.

% of cells with markers fully mixed after 40 minutes

C E LL B I O LO G Y

100

1 1 1 1 1 1

50

0

1 1

1 1

1 1

5

15 25 35 incubation temperature (C)

 Figure 7

Eect o temperature on the rate o diusion o fuorescent markers in membranes

[1 ]

Membrane proteins Membrane proteins are diverse in terms o structure, position in the membrane and unction. C ell membranes have a wide range o unctions. The primary unction is to orm a barrier through which ions and hydrophilic molecules cannot easily pass. This is carried out by the phospholipid bilayer. Almost all other unctions are carried out by proteins in the membrane. Six examples are listed in table 1 .

functions o membrane proteins Hormone binding sites (also called hormone receptors) , or example the insulin receptor. Figure 8 shows an example. Immobilized enzymes with the active site on the outside, or example in the small intestine. Cell adhesion to orm tight junctions between groups o cells in tissues and organs. Cell-to-cell communication, or example receptors or neurotransmitters at synapses. Channels or passive transport to allow hydrophilic particles across by acilitated difusion. Pumps or active transport which use ATP to move particles across the membrane.  Table 1

 Figure 8

Hormone receptor ( purple) embedded in phospholipid bilayer (grey) . The hormone (blue/red) is thyroid stimulating hormone. G-protein (brown) conveys the hormone's message to the interior o the cell

30

B ecause o these varied unctions, membrane proteins are very diverse in structure and in their position in the membrane. They can be divided into two groups. 

Integral proteins are hydrophobic on at least part o their surace and they are thereore embedded in the hydrocarbon chains in the centre o the membrane. Many integral proteins are transmembrane  they extend across the membrane, with hydrophilic parts proj ecting through the regions o phosphate heads on either side.

1 . 3 M e M b rAn e s tru ctu r e



Peripheral proteins are hydrophilic on their surace, so are not embedded in the membrane. Most o them are attached to the surace o integral proteins and this attachment is oten reversible. Some have a single hydrocarbon chain attached to them which is inserted into the membrane, anchoring the protein to the membrane surace.

Figure 9 includes examples o both types o membrane protein. Membranes all have an inner ace and an outer ace and membrane proteins are orientated so that they can carry out their unction correctly. For example, pump proteins in the plasma membranes o root cells in plants are orientated so that they pick up potassium ions rom the soil and pump them into the root cell. The protein content o membranes is very variable, because the unction o membranes varies. The more active a membrane, the higher is its protein content. Membranes in the myelin sheath around nerve fbres j ust act as insulators and have a protein content o only 1 8% . The protein content o most plasma membranes on the outside o the cell is about 5 0% . The highest protein contents are in the membranes o chloroplasts and mitochondria, which are active in photosynthesis and respiration. These have protein contents o about 75 % .

dawig mma  Draw the fuid mosaic model o membrane structure. The structure o membranes is ar too complicated or us to show all o it in ull detail in a drawing, but we can show our understanding o it using symbols to represent the molecules present. A diagram o membrane structure is shown in fgure 9.

 Figure 9

The diagram shows these components o a membrane: 

phospholipids;



integral proteins;



peripheral proteins;



cholesterol.

Membrane structure

31

1

C E LL B I O LO G Y

Identiy which each component in the diagram is. Using similar symbols to represent the components draw the structure o a membrane, according to the fuid mosaic model, that contains these proteins: channels or acilitated diusion, pumps or active transport, immobilized enzymes and receptors or hormones or neurotransmitters. It is worth thinking about what you have been doing when you draw the fuid mosaic model o membrane structure. D rawings simpliy and interpret a structure or process. They are used in science as visual explanations. They show our understanding o a structure or process and not merely what it looks like. D rawings are based on models, hypotheses or theories. For example, when we show an animal tissue as a group o cells with lines to represent the plasma membranes, we are basing our drawing on the cell theory. A diagram in a book or scientic paper usually starts out as a drawing on paper by the author, which is tidied up to make it suitable or printing. It is now possible to use computer sotware, but a pencil and paper are perhaps still the best way to draw. No artistic ability is needed or scientic drawing, and all biologists can develop and improve their drawing skills. O  course some biologists produce particularly good drawings. Some examples are shown in gure 1 0.

 Figure 10 Anatomical

drawings by Leonardo da Vinci

Cholesterol in membranes Cholesterol is a component of animal cell membranes. The two main components o cell membranes are phospholipids and proteins. Animal cell membranes also contain cholesterol. CH 3 CH 2 CH 2 CH 3 cholesterol CH 3

CH CH 2 CH

CH 3

CH 3

C holesterol is a type o lipid, but it is not a at or oil. Instead it belongs to a group o substances called steroids. Most o a cholesterol molecule is hydrophobic so it is attracted to the hydrophobic hydrocarbon tails in the centre o the membrane, but one end o the cholesterol molecule has a hydroxyl ( - O H) group which is hydrophilic. This is attracted to the phosphate heads on the periphery o the membrane. C holesterol molecules are thereore positioned between phospholipids in the membrane.

HO hydrophilic  Figure 11

32

hydrophobic

The structure of cholesterol

The amount o cholesterol in animal cell membranes varies. In the membranes o vesicles that hold neurotransmitters at synapses as much o 3 0% o the lipid in the membrane is cholesterol.

1 . 4 M e M b r An e trAn s Po r t

The role of cholesterol in membranes Cholesterol in mammalian membranes reduces membrane fuidity and permeability to some solutes. C ell membranes do not correspond exactly to any o the three states o matter. The hydrophobic hydrocarbon tails usually behave as a liquid, but the hydrophilic phosphate heads act more like a solid. O verall the membrane is fuid as components o the membrane are ree to move. The fuidity o animal cell membranes needs to be careully controlled. I they were too fuid they would be less able to control what substances pass through, but i they were not fuid enough the movement o the cell and substances within it would be restricted. C holesterol disrupts the regular packing o the hydrocarbon tails o phospholipid molecules, so prevents them crystallizing and behaving as a solid. However it also restricts molecular motion and thereore the fuidity o the membrane. It also reduces the permeability to hydrophilic particles such as sodium ions and hydrogen ions. D ue to its shape cholesterol can help membranes to curve into a concave shape, which helps in the ormation o vesicles during endocytosis.

1.4 Mma ap Understanding  Particles move across membranes by simple

diusion, acilitated diusion, osmosis and active transport.  The fuidity o membranes allows materials to be taken into cells by endocytosis or released by exocytosis.  Vesicles move materials within cells.

Nature of science  Experimental design: accurate quantitative

measurements in osmosis experiments are essential.

Applications  Structure and unction o sodiumpotassium

pumps or active transport and potassium channels or acilitated diusion in axons.  Tissues or organs to be used in medical procedures must be bathed in a solution with the same osmolarity as the cytoplasm to prevent osmosis.

Skills  Estimation o osmolarity in tissues by bathing

samples in hypotonic and hypertonic solutions.

33

1

C E LL B I O LO G Y

outside of cell

endocytosis

Endocytosis The fuidity o membranes allows materials to be taken into cells by endocytosis or released by exocytosis.

cell interior

A vesicle is a small sac o membrane with a droplet o fuid inside. Vesicles are spherical and are normally present in eukaryotic cells. They are a very dynamic eature o cells. They are constructed, moved around and then deconstructed. This can happen because o the fuidity o membranes, which allows structures surrounded by a membrane to change shape and move. To orm a vesicle, a small region o a membrane is pulled rom the rest o the membrane and is pinched o. Proteins in the membrane carry out this process, using energy rom ATP. Vesicles can be ormed by pinching o a small piece o the plasma membrane o cells. The vesicle is ormed on the inside o the plasma membrane. It contains material that was outside the cell, so this is a method o taking materials into the cell. It is called endocytosis. Figure 1 shows how the process occurs.

vesicle

 Figure 1

Endocytosis

Vesicles taken in by endocytosis contain water and solutes rom outside the cell but they also oten contain larger molecules needed by the cell that cannot pass across the plasma membrane. For example, in the placenta, proteins rom the mothers blood, including antibodies, are absorbed into the etus by endocytosis. S ome cells take in large undigested ood particles by endocytosis. This happens in unicellular organisms including Amoeba and Paramecium. S ome types o white blood cells take in pathogens including bacteria and viruses by endocytosis and then kill them, as part o the bodys response to inection.

Vesicle movement in cells Vesicles move materials within cells. Vesicles can be used to move materials around inside cells. In some cases it is the contents o the vesicle that need to be moved. In other cases it is proteins in the membrane o the vesicle that are the reason or vesicle movement. An example o moving the vesicle contents occurs in secretory cells. Protein is synthesized by ribosomes on the rough endoplasmic reticulum ( rE R) and accumulates inside the rE R. Vesicles containing the proteins bud o the rE R and carry them to the Golgi apparatus. The vesicles use with the Golgi apparatus, which processes the protein into its nal orm. When this has been done, vesicles bud o the Golgi apparatus and move to the plasma membrane, where the protein is secreted. In a growing cell, the area o the plasma membrane needs to increase. Phospholipids are synthesized next to the rER and become inserted into the rER membrane. Ribosomes on the rER synthesize membrane proteins which also become inserted into the membrane. Vesicles bud o the rE R and move to the plasma membrane. They use with it, each

34

1 . 4 M e M b r An e trAn s Po r t

increasing the area of the plasma membrane by a very small amount. This method can also be used to increase the size of organelles in the cytoplasm such as lysosomes and mitochondria. Proteins are synthesized by ribosomes and then enter the rough endoplasmic reticulum

ENDOCYTOSIS Part of the plasma membrane is pulled inwards A droplet of uid becomes enclosed when a vesicle is pinched o Vesicles can then move through the cytoplasm carrying their contents

Vesicles bud o from the rER and carry the proteins to the Golgi apparatus

The Golgi apparatus modies the proteins

outside of cell exocytosis

Vesicles bud o from the Golgi apparatus and carry the modied proteins to the plasma membrane

vesicle

EXOCYTOSIS Vesicles fuse with the plasma membrane The contents of the vesicle are expelled The membrane then attens out again

 Figure 2

Exocytosis The fuidity o membranes allows materials to be taken into cells by endocytosis or released by exocytosis. Vesicles can be used to release materials from cells. If a vesicle fuses with the plasma membrane, the contents are then outside the membrane and therefore outside the cell. This process is called exocytosis. D igestive enzymes are released from gland cells by exocytosis. The polypeptides in the enzymes are synthesized by the rER, processed in the Golgi apparatus and then carried to the membrane in vesicles for exocytosis. In this case the release is referred to as secretion, because a useful substance is being released, not a waste product. E xocytosis can also be used to expel waste products or unwanted materials. An example is the removal of excess water from the cells of unicellular organisms. The water is loaded into a vesicle, sometimes called a contractile vacuole, which is then moved to the plasma membrane for expulsion by exocytosis. This can be seen quite easily in Paramecium, using a microscope. Figure 4 shows a drawing of Paramecium showing a contractile vesicle at each end of the cell.

cell interior 

Figure 3 Exocytosis

contractile vesicle

Simple difusion

mouth

Particles move across membranes by simple diusion, acilitated diusion, osmosis and active transport.

endoplastule

S imple diffusion is one of the four methods of moving particles across membranes. D iffusion is the spreading out of particles in liquids and gases that happens because the particles are in continuous random motion. More particles move from an area of higher concentration to an area of lower concentration than move in the opposite direction. There is therefore a net movement from the higher to the lower concentration  a movement down the concentration gradient. Living

endoplast contractile vesicle

 Figure 4 Drawing of Paramecium

35

1

C E LL B I O LO G Y

toK can he same aa jusify muually exlusive nlusins? In an experiment to test whether NaCl can difuse through dialysis tubing, a 1% solution o NaCl was placed inside a dialysis tube and the tube was clamped shut. The tube containing the solution was immersed in a beaker containing water. A conductivity meter was inserted into the water surrounding the tubing. I the conductivity o the solution increases, then the NaCl is difusing out o the tubing. time /s  1 cnuiviy  10 mg l - 1 0 81.442 30 84.803 60 88.681 90 95.403 120 99.799 Noting the uncertainty o the conductivity probe, discuss whether the data supports the conclusion that NaCl is difusing out o the dialysis tubing.

organisms do not have to use energy to make diusion occur so it is a passive process. S imple diusion across membranes involves particles passing between the phospholipids in the membrane. It can only happen i the phospholipid bilayer is permeable to the particles. Non- polar particles such as oxygen can diuse through easily. I the oxygen concentration inside a cell is reduced due to aerobic respiration and the concentration outside is higher, oxygen will pass into the cell through the plasma membrane by passive diusion. An example is shown in fgure 6 .

 Figure 5 Model

o difusion with dots representing particles

The centre o membranes is hydrophobic, so ions with positive or negative charges cannot easily pass through. Polar molecules, which have partial positive and negative charges over their surace, can diuse at low rates between the phospholipids o the membrane. Small polar particles such as urea or ethanol pass through more easily than large particles. the cornea has no blood supply so its cells obtain oxygen by simple diusion from the air high concentration of oxygen in the air

air

high concentration of oxygen in the tears that coat the cornea

uid (tears) cell on outer surface of the cornea oxygen passes through the plasma membrane by simple diusion

lower concentration of oxygen in the cornea cells due to aerobic respiration

 Figure 6 Passive difusion

daa-base quesins: Difusion o oxygen in the cornea Oxygen concentrations were measured in the cornea o anesthetized rabbits at dierent distances rom the outer surace. These measurements were continued into the aqueous humor behind the cornea. The rabbits cornea is 400 micrometres (400 m) thick. The graph (fgure 7) shows the measurements. You may need to look at a diagram o eye structure beore answering the questions. The oxygen concentration in normal air is 2 0 kilopascals ( 2 0 kPa) .

36

1

C alculate the thickness o the rabbit cornea in millimetres. [1 ]

2

a) D escribe the trend in oxygen concentrations in the cornea rom the outer to the inner surace.

[2 ]

b) Suggest reasons or the trend in oxygen concentration in the cornea. [2 ] 3

a) C ompare the oxygen concentrations in the aqueous humor with the concentrations in the cornea. [2 ]

1 . 4 M e M b r An e trAn s Po r t

20

[2 ]

4

Using the data in the graph, evaluate diffusion as a method of moving substances in large multicellular organisms. [2 ]

5

a) Predict the effect of wearing contact lenses on oxygen concentrations in the cornea.

[1 ]

b) S uggest how this effect could be minimized.

[1 ]

6

The range bars for each data point indicate how much the measurements varied. Explain the reason for showing range bars on the graph. [2 ]

Concentration of oxygen/kPa

b) Using the data in the graph, deduce whether oxygen diffuses from the cornea to the aqueous humor.

15

10

5

0 0

100 200 300 400 distance from outer surface of cornea/m

 Figure 7

Facilitated difusion Particles move across membranes by simple difusion, acilitated difusion, osmosis and active transport. Facilitated diffusion is one of the four methods of moving particles across membranes. Ions and other particles that cannot diffuse between phospholipids can pass into or out of cells if there are channels for them through the plasma membrane. These channels are holes with a very narrow diameter. The walls of the channel consist of protein. The diameter and chemical properties of the channel ensure that only one type of particle passes through, for example sodium ions, or potassium ions, but not both. B ecause these channels help particles to pass through the membrane, from a higher concentration to a lower concentration, the process is called facilitated diffusion. C ells can control which types of channel are synthesized and placed in the plasma membrane and in this way they can control which substances diffuse in and out. Figure 8 shows the structure of a channel for magnesium ions, viewed from the side and from the outside of the membrane. The structure of the protein making up the channel ensures that only magnesium ions are able to pass through the hole in the centre.

(a)

(b) Membrane Cytoplasm

Osmosis Particles move across membranes by simple difusion, acilitated difusion, osmosis and active transport. Osmosis is one of the four methods of moving particles across membranes.

 Figure 8 Magnesium

channel

37

1

C E LL B I O LO G Y Water is able to move in and out o most cells reely. S ometimes the number o water molecules moving in and out is the same and there is no net movement, but at other times more molecules move in one direction or the other. This net movement is osmosis.

 Figure 9

O smosis is due to dierences in the concentration o substances dissolved in water ( solutes) . Substances dissolve by orming intermolecular bonds with water molecules. These bonds restrict the movement o the water molecules. Regions with a higher solute concentration thereore have a lower concentration o water molecules ree to move than regions with a lower solute concentration. B ecause o this there is a net movement o water rom regions o lower solute concentration to regions with higher solute concentration. This movement is passive because no energy has to be expended directly to make it occur. O smosis can happen in all cells because water molecules, despite being hydrophilic, are small enough to pass though the phospholipid bilayer. Some cells have water channels called aquaporins, which greatly increase membrane permeability to water. E xamples are kidney cells that reabsorb water and root hair cells that absorb water rom the soil. At its narrowest point, the channel in an aquaporin is only slightly wider than water molecules, which thereore pass through in single fle. Positive charges at this point in the channel prevent protons (H+ ) rom passing through.

Active transport Particles move across membranes by simple difusion, acilitated difusion, osmosis and active transport. Active transport is one o the our methods o moving particles across membranes. C ells sometimes take in substances, even though there is already a higher concentration inside than outside. The substance is absorbed against the concentration gradient. Less commonly, cells sometimes pump substances out, even though there is already a larger concentration outside. This type o movement across membranes is not diusion and energy is needed to carry it out. It is thereore called active transport. Most active transport uses a substance called ATP as the energy supply or this process. E very cell produces its own supply o ATP by cell respiration. Active transport is carried out by globular proteins in membranes, usually called pump proteins. The membranes o cells contain many dierent pump proteins allowing the cell to control the content o its cytoplasm precisely.

 Figure 10

38

Action of a pump protein

Figure 1 0 illustrates how a pump protein works. The molecule or ion enters the pump protein and can reach as ar as a central chamber. A conormational change to the protein takes place using energy rom ATP. Ater this, the ion or molecule can pass to the opposite side o the membrane and the pump protein returns to its original conormation. The pump protein shown transports Vitamin B 1 2 into E. coli.

1 . 4 M e M b r An e trAn s Po r t

daa-a qui: Phosphate absorption in barley roots Roots were cut off from barley plants and were used to investigate phosphate absorption. Roots were placed in phosphate solutions and air was bubbled through. The phosphate concentration was the same in each case, but the percentage of oxygen and nitrogen was varied in the air bubbled through. The rate of phosphate absorption was measured. Table 1 shows the results. 1

2

Describe the effect of reducing the oxygen concentration below 21 .0% on the rate of phosphate absorption by roots. You should only use information from the table in your answer. [3 ] Explain the effect of reducing the oxygen percentage from 2 1 .0 to 0.1 on phosphate absorption. In your answer you should use as much biological understanding as possible of how cells absorb mineral ions.

4

 Table 1 0 .4 0 .3

[3 ]

Phosphate absorption /mol g2 1 h 2 1

0 .2 0 .1 0

An experiment was done to test which method of membrane transport was used by the roots to absorb phosphate. Roots were placed in the phosphate solution as before, with 2 1 .0% oxygen bubbling through. Varying concentrations of a substance called D NP were added. D NP blocks the production of ATP by aerobic cell respiration. Figure 1 1 shows the results of the experiment. 3

oxyg nig Phpha /% /% api/ml g1 h 1 0.1 99.9 0.07 0.3 99.7 0.15 0.9 99.1 0.27 2.1 97.1 0.32 21.0 79.0 0.33

0

2

4

6

8

10

DNP concentration / mmol dm 2 3  Figure 11

Efect o DNP concentration on phosphate absorption

D educe, with a reason, whether the roots absorbed the phosphate by diffusion or active transport.

[2 ]

D iscuss the conclusions that can be drawn from the data in the graph about the method of membrane transport used by the roots to absorb phosphate.

[2 ]

Active transport of sodium and potassium in axons Structure and function of sodiumpotassium pumps for active transport. An axon is part of a neuron ( nerve cell) and consists of a tubular membrane with cytoplasm inside. Axons can be as narrow as one micrometre in diameter, but as long as one metre. Their function is to convey messages rapidly from one part of the body to another in an electrical form called a nerve impulse. A nerve impulse involves rapid movements of sodium and then potassium ions across the axon membrane. These movements occur by facilitated diffusion through sodium and potassium channels. They occur because of concentration gradients between the inside and outside of the axon. The concentration gradients are built up by active transport, carried out by a sodium potassium pump protein.

The sodiumpotassium pump follows a repeating cycle of steps that result in three sodium ions being pumped out of the axon and two potassium ions being pumped in. Each time the pump goes round this cycle it uses one ATP. The cycle consists of these steps: 1

The interior of the pump is open to the inside of the axon; three sodium ions enter the pump and attach to their binding sites.

2

ATP transfers a phosphate group from itself to the pump; this causes the pump to change shape and the interior is then closed.

3

The interior of the pump opens to the outside of the axon and the three sodium ions are released.

39

1

C E LL B I O LO G Y

4

Two potassium ions from outside can then enter and attach to their binding sites.

5

B inding of potassium causes release of the phosphate group; this causes the pump to change shape again so that it is again only open to the inside of the axon. 1

6

The interior of the pump opens to the inside of the axon and the two potassium ions are released; sodium ions can then enter and bind to the pump again ( stage 1 ) .

2

3

p

p

ATP ADP

4

5

6

p p  Figure 12

Active transport in axons

Facilitated difusion o potassium in axons Structure and unction o sodiumpotassium pumps or active transport and potassium channels or acilitated difusion in axons. A nerve impulse involves rapid movements of sodium and then potassium ions across the axon membrane. These movements occur by facilitated diffusion through sodium and potassium channels. Potassium channels will be described here as a special example of facilitated diffusion. E ach potassium channel consists of four protein subunits with a narrow pore between them that allows potassium ions to pass in either direction. The pore is 0.3 nm wide at its narrowest.

40

Potassium ions are slightly smaller than 0 . 3 nm, but when they dissolve they become bonded to a shell of water molecules that makes them too large to pass through the pore. To pass through, the bonds between the potassium ion and the surrounding water molecules are broken and bonds form temporarily between the ion and a series of amino acids in the narrowest part of the pore. After the potassium ion has passed through this part of the pore,

1 . 4 M e M b r An e trAn s Po r t

it can again become associated with a shell o water molecules. Other positively charged ions that we might expect to pass through the pore are either too large to t through or are too small to orm bonds with the amino acids in the narrowest part o the pore, so they cannot shed their shell o water molecules. This explains the specicity o the pump. Potassium channels in axons are voltage gated. Voltages across membranes are due to an imbalance o positive and negative charges across the membrane. I an axon has relatively more

positive charges outside than inside, potassium channels are closed. At one stage during a nerve impulse there are relatively more positive charges inside. This causes potassium channels to open, allowing potassium ions to diuse through. However, the channel rapidly closes again. This seems to be due to an extra globular protein subunit or ball, attached by a fexible chain o amino acids. The ball can t inside the open pore, which it does within milliseconds o the pore opening. The ball remains in place until the potassium channel returns to its original closed state. This is shown in gure 1 3 .

1 channel closed +

+

+

2 channel briey open - - - + + + +

+ +++ - - -

-

-

+ + + +

+ + + +

+

++ + + - - - chain ball

net negative charge inside the axon and net positive charge outside

net negative charge

K+ ions

- - - + + + +

+ + + inside of axon

outside

net positive charge

3 channel closed by ball and chain -

-

- + + + +

+

+

+

+

hydrophobic core of the membrane

- + + + +

+

+

-

+

-

+

hydrophilic outer parts of the membrane

 Figure 13

eimai f mlaiy Estimation of osmolarity in tissues by bathing samples in hypotonic and hypertonic solutions. O smosis is due to solutes that orm bonds with water. These solutes are osmotically active. Glucose, sodium ions, potassium ions and chloride ions are all osmotically active and solutions o them are oten used in osmosis experiments. C ells contain many dierent osmotically active solutes. The osmolarity o a solution is the total concentration o osmotically active solutes. The

units or measuring it are osmoles or milliosmoles ( mO sm) . The normal osmolarity o human tissue is about 3 00 mO sm. An isotonic solution has the same osmolarity as a tissue. A hypertonic solution has a higher osmolarity and a hypotonic solution has a lower osmolarity. I samples o a tissue are bathed in hypertonic and hypotonic solutions, and

41

1

C E LL B I O LO G Y

measurements are taken to fnd out whether water enters or leaves the tissue, it is possible to deduce what concentration o solution would be

data-base questions: Osmosis in plant tissues

isotonic and thereore fnd out the osmolarity o the tissue. The data- based questions below give the results rom an experiment o this type. 4

I samples o plant tissue are bathed in salt or sugar solutions or a short time, any increase or decrease in mass is due almost entirely to water entering or leaving the cells by osmosis. Figure 1 4 shows the percentage mass change o our tissues, when they were bathed in salt solutions o dierent concentrations. 1

a) S tate whether water moved into or out o the tissues at 0.0 mol dm 3 sodium chloride solution. [1 ]

40 +

3

The experiment in the data- based question can be repeated using potato tubers, or any other plant tissue rom around the world that is homogeneous and tough enough to be handled without disintegrating. D iscuss with a partner or group how you could do the ollowing things:

42

1

D ilute a 1 mol dm 3 sodium chloride solution to obtain the concentrations shown on the graph.

2

O btain samples o a plant tissue that are similar enough to each other to give comparable results.

3

Ensure that the surace o the tissue samples is dry when fnding their mass, both at the start and end o the experiment.

4

Ensure that all variables are kept constant, apart rom salt concentration o the bathing solution.

+

+

+ +

+

+

20

% Mass change

PINE KERNEL

+

Sodium chloride concentration / mol dm 2 3

10

0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

0 .9

1.0

BUTTERNUT SQUASH SWEET POTATO

2 10

2 20

2 30

D educe which tissue had the lowest solute concentration in its cytoplasm. Include how you reached your conclusion in your answer. [2 ] S uggest reasons or the dierences in solute concentration between the tissues. [3 ]

+

30

b) State whether water moved into or out o the tissues at 1 .0 mol dm 3 sodium chloride solution. [2 ] 2

Explain the reasons or using percentage mass change rather than the actual mass change in grams in this type o experiment. [2 ]

2 40

CACTUS

2 50

 Figure 14 Mass changes in

plant tissues bathed in

salt solutions

5

Leave the tissue in the solutions or long enough to get a signifcant mass change, but not so long that another actor aects the mass, such as decomposition!

6

You might choose to be more inventive in your experimental approach. Figure 1 5 gives one idea or measuring changes to the turgidity o plant tissue, but other methods could be used.

 plant tissue

angle gives measure of turgidity

weight  Figure 15 Method

of plant tissue

of assessing turgidity

1 . 4 M e M b r An e trAn s Po r t

expimal dig Experimental design: accurate quantitative measurements in osmosis experiments are essential. An ideal experiment gives results that have only one reasonable interpretation. C onclusions can be drawn from the results without any doubts or uncertainties. In most experiments there are some doubts and uncertainties, but if the design of an experiment is rigorous, these can be minimized. The experiment then provides strong evidence for or against a hypothesis. This checklist can be used when designing an experiment: 

Results should if possible be quantitative as these give stronger evidence than descriptive results.



Measurements should be as accurate as possible, using the most appropriate and best quality meters or other apparatus.



Repeats are needed, because however accurately quantitative measurements are taken biological samples are variable.



All factors that might affect the results of the experiment must be controlled, with only the factors under investigation being allowed to vary and all other factors remaining constant.

After doing an experiment the design can be evaluated using this checklist. The evaluation might lead to improvements to the design that would have made the experiment more rigorous.

 Figure 16 Replicates are needed

for each treatment in a rigorous experiment

If you have done an osmosis experiment in which samples of plant tissue are bathed in solutions of varying solute concentration, you can evaluate its design. If you did repeats for each concentration of solution, and the results were very similar to each other, your results were probably reliable.

Designing osmosis experiments Rigorous experimental design is needed to produce reliable results: how can accurate quantitative measurements be obtained in osmosis experiments? The osmolarity of plant tissues can be investigated in many ways. Figure 1 7 shows some red onion cells that had been placed in a sodium chloride solution. The following method can be used to observe the consequences of osmosis in red onion cells. 1

Peel off some epidermis from the scale of a red onion bulb.

2

C ut out a sample of it, about 5  5 mm.

3

Mount the sample in a drop of distilled water on a microscope slide, with a cover slip.

 Figure 17

Micrograph of red onion cells placed in salt solution

43

1

C E LL B I O LO G Y

4

O bserve using a microscope. The cytoplasm should fll the space inside the cell wall, with the plasma membrane pushed up against it.

5

Mount another sample o epidermis in sodium chloride solutions with concentration o 0.5 mol dm - 3 or 3 % . I water leaves the cells by osmosis and the volume o cytoplasm is reduced, the plasma membrane pulls away rom the cell wall, as shown in Figure 1 7. Plant cells with their membranes pulled away rom their cell walls are plasmolysed and the process is plasmolysis.

This method can be used to help design an experiment to fnd out the osmolarity o onion cells or other cells in which the area occupied by the cytoplasm can easily be seen. The checklist in the previous section can be used to try to ensure that the design is rigorous.

Preventing osmosis in excised tissues and organs Tissues or organs to be used in medical procedures must be bathed in a solution with the same osmolarity as the cytoplasm to prevent osmosis. Animal cells can be damaged by osmosis. Figure 1 8 shows blood cells that have been a)

b)

 Figure 18

c)

Blood cells bathed in solutions o diferent solute concentration

In a solution with higher osmolarity ( a hypertonic solution) , water leaves the cells by osmosis so their cytoplasm shrinks in volume. The area o plasma membrane does not change, so it develops indentations, which are sometimes called crenellations. In a solution with lower osmolarity ( hypotonic) , the cells take in water by osmosis and swell up. They may eventually burst, leaving ruptured plasma membranes called red cell ghosts. B oth hypertonic and hypotonic solutions thereore damage human cells, but in a solution with same osmolarity as the cells ( isotonic) , water molecules enter and leave the cells at the same rate so they remain healthy. It is thereore important or any human tissues and organs to be bathed in an isotonic solution during medical procedures. Usually an isotonic sodium chloride solution is

44

bathed in solutions with ( a) the same osmolarity, ( b) higher osmolarity and ( c) lower osmolarity.

used, which is called normal saline. It has an osmolarity o about 3 00 mO sm ( milliO smoles) . Normal saline is used in many medical procedures. It can be: 

saely introduced to a patients blood system via an intravenous drip.



used to rinse wounds and skin abrasions.



used to keep areas o damaged skin moistened prior to skin grats.



used as the basis or eye drops.



rozen to the consistency o slush or packing hearts, kidneys and other donor organs that have to be transported to the hospital where the transplant operation is to be done.

1 . 5 tH e o rI GI n o f ce lls

 Figure 19

Donor liver packed in an isotonic medium, surrounded by isotonic slush. There is a worldwide shortage of donor organs  in most countries it is possible to register as a possible future donor

1.5 th igi   Understanding  Cells can only be ormed by division o

pre-existing cells.  The frst cells must have arisen rom non-living material.  The origin o eukaryotic cells can be explained by the endosymbiotic theory.

Applications  Evidence rom Pasteurs experiments that

spontaneous generation o cells and organisms does not now occur on Earth.

Nature of science  Testing the general principles that underlie the

natural world: the principle that cells only come rom pre-existing cells needs to be verifed.

Cell division and the origin of cells Cells can only be ormed by division o pre-existing cells. Since the 1 880s there has been a theory in biology that cells can only be produced by division of a pre-existing cell. The evidence for this hypothesis is very strong and is discussed in the nature of science panel below. The implications of the hypothesis are remarkable. If we consider the trillions of cells in our bodies, each one was formed when a previously

45

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C E LL B I O LO G Y

toK Wha d we gain, and wha d we le, when we name mehing? When Dr Craig Venters team announced that they had succeeded in transplanting the synthetic genome rom one bacterium into another bacterium in the journal Science some ethicists responded by questioning the language o calling it the creation o a synthetic cell: The science is ying 30,000 eet over the publics understanding ... Scientists can be their own worst enemy by using words like clone or synthetic lie. Glenn Mcgee, funde f Ameican Junal f biehic Frankly, hes describing it in a way thats drumming up controversy more than characterising it accurately. His claim that weve got the frst selreplicating lie orm whose parent is a computer, thats just silly. It misuses the word parent. The advance here needs to be described in sane and accurate ways. What he's managed to do is synthesise a genome much larger than any genome thats been synthesised rom scratch beore. Gegy Kaenick, Haing Iniue reeach schla

existing cell divided in two. B eore that all o the genetic material in the nucleus was copied so that both cells ormed by cell division had a nucleus with a ull complement o genes. We can trace the origin o cells in the body back to the frst cell  the zygote that was the start o our lives, produced by the usion o a sperm and an egg. S perm and egg cells were produced by cell division in our parents. We can trace the origins o all cells in our parents bodies back to the zygote rom which they developed, and then continue this process over the generations o our human ancestors. I we accept that humans evolved rom pre- existing ancestral species, we can trace the origins o cells back through hundreds o millions o years to the earliest cells on Earth. There is thereore a continuity o lie rom its origins on Earth to the cells in our bodies today. In 2 01 0 there were reports that biologists had created the frst artifcial cell, but this cell was not entirely new. The base sequence o the D NA o a bacterium ( Mycoplasma mycoides) was synthesized artifcially, with a ew deliberate changes. This D NA was transerred to pre- existing cells o a dierent type o bacterium ( Mycoplasma capricolum) , which was eectively converted into Mycoplasma mycoides. This process was thereore an extreme orm o genetic modifcation and the creation o entirely new cells remains an insuperable challenge at the moment.

Aciviy the l f silphium The Greek coin in fgure 2 depicts a Silphium plant, which grew in a small part o what is now Libya and was highly prized or its medicinal uses, especially as a birth control agent. It seems to have been so widely collected that within a ew hundred years o the ancient Greeks colonizing North Arica it had become extinct. Rather than arising again spontaneously, Silphium has remained extinct and we cannot now test its contraceptive properties scientifcally. How can we prevent the loss o other plants that could be o use to us?

 Figure 2  Figure 1

46

Synthetic Mycoplasma bacteria

An ancient Greek coin, showing Silphium

1 . 5 tH e o rI GI n o f ce lls

Spontaneous generation and the origin of cells Veriying the general principles that underlie the natural world: the principle that cells only come rom pre-existing cells needs to be verifed. Spontaneous generation is the ormation o living organisms rom non-living matter. The Greek philosopher and botanist Theophrastus reported that a plant called Silphium had sprung up rom soil where it was not previously present and described this as an example o spontaneous generation. Aristotle wrote about insects being ormed rom the dew alling on leaves or rom the hair, fesh or aeces o animals. In the 1 6th century the GermanSwiss botanist and astrologer Paracelsus quoted observations o spontaneous generation o mice, rogs and eels rom water, air or decaying matter. It is easy to see how ideas o spontaneous generation could have persisted when cells and microorganisms had not been discovered and the nature o sexual reproduction was not understood. From the 1 7th century onwards biologists carried out experiments to test the theory that lie could arise rom non-living matter. Francesco Redi showed that maggots only developed in rotting meat i fies were allowed to come into contact with it. Lazzaro Spallanzani boiled soup in eight containers, then sealed our o them and let the others open to the air. Organisms grew in the containers let open but not in the others.

S ome biologists remained convinced that spontaneous generation could occur i there was access to the air. Louis Pasteur responded by carrying out careully designed experiments with swan- necked fasks, which established beyond reasonable doubt that spontaneous generation o lie does not now occur. Pasteurs experiments are described in the next section o this sub- topic. Apart rom the evidence rom the experiments o Pasteur and others, there are other reasons or biologists universally accepting that cells only come rom pre- existing cells: 

A cell is a highly complex structure and no natural mechanism has been suggested or producing cells rom simpler subunits.



No example is known o increases in the number o cells in a population, organism or tissue without cell division occurring.



Viruses are produced rom simpler subunits but they do not consist o cells, and they can only be produced inside the host cells that they have inected.

Spontaneous generation and Pasteurs experiments Evidence rom Pasteurs experiments that spontaneous generation o cells and organisms does not now occur on Earth. Louis Pasteur made a nutrient broth by boiling water containing yeast and sugar. He showed that i this broth was kept in a sealed fask, it remained unchanged, and no ungi or other organisms appeared. He then passed air though a pad o cotton wool in a tube, to lter out microscopic particles rom the air, including bacteria and the spores o ungi. I the pad o cotton wool was placed in broth in a sealed fask, within 3 6 hours, there were large number o microorganisms in the broth and mould grew over its surace. The most amous o Pasteurs experiments involved the use o swan-necked fasks. He placed samples o broth in fasks with long necks and

then melted the glass o the necks and bent it into a variety o shapes, shown in gure 3 . Pasteur then boiled the broth in some o the fasks to kill any organisms present but let others unboiled as controls. Fungi and other organisms soon appeared in the unboiled fasks but not in the boiled ones, even ater long periods o time. The broth in the fasks was in contact with air, which it had been suggested was needed or spontaneous generation, yet no spontaneous generation occurred. Pasteur snapped the necks o some o the fasks to leave a shorter vertical neck. Organisms were soon apparent in these fasks and decomposed the broth.

47

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C E LL B I O LO G Y

Pasteur published his results in 1 860 and subsequently repeated them with other liquids including urine and milk, with the same results. He concluded that the swan necks prevented organisms

rom the air getting into the broth or other liquids and that no organisms appeared spontaneously. His experiments convinced most biologists, both at the time o publication and since then.

Origin o the frst cells The frst cells must have arisen rom non-living material. I we trace back the ancestry o cells over billions o years, we must eventually reach the earliest cells to have existed. These were the frst living things on Earth. Unless cells arrived on E arth rom somewhere else in the universe, they must have arisen rom non- living material. This is a logical conclusion, but it gives perhaps the hardest question o all or biologists to answer: how could a structure as complex as the cell have arisen by natural means rom non-living material? It has sometimes been argued that complex structures cannot arise by evolution, but there is evidence that this can happen in a series o stages over long periods o time. Living cells may have evolved over hundreds o millions o years. There are hypotheses or how some o the main stages could have occurred.

 Figure 3

Drawings o Pasteurs swan-necked fasks

1. Production of carbon compounds such as sugars and amino acids

2. Assembly of carbon compounds into polymers

S tanley Miller and Harold Urey passed steam through a mixture o methane, hydrogen and ammonia. The mixture was thought to be representative o the atmosphere o the early E arth. E lectrical discharges were used to simulate lightning. They ound that amino acids and other carbon compounds needed or lie were produced.

A possible site or the origin o the frst carbon compounds is around deep- sea vents. These are cracks in the Earths surace, characterized by gushing hot water carrying reduced inorganic chemicals such as iron sulphide. These chemicals represent readily accessible supplies o energy, a source o energy or the assembly o these carbon compounds into polymers.

water vapour

ammonia (NH 3 ) methane (CH 4) hydrogen (H 2 )

electrode

condenser

cold water in cooled water containing organic compounds  Figure 5 Deep sea

sample taken for chemical analysis  Figure 4 Miller and

48

Ureys apparatus

vents

1 . 5 tH e o rI GI n o f ce lls

3. Formation of membranes I phospholipids or other amphipathic carbon compounds were among the frst carbon compounds, they would have naturally assembled into bilayers. Experiments have shown that these bilayers readily orm vesicles resembling the plasma membrane o a small cell. This would have allowed dierent internal chemistry rom that o the surroundings to develop.

4. Development of a mechanism for inheritance Living organisms currently have genes made o D NA and use enzymes as catalysts. To replicate D NA and be able to pass genes on to ospring, enzymes are needed. However, or enzymes to be made, genes are needed. The solution to this conundrum may have been an earlier phase in evolution when RNA was the genetic material. It can store inormation in the same way as D NA but it is both sel- replicating and can itsel act as a catalyst.

 Figure 6 Liposomes

Endosymbiosis and eukaryotic cells The origin o eukaryotic cells can be explained by the endosymbiotic theory. The theory o endosymbiosis helps to explain the evolution o eukaryotic cells. It states that mitochondria were once ree- living prokaryotic organisms that had developed the process o aerobic cell respiration. Larger prokaryotes that could only respire anaerobically took them in by endocytosis. Instead o killing and digesting the smaller prokaryotes they allowed them to continue to live in their cytoplasm. As long as the smaller prokaryotes grew and divided as ast as the larger ones, they could persist indefnitely inside the larger cells. According to the theory o endosymbiosis they have persisted over hundreds o millions o years o evolution to become the mitochondria inside eukaryotic cells today. The larger prokaryotes and the smaller aerobically respiring ones were in a symbiotic relationship in which both o them benefted. This is known as a mutualistic relationship. The smaller cell would have been supplied with ood by the larger one. The smaller cell would have carried out aerobic respiration to supply energy efciently to the larger cell. Natural selection thereore avoured cells that had developed this endosymbiotic relationship. The endosymbiotic theory also explains the origin o chloroplasts. I a prokaryote that had developed photosynthesis was taken in by a larger cell and was allowed to survive, grow and divide, it could have developed into the chloroplasts o photosynthetic eukaryotes. Again, both o the organisms in the endosymbiotic relationship would have benefted.

Aiviy Wh did i bgi? Erasmus Darwin was Charles Darwins grandather. In a poem entitled The Temple o Nature, published in 1803, he tells us how and where he believed lie to have originated: Organic Lie began beneath the waves ... Hence without parent by spontaneous birth Rise the frst specks o animated earth Has Erasmus Darwins hypothesis that lie began in the sea been alsifed?

49

1

C E LL B I O LO G Y original ancestral prokaryote

Activity

evolution of the nucleus

Bangiomorpha and the origins of sex. The frst known eukaryote and frst known multicellular organism is Bangiomorpha pubescens. Fossils o this red alga were discovered in 1,200 million year old rocks rom northern Canada. It is the frst organism known to produce two dierent types o gamete a larger sessile emale gamete and a smaller motile male gamete. Bangiomorpha is thereore the frst organism known to reproduce sexually. It seems unlikely that eukaryote cell structure, multicellularity and sexual reproduction evolved simultaneously. What is the most likely sequence or these landmarks in evolution?

evolution of photosynthesis

evolution of aerobic respiration

evolution of linear chromosomes, mitosis and meiosis

endocytosis produces mitochondria endocytosis to produce chloroplasts

evolution of plant cells

plant cell (eukaryotic)  Figure 7

evolution of animal cells

animal cell (eukaryotic)

Endosymbiosis

Although no longer capable of living independently, chloroplasts and mitochondria both have features that suggest they evolved from independent prokaryotes:

50



They have their own genes, on a circular D NA molecule like that of prokaryotes.



They have their own 70S ribosomes of a size and shape typical of some prokaryotes.



They transcribe their D NA and use the mRNA to synthesize some of their own proteins.



They can only be produced by division of pre- existing mitochondria and chloroplasts.

1 . 6 ce ll d I VI s I o n

1.6 c ivii Understanding  Mitosis is division o the nucleus into two   

 

genetically identical daughter nuclei. Chromosomes condense by supercoiling during mitosis. Cytokinesis occurs ater mitosis and is dierent in plant and animal cells. Interphase is a very active phase o the cell cycle with many processes occurring in the nucleus and cytoplasm. Cyclins are involved in the control o the cell cycle. Mutagens, oncogenes and metastasis are involved in the development o primary and secondary tumours.

Applications  The correlation between smoking and incidence

o cancers.

Skills  Identifcation o phases o mitosis in cells

viewed with a microscope.  Determination o a mitotic index rom a micrograph.

Nature of science  Serendipity and scientifc discoveries: the

discovery o cyclins was accidental.

The role of mitosis Mitosis is division o the nucleus into two genetically identical daughter nuclei. The nucleus of a eukaryotic cell can divide to form two genetically identical nuclei by a process called mitosis. Mitosis allows the cell to divide into two daughter cells, each with one of the nuclei and therefore genetically identical to the other. B efore mitosis can occur, all of the D NA in the nucleus must be replicated. This happens during interphase, the period before mitosis. E ach chromosome is converted from a single D NA molecule into two identical D NA molecules, called chromatids. D uring mitosis, one of these chromatids passes to each daughter nucleus. Mitosis is involved whenever cells with genetically identical nuclei are required in eukaryotes: during embryonic development, growth, tissue repair and asexual reproduction. Although mitosis is a continuous process, cytologists have divided the events into four phases: prophase, metaphase, anaphase and telophase. The events that occur in these phases are described in a later section of this sub- topic.

Hydra viridissima with a small new polyp attached, produced by asexual reproduction involving mitosis

 Figure 1

51

1

C E LL B I O LO G Y

Interphase

Activity There is a limit to how many times most cells in an organism can undergo mitosis. Cells taken rom a human embryo will only divide between 40 and 60 times, but given that the number o cells doubles with each division, it is easily enough to produce an adult human body. There are exceptions where much greater numbers o divisions can occur, such as the germinal epithelium in the testes. This is a layer o cells that divides to provide cells used in sperm production. Discuss how many times the cells in this layer might need to divide during a man's lie.

Mitosis in esi Cy t o k I N TE

R

PH A SE

s

Each of the 1 chromosomes is duplicated Cellular contents, apart from the chromosomes are duplicated.

G

G0

 Figure 2

The cell cycle is the seque nce o events b etween one cell division and the next. It has two main phases: interphase and cell division. Interphase is a very active phase in the lie o a cell when many metabolic reactions occur. S ome o these, such as the reactions o cell respiration, also occur during cell division, b ut D NA replication in the nucleus and protein synthesis in the cytoplasm only happen during interphase. During interphase the numbers o mitochondria in the cytoplasm increase. This is due to the growth and division o mitochondria. In plant cells and algae the numbers o chloroplasts increase in the same way. They also synthesize cellulose and use vesicles to add it to their cell walls. Interphase consists o three phases, the G 1 phase, S phase and G 2 phase. In the S phase the cell replicates all the genetic material in its nucleus, so that ater mitosis both the new cells have a complete set o genes. Some do not progress beyond G 1 , because they are never going to divide so do not need to prepare or mitosis. They enter a phase called G 0 which may be temporary or permanent.

Supercoiling of chromosomes

G2

S

Interphase is a very active phase o the cell cycle with many processes occurring in the nucleus and cytoplasm.

The cell cycle

Chromosomes condense by supercoiling during mitosis. During mitosis, the two chromatids that make up each chromosome must be separated and moved to opposite poles o the cell. The DNA molecules in these chromosomes are immensely long. Human nuclei are on average less than 5 m in diameter but D NA molecules in them are more than 5 0,000 m long. It is thereore essential to package chromosomes into much shorter structures. This process is known as condensation o chromosomes and it occurs during the frst stage o mitosis. Condensation occurs by means repeatedly coiling the DNA molecule to make the chromosome shorter and wider. This process is called supercoiling. Proteins called histones that are associated with DNA in eukaryote chromosomes help with supercoiling and enzymes are also involved.

Phases of mitosis Identifcation o phases o mitosis in cells viewed with a microscope. There are large numbers o dividing cells in the tips o growing roots. I root tips are treated chemically to allow the cells to be separated, they can be squashed to orm a single layer o cells on a microscope slide. S tains that bind to D NA are used to make the chromosomes visible and stages o mitosis can then be observed using a microscope.

52

To be able to identiy the our stages o mitosis, it is necessary to understand what is happening in them. Ater studying the inormation in this section you should be able to observe dividing cells using a microscope or in a micrograph and assign them to one o the phases.

1 . 6 ce ll d I VI s I o n

Prophase The chromosomes become shorter and atter by coiling. To become short enough they have to coil repeatedly. This is called supercoiling. The nucleolus breaks down. Microtubules grow rom structures called microtubule organizing centres (MTOC) to orm a spindle-shaped array that links the poles o the cell. At the end o prophase the nuclear membrane breaks down.

 Interphase  chromosomes are

 Prophase  nucleoli visible

visible inside the nuclear membrane centromere

MTOC

in the nucleus but no individual chromosomes

microtubules nuclear envelope disintegrates chromosome consisting of two sister chromatids  Early

prophase

spindle microtubules  Late prophase

Metaphase Microtubules continue to grow and attach to the centromeres on each chromosome. The two attachment points on opposite sides o each centromere allow the chromatids o a chromosome to attach to microtubules rom diferent poles. The microtubules are all put under tension to test whether the attachment is correct. This happens by shortening o the microtubules at the centromere. I the attachment is correct, the chromosomes remain on the equator o the cell.

Metaphase plate equator

mitotic spindle

 Metaphase  chromosomes

 Metaphase

aligned on the equator and not inside a nuclear membrane

Anaphase At the start o anaphase, each centromere divides, allowing the pairs o sister chromatids to separate. The spindle microtubules pull them rapidly towards the poles o the cell. Mitosis produces two genetically identical nuclei because sister chromatids are pulled to opposite poles. This is ensured by the way that the spindle microtubules were attached in metaphase.

Daughter chromosomes separate  Anaphase  two groups of V-shaped

chromatids pointing to the two poles

 Anaphase

53

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C E LL B I O LO G Y

Telophase The chromatids have reached the poles and are now called chromosomes. At each pole the chromosomes are pulled into a tight group near the MTOC and a nuclear membrane reforms around them. The chromosomes uncoil and a nucleolus is formed. By this stage of mitosis the cell is usually already dividing and the two daughter cells enter interphase again.

 Telophase  tight groups of

chromosomes at each pole, new cell wall forming at the equator

 Interphase  nucleoli visible

inside the nuclear membranes but not individual chromosomes

Cleavage furrow Nuclear envelope forming  Telophase

data-base questions: Centromeres and telomeres Figure 3 and the other micrographs on the preceeding pages show cells undergoing mitosis. In gure 3 , D NA has been stained blue. The centromeres have been stained with a red fuorescent dye. At the ends o the chromosomes there are structures called telomeres. These have been stained with a green fuorescent dye. 1

D educe the stage o mitosis that the cell was in, giving reasons or your answer. [3 ]

2

The cell has an even number o chromosomes. a)

S tate how many chromosomes there are in this cell.

b) E xplain the reason or body cells in plants and animals having an even number o chromosomes.  Figure 3

Cell in mitosis

c)

[1 ] [2 ]

In the micrograph o a cell in interphase, the centromeres are on one side o the nucleus and the telomeres are on the other side. S uggest reasons or this. [2 ]

d) An enzyme called telomerase lengthens the telomeres, by adding many short repeating base sequences o DNA. This enzyme is only active in the germ cells that are used to produce gametes. When DNA is replicated during the cell cycle in body cells, the end o the telomere cannot be replicated, so the telomere becomes shorter. Predict the consequences or a plant or animal o the shortening o telomeres. [2 ]

54

1 . 6 ce ll d I VI s I o n

The mitotic index Determination o a mitotic index rom a micrograph. The mitotic index is the ratio between the number o cells in mitosis in a tissue and the total number o observed cells. It can be calculated using this equation: number o cells in mitosis Mitotic index = ___ total number o cells Figure 4 is a micrograph o cells rom a tumour that has developed rom a Leydig cell in the testis. The mitotic index or this tumour can be calculated i the total number o cells in the micrograph is counted and also the number o cells in meiosis. To fnd the mitotic index o the part o a root tip where cells are prolierating rapidly, these instructions can be used: 

Obtain a prepared slide o an onion or garlic root tip. Find and examine the meristematic region, i.e. a region o rapid cell division.



C reate a tally chart. C lassiy each o about a hundred cells in this region as being either in interphase or in any o the stages o mitosis.



Use this data to calculate the mitotic index.

Figure 4 Cells undergoing mitosis in a Leydig cell tumour

Cytokinesis Cytokinesis occurs ater mitosis and is diferent in plant and animal cells. C ells can divide ater mitosis when two genetically identical nuclei are present in a cell. The process o cell division is called cytokinesis. It usually begins beore mitosis has actually been completed and it happens in a dierent way in plant and animal cells. In animal cells the plasma membrane is pulled inwards around the equator o the cell to orm a cleavage urrow. This is accomplished using a ring o contractile protein immediately inside the plasma membrane at the equator. The proteins are actin and myosin and are similar to proteins that cause contraction in muscle. When the cleavage urrow reaches the centre, the cell is pinched apart into two daughter cells. In plant cells vesicles are moved to the equator where they use to orm tubular structures across the equator. With the usion o more vesicles these tubular structures merge to orm two layers o membrane across the whole o the equator, which develop into the plasma membranes o the two daughter cells and are connected to the existing plasma membranes at the sides o the cell, completing the division o the cytoplasm. The next stage in plants is or pectins and other substances to be brought in vesicles and deposited by exocytosis between the two new membranes. This orms the middle lamella that will link the new cell walls. B oth o the daughter cells then bring cellulose to the equator and deposit it by exocytosis adj acent to the middle lamella. As a result, each cell builds its own cell wall adj acent to the equator.

 Figure 5 Cytokinesis in

(a) fertilized sea urchin egg (b) cell from shoot tip of Coleus plant

55

1

C E LL B I O LO G Y

Cyclins and the control of the cell cycle Cyclins are involved in the control o the cell cycle. Each o the phases o the cell cycle involves many important tasks. A group o proteins called cyclins is used to ensure that tasks are perormed at the correct time and that the cell only moves on to the next stage o the cycle when it is appropriate. C yclins bind to enzymes called cyclin- dependent kinases. These kinases then become active and attach phosphate groups to other proteins in the cell. The attachment o phosphate triggers the other proteins to become active and carry out tasks specifc to one o the phases o the cell cycle.

concentration

There are our main types o cyclin in human cells. The graph in fgure 6 shows how the levels o these cyclins rise and all. Unless these cyclins reach a threshold concentration, the cell does not progress to the next stage o the cell cycle. Cyclins thereore control the cell cycle and ensure that cells divide when new cells are needed, but not at other times.

G 1 phase

S phase

G 2 phase

mitosis

Cyclin D triggers cells to move from G 0 to G 1 and from G 1 into S phase. Cyclin E prepares the cell for DNA replication in S phase. Cyclin A activates DNA replication inside the nucleus in S phase. Cyclin B promotes the assembly of the mitotic spindle and other tasks in the cytoplasm to prepare for mitosis.  Figure 6

Discovery of cyclins Serendipity and scientifc discoveries: the discovery o cyclins was accidental. During research into the control o protein synthesis in sea urchin eggs, Tim Hunt discovered a protein that increased in concentration ater ertilization then decreased in concentration, unlike other proteins which continued to increase. The protein was being synthesized over a period o about 30 minutes and then soon ater was being broken down. Further experiments showed that the protein went through repeated increases and decreases in concentration that coincided with the phases o the cell cycle. The breakdown occurred about ten minutes ater the start o mitosis. Hunt named the protein cyclin.

56

Further research revealed other cyclins and confrmed what Hunt had suspected rom an early stage  that cyclins are a key actor in the control o the cell cycle. Tim Hunt was awarded a Nobel Prize or Physiology in 2 001 to honour his work in the discovery o cyclins. His Nobel Lecture can be downloaded rom the internet and viewed. In it he mentions the importance o serendipity several times because he had not set out to discover how the cell cycle is controlled. This discovery is an example o serendipity  a happy and unexpected discovery made by accident.

1 . 6 ce ll d I VI s I o n

tumur frmai a ar Mutagens, oncogenes and metastasis are involved in the development o primary and secondary tumours. Tumours are abnormal groups o cells that develop at any stage o lie in any part o the body. In some cases the cells adhere to each other and do not invade nearby tissues or move to other parts o the body. These tumours are unlikely to cause much harm and are classifed as benign. In other tumours the cells can become detached and move elsewhere in the body and develop into secondary tumours. These tumours are malignant and are very likely to be lie- threatening. D iseases due to malignant tumours are commonly known as cancer and have diverse causes. C hemicals and agents that cause cancer are known as carcinogens, because carcinomas are malignant tumours. There are various types o carcinogens including some viruses. All mutagens are carcinogenic, both chemical mutagens and also high energy radiation such as X- rays and short- wave ultraviolet light. This is because mutagens are agents that cause gene mutations and mutations can cause cancer.

Aiviy car rarh Tumours can orm in any tissue at any age, but the skin, lung, large intestine (bowel) , breast and prostate gland are particularly vulnerable. Cancer is a major cause o death in most human populations so there is a pressing need to fnd methods o prevention and treatment. This involves basic research into the control o the cell cycle. Great progress has been made but more is needed. Who should pay or research into cancer?

Mutations are random changes to the base sequence o genes. Most genes do not cause cancer i they mutate. The ew genes that can become cancer-causing ater mutating are known as oncogenes. In a normal cell oncogenes are involved in the control o the cell cycle and cell division. This is why mutations in them can result in uncontrolled cell division and thereore tumour ormation. S everal mutations must occur in the same cell or it to become a tumour cell. The chance o this happening is extremely small, but because there are vast numbers o cells in the body, the total chance o tumour ormation during a lietime is signifcant. When a tumour cell has been ormed it divides repeatedly to orm two, then our, then eight cells and so on. This group o cells is called a primary tumour. Metastasis is the movement o cells rom a primary tumour to set up secondary tumours in other parts o the body.

Smoking and cancer The correlation between smoking and incidence o cancers. A correlation in science is a relationship between two variable actors. The relationship between smoking and cancer is an example o a correlation. There are two types o correlation. With a positive correlation, when one actor increases the other one also increases; they also decrease together. With a negative correlation, when one actor increases the other decreases. There is a positive correlation between cigarette smoking and the death rate due to cancer. This has been shown repeatedly in surveys. table 1 shows the results o one o the largest surveys, and the longest

57

1

C E LL B I O LO G Y

continuous one. The data shows that the more cigarettes smoked per day, the higher the death rate due to cancer. They also show a higher death rate among those who smoked at one time but had stopped. The results o the survey also show huge increases in the death rate due to cancers o the mouth, pharynx, larynx and lung. This is expected as smoke rom cigarettes comes into contact with each o these parts o the body, but there is also a positive correlation between smoking and cancers o the esophagus, stomach, kidney, bladder, pancreas and cervix. Although the death rate due to other cancers is not signifcantly dierent in smokers and non- smokers, table 1 shows smokers are several times more likely to die rom all cancers than non- smokers.

It is important in science to distinguish between a correlation and a cause. Finding that there is a positive correlation between smoking and cancer does not prove that smoking causes cancer. However, in this case the causal links are well established. C igarette smoke contains many dierent chemical substances. Twenty o these have been shown in experiments to cause tumours in the lungs o laboratory animals or humans. There is evidence that at least orty other chemicals in cigarette smoke are carcinogenic. This leaves little doubt that smoking is a cause o cancer.

caue o death etween 1951 and 2001

current moker (igarette/day)

lieong non-moker

former igarette moker

114

1524

25

All cancers

360

466

588

747

1,061

Lung cancer

17

68

131

233

417

Cancer of mouth, pharynx, larynx and esophagus

9

26

36

47

106

334

372

421

467

538

(sampe ize: 34,439 mae dotor in britain)

All other cancers  Table 1

58

Mortaity rate per 100,000 men/year

from British Medical Journal 328(7455) June 24 2004

1 . 6 ce ll d I VI s I o n

daa-ba qui: The efect o smoking on health One o the largest ever studies o the eect o smoking on health involved 34,439 male British doctors. Inormation was collected on how much they smoked rom 1 951 to 2001 and the cause o

n-mkr

114 igar pr ay

1524 igar pr ay

>25 igar pr ay

107

237

310

471

1,037

1,447

1,671

1,938

Stomach and duodenal ulcers

8

11

33

34

Cirrhosis o the liver

6

13

22

68

Parkinsons disease

20

22

6

18

typ f ia Respiratory (diseases o the lungs and airways) Circulatory (diseases o the heart and blood vessels)

1

death was recorded or each o the doctors who died during this period. The table below shows some o the results. The fgures given are the number o deaths per hundred thousand men per year.

D educe whether there is a positive correlation between smoking and the mortality rate due to all types o disease. [2 ]

4

2

Using the data in the table, discuss whether the threat to health rom smoking is greater with respiratory or with circulatory diseases. [4]

5

3

Discuss whether the data suggests that smoking a small number o cigarettes is sae. [3]

D iscuss whether the data p roves that smoking is a cause o cirrhosis o the liver.

[3 ]

The table does not include deaths due to cancer. The survey showed that seven types o cancer are linked with smoking. Suggest three cancers that you would expect smoking to cause. [3 ]

59

1

C E LL B I O LO G Y

Questions 1

c) E xplain the dierence in area o the inner and outer mitochondrial membranes. [3 ]

Figure 7 represents a cell rom a multicellular organism.

d) Using the data in the table, identiy two o the main activities o liver cells. [2 ]

3

In human secretory cells, or example in the lung and the pancreas, positively charged ions are pumped out, and chloride ions ollow passively through chloride channels. Water also moves rom the cells into the liquid that has been secreted.

prokaryotic or eukaryotic;

[1 ]

In the genetic disease cystic brosis, the chloride channels malunction and too ew ions move out o the cells. The liquid secreted by the cells becomes thick and viscous, with associated health problems.

( ii) part o a root tip or a nger tip;

[1 ]

a) S tate the names o the processes that:

 Figure 7

a) Identiy, with a reason, whether the cell is

( i)

( iii) in a phase o mitosis or in interphase. [1 ] b) The magnication o the drawing is 2 ,5 00  . ( i)

C alculate the actual size o the cell.

( ii) move chloride ions out o the secretory cells.

[2 ]

( ii) C alculate how long a 5 m scale bar should be i it was added to the drawing. [1 ] c) Predict what would happen to the cell i it was placed in a concentrated salt solution or one hour. Include reasons or your answer. [3]

Plasma membrane

4

The amount o D NA present in each cell nucleus was measured in a large number o cells taken rom two dierent cultures o human bone marrow ( gure 8) . a) For each label ( I, II and III) in the S ample B graph, deduce which phase o the cell cycle the cells could be in; i.e. G1 , G2 or S . [3 ]

Area (m 2 ) 1,780

Rough endoplasmic reticulum

30,400

Mitochondrial outer membrane

7,470

Mitochondrial inner membrane

39,600

Nucleus

280

Lysosomes

100

Other components

18,500

 Table 2

a) C alculate the total area o membranes in the liver cell. [2 ] b) C alculate the area o plasma membrane as a percentage o the total area o membranes in the cell. S how your working. [3 ]

60

b) Explain why the fuid secreted by people with cystic brosis is thick and viscous. [4]

Table 2 shows the area o membranes in a rat liver cell.

Membrane component

[1 ]

( iii) move water out o the secretory cells. [1 ]

b) Estimate the approximate amount o D NA per nucleus that would be expected in the ollowing human cell types: ( i) bone marrow at prophase ( ii) bone marrow at telophase. Number of cells (in thousands)

2

move positively charged ions out o the secretory cells [1 ]

3

Sample A (non-dividing cell culture)

2 1

5 10 15 DNA/pg per nucleus  Figure 8

Number of cells (in thousands)

( i)

[2 ]

Sample B 3 (rapidly dividing cell culture) I 2 III 1 II 5 10 15 DNA/pg per nucleus

2

M o le cu lar B I o lo GY

Intdtin Water is the medium for life. Living organisms control their composition by a complex web of chemical reactions that occur within this medium. Photosynthesis uses the energy in sunlight to supply the chemical energy needed for life and cell respiration releases this energy when it is needed. C ompounds of carbon,

hydrogen and oxygen are used to supply and store energy. Many proteins act as enzymes to control the metabolism of the cell and others have a diverse range of biological functions. Genetic information is stored in D NA and can be accurately copied and translated to make the proteins needed by the cell.

2.1 Molecules to metabolism undstnding  Molecular biology explains living processes in  

 



terms o the chemical substances involved. Carbon atoms can orm our bonds allowing a diversity o compounds to exist. Lie is based on carbon compounds including carbohydrates, lipids, proteins and nucleic acids. Metabolism is the web o all the enzyme catalysed reactions in a cell or organism. Anabolism is the synthesis o complex molecules rom simpler molecules including the ormation o macromolecules rom monomers by condensation reactions. Catabolism is the breakdown o complex molecules into simpler molecules including the hydrolysis o macromolecules into monomers.

appitins  Urea as an example o a compound that is

produced by living organisms but can also be artifcially synthesized.

Skis  Drawing molecular diagrams o glucose, ribose, a

saturated atty acid and a generalized amino acid.  Identifcation o biochemicals such as carbohydrate, lipid or protein rom molecular diagrams.

Nt f sin  Falsifcation o theories: the artifcial synthesis

o urea helped to alsiy vitalism.

61

2

M O L E C U L AR B I O LO G Y

Molecular biology Molecular biology explains living processes in terms o the chemical substances involved.

 Figure 1

A molecular biologist at work in the laboratory

The discovery o the structure o D NA in 1 95 3 started a revolution in biology that has transormed our understanding o living organisms. It raised the possibility o explaining biological processes rom the structure o molecules and how they interact with each other. The structures are diverse and the interactions are very complex, so although molecular biology is more than 5 0 years old, it is still a relatively young science. Many molecules are important in living organisms including one as apparently simple as water, but the most varied and complex molecules are nucleic acids and proteins. Nucleic acids comprise D NA and RNA. They are the chemicals used to make genes. Proteins are astonishingly varied in structure and carry out a huge range o tasks within the cell, including controlling chemical reactions o the cell by acting as enzymes. The relationship between genes and proteins is at the heart o molecular biology. The approach o the molecular biologist is reductionist as it involves considering the various biochemical processes o a living organism and breaking down into its component parts. This approach has been immensely productive in biology and has given us insights into whole organisms that we would not otherwise have. Some biologists argue that the reductionist approach o the molecular biologist cannot explain everything though, and that when component parts are combined there are emergent properties that cannot be studied without looking at the whole system together.

Synthesis of urea Urea as an example o a compound that is produced by living organisms but can also be artifcially synthesized. Urea is a nitrogen- containing compound with a relatively simple molecular structure ( fgure 2 ) . It is a component o urine and this was where it was frst discovered. It is produced when there is an excess o amino acids in the body, as a means o excreting the nitrogen rom the amino acids. A cycle o reactions, catalysed by enzymes, is used to produce it ( fgure 3 ) . This happens in the liver. Urea is then transported by the blood stream to the kidneys where it is fltered out and passes out o the body in the urine. Urea can also be synthesized artifcially. The chemical reactions used are dierent rom those in the liver and enzymes are not involved, but the urea that is produced is identical.

O

ammonia + carbon dioxide  ammonium carbamate  urea + water

C H 2N  Figure 2

62

NH 2

Molecular diagram of urea

About 1 00 million tonnes are produced annually. Most o this is used as a nitrogen ertilizer on crops.

2 .1 M o le c u le s to M e tab o li s M

CO 2 + NH 3 enzyme 1 carbamoyl phosphate ornithine urea enzyme 2 arginase

citrulline

arginine

aspartate

fumarate

enzyme 3

enzyme 4 argininosuccinate

 Figure 3

The cycle of reactions occurring in liver cells that is used to synthesize urea

urea and the alsifcation o vitalism Falsifcation o theories: the artifcial synthesis o urea helped to alsiy vitalism. Urea was discovered in urine in the 1 720s and was assumed to be a product o the kidneys. At that time it was widely believed that organic compounds in plants and animals could only be made with the help o a vital principle. This was part o vitalism  the theory that the origin and phenomena o lie are due to a vital principle, which is dierent rom purely chemical or physical orces. Aristotle used the word psyche or the vital principle  a Greek word meaning breath, lie or soul. In 1 82 8 the German chemist Friedrich Whler synthesized urea artifcially using silver isocyanate and ammonium chloride. This was the frst organic compound to be synthesized artifcially. It was a very signifcant step, because no vital principle had been involved in the synthesis. Whler wrote this excitedly to the S wedish chemist Jns Jacob B erzelius: In a manner of speaking, I can no longer hold my chemical water. I must tell you that I can make urea without the kidneys of any animal, be it man or dog. An obvious deduction was that i urea had been synthesized without a vital principle, other

organic compounds could be as well. Whlers achievement was evidence against the theory o vitalism. It helped to alsiy the theory, but it did not cause all biologists to abandon vitalism immediately. It usually requires several pieces o evidence against a theory or most biologists to accept that it has been alsifed and sometimes controversies over a theory continue or decades. Although biologists now accept that processes in living organisms are governed by the same chemical and physical orces as in non- living matter, there remain some organic compounds that have not been synthesized artifcially. It is still impossible to make complex proteins such as hemoglobin, or example, without using ribosomes and other components o cells. Four years ater his synthesis o urea, Whler wrote this to B erzelius: Organic chemistry nowadays almost drives one mad. To me it appears like a primeval tropical forest full of the most remarkable things; a dreadful endless jungle into which one dare not enter, for there seems no way out.

63

2

M O L E C U L AR B I O LO G Y

carbon ompounds

ativity

Carbon atoms can orm our bonds allowing a diversity o compounds to exist.

crbon ompounds Can you fnd an example o a biological molecule in which a carbon atom is bonded to atoms o three other elements or even our other elements?

C arbon is only the 1 5 th most abundant element on Earth, but it can be used to make a huge range of different molecules. This has given living organisms almost limitless possibilities for the chemical composition and activities of their cells. The diversity of carbon compounds is explained by the properties of carbon.

Titin is a giant protein that acts as a molecular spring in muscle. The backbone o the titin molecule is a chain o 100,000 atoms, linked by single covalent bonds.

Carbon atoms form covalent bonds with other atoms. A covalent bond is formed when two adjacent atoms share a pair of electrons, with one electron contributed by each atom. Covalent bonds are the strongest type of bond between atoms so stable molecules based on carbon can be produced. Each carbon atom can form up to four covalent bonds  more than most other atoms, so molecules containing carbon can have complex structures. The bonds can be with other carbon atoms to make rings or chains of any length. Fatty acids contain chains of up to 2 0 carbon atoms for example. The bonds can also be with other elements such as hydrogen, oxygen, nitrogen or phosphorus.

Can you fnd an example o a molecule in your body with a chain o over 1,000,000,000 atoms?

C arbon atoms can bond with just one other element, such as hydrogen in methane, or they can bond to more than one other element as in ethanol ( alcohol found in beer and wine) . The four bonds can all be single covalent bonds or there can be two single and one double covalent bond, for example in the carboxyl group of ethanoic acid (the acid in vinegar) .

H H

C

H

methane

classifying arbon ompounds

H

H

H

H

C

C

H

H

H H

C

H

ethanol

Living organisms use four main classes of carbon compound. They have different properties and so can be used for different purposes. O

C

Carbohydrates are characterized by their composition. They are composed of carbon, hydrogen and oxygen, with hydrogen and oxygen in the ratio of two hydrogen atoms to one oxygen, hence the name carbohydrate.

ethanoic acid O

H

H

O

Lie is based on carbon compounds including carbohydrates, lipids, proteins and nucleic acids.

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

H

H

H

H

H

H

H

H

H

H

H

linolenic acid  an omega-3 fatty acid  Figure 4 Some common

naturally-occurring carbon compounds

O C OH

Lip ids are a broad class of molecules that are insoluble in water, including steroids, waxes, fatty acids and triglycerides. In common language, triglycerides are fats if they are solid at room temperature or oils if they are liquid at room temperature.

Proteins are composed of one or more chains of amino acids. All of the amino acids in these chains contain the elements carbon, hydrogen, oxygen and nitrogen, but two of the twenty amino acids also contain sulphur. Nucleic acids are chains of subunits called nucleotides, which contain carbon, hydrogen, oxygen, nitrogen and phosphorus. There are two types of nucleic acid: ribonucleic acid ( RNA) and deoxyribonucleic acid ( D NA) .

64

2 .1 M o le c u le s to M e tab o li s M

Drawing molecules Drawing molecular diagrams of glucose, ribose, a saturated fatty acid and a generalized amino acid. There is no need to memorize the structure o many dierent molecules but a biologist should be able to draw diagrams o a ew o the most important molecules. Each atom in a molecule is represented using the symbol o the element. For example a carbon

Name of group hydroxyl

Full structure O

atom is represented with C and an oxygen atom with O . S ingle covalent bonds are shown with a line and double bonds with two lines. S ome chemical groups are shown with the atoms together and bonds not indicated. Table 1 gives examples.

Simplied notation OH

H H

amine

NH 2

N H O

carboxyl

COOH

C O

H

H

methyl

C

H

CH 3

H 

Table 1

Ribose 

The ormula or ribose is C 5 H 1 0 O 5



The molecule is a fve- membered ring with a side chain.

OH 5

H

C 4



Four carbon atoms are in the ring and one orms the side chain.



The carbon atoms can be numbered starting with number 1 on the right.



The hydroxyl groups ( O H) on carbon atoms 1 , 2 and 3 point up, down and down respectively.

H O H C1

C

C2

OH

OH

H

H 3



Ribose

Glucose 

The ormula or glucose is C 6 H 1 2 O 6



The molecule is a six- membered ring with a side chain.

6

CH 2 OH

5

C

O

H OH

H

C

C

H 4C

HO 3



Five carbon atoms are in the ring and one orms the side chain.



The carbon atoms can be numbered starting with number 1 on the right.



The hydroxyl groups ( O H) on carbon atoms 1 , 2 , 3 and 4 point down, down, up and down respectively, although in a orm o glucose used by plants to make cellulose the hydroxyl group on carbon atom 1 points upwards.

OH

CH

1

C OH

2

H 

C

OH

Glucose

65

2

M O L E C U L AR B I O LO G Y

O

Saturated fatty acids

OH C



The carbon atoms form an unbranched chain.



In saturated fatty acids they are bonded to each other by single bonds.

H C H H C H



The number of carbon atoms is most commonly between 1 4 and 2 0.

H C H



At one end of the chain the carbon atom is part of a carboxyl group

H C H



At the other end the carbon atom is bonded to three hydrogen atoms.



All other carbon atoms are bonded to two hydrogen atoms.

H C H H C H H C H H C H

Amino acids 

H C H

A carbon atom in the centre of the molecule is bonded to four different things: 

an amine group, hence the term amino acid;



a carboxyl group which makes the molecule an acid;



a hydrogen atom;



the R group, which is the variable part of amino acids.

H C H H C H H C H H C H H C H H C H H O

R N H

C

C

O H full molecular diagram 

R

O

H

N 2N

C

COOH

CH 3

(CH 2 ) n

H H simplied molecular diagram

C



Full molecular diagram o a saturated atty acid

OH 

Molecular diagrams o an amino acid

Simplifed molecular diagram o a saturated atty acid

Identifying molecules Identifcation o biochemicals as carbohydrate, lipid or protein rom molecular diagrams. The molecules of carbohydrates, lipids and proteins are so different from each other that it is usually quite easy to recognize them.

66



Proteins contain C , H, O and N whereas carbohydrates and lipids contain C , H and O but not N.



Many proteins contain sulphur ( S ) but carbohydrates and lipids do not.



C arbohydrates contain hydrogen and oxygen atoms in a ratio of 2 :1 , for example glucose is C 6 H 1 2 O 6 and sucrose ( the sugar commonly used in baking) is C 1 2 H 22 O 1 1



Lipids contain relatively less oxygen than carbohydrates, for example oleic acid ( an unsaturated fatty acid) is C 1 8 H 34O 2 and the steroid testosterone is C 1 9 H 28 O 2



Figure 5 A commonly-occurring biological molecule

2 .1 M o le c u le s to M e tab o li s M

Metbolism Metabolism is the web of all the enzyme catalysed reactions in a cell or organism. All living organisms carry out large numbers o dierent chemical reactions. These reactions are catalysed by enzymes. Most o them happen in the cytoplasm o cells but some are extracellular, such as the reactions used to digest ood in the small intestine. Metabolism is the sum o all reactions that occur in an organism. Metabolism consists o pathways by which one type o molecule is transormed into another, in a series o small steps. These pathways are mostly chains o reactions but there are also some cycles. An example is shown in fgure 3. E ven in relatively simple prokaryote cells, metabolism consists o over 1 , 000 dierent reactions. Global maps showing all reactions are very complex. They are available on the internet, or example in the Kyoto E ncyclopedia o Genes and Genomes.

anbolism Anabolism is the synthesis of complex molecules from simpler molecules including the formation of macromolecules from monomers by condensation reactions. Metabolism is oten divided into two parts, anabolism and catabolism. Anabolism is reactions that build up larger molecules rom smaller ones. An easy way to remember this is by recalling that anabolic steroids are hormones that promote body building. Anabolic reactions require energy, which is usually supplied in the orm o ATP. Anabolism includes these processes: 

Protein synthesis using ribosomes.



D NA synthesis during replication.



Photosynthesis, including production o glucose rom carbon dioxide and water.



Synthesis o complex carbohydrates including starch, cellulose and glycogen.

ctbolism Catabolism is the breakdown of complex molecules into simpler molecules including the hydrolysis of macromolecules into monomers. C atabolism is the part o metabolism in which larger molecules are broken down into smaller ones. C atabolic reactions release energy and in some cases this energy is captured in the orm o ATP, which can then be used in the cell. C atabolism includes these processes: 

D igestion o ood in the mouth, stomach and small intestine.



C ell respiration in which glucose or lipids are oxidized to carbon dioxide and water.



D igestion o complex carbon compounds in dead organic matter by decomposers.

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2.2 Water understnding  Water molecules are polar and hydrogen bonds

form between them.  Hydrogen bonding and dipolarity explain the adhesive, cohesive, thermal and solvent properties of water.  Substances can be hydrophilic or hydrophobic.

applictions  Comparison of the thermal properties of water

with those of methane.  Use of water as a coolant in sweat.  Methods of transport of glucose, amino acids, cholesterol, fats, oxygen and sodium chloride in blood in relation to their solubility in water.

Ntre of science  Use theories to explain natural phenomena:

the theory that hydrogen bonds form between water molecules explains waters properties.

H

H

Water molecules are polar and hydrogen bonds form between them.

O

tends to small pull the positive electrons charge  + on each slightly hydrogen in this atom direction Corresponding negative charge 2 - on oxygen atom  Figure 1

Water molecules

water molecule hydrogen bond  Figure 2

The dotted line indicates the presence of an intermolecular force between the molecules. This is called a hydrogen bond

68

Hydrogen bonding in wter A water molecule is ormed by covalent bonds between an oxygen atom and two hydrogen atoms. The bond between hydrogen and oxygen involves unequal sharing o electrons  it is a polar covalent bond. This is because the nucleus o the oxygen atom is more attractive to electrons than the nuclei o the hydrogen atoms ( fgure 1 ) . B ecause o the unequal sharing o electrons in water molecules, the hydrogen atoms have a partial positive charge and oxygen has a partial negative charge. B ecause water molecules are bent rather than linear, the two hydrogen atoms are on the same side o the molecule and orm one pole and the oxygen orms the opposite pole. Positively charged particles ( positive ions) and negatively charged particles ( negative ions) attract each other and orm an ionic bond. Water molecules only have partial charges, so the attraction is less but it is still enough to have signifcant eects. The attraction between water molecules is a hydrogen bond. S trictly speaking it is an intermolecular orce rather than a bond. A hydrogen bond is the orce that orms when a hydrogen atom in one polar molecule is attracted to a slightly negative atom o another polar covalent molecule. Although a hydrogen bond is a weak intermolecular orce, water molecules are small, so there are many o them per unit volume o water and large numbers o hydrogen bonds ( fgure 2 ) . C ollectively they give water its unique properties and these properties are, in turn, o immense importance to living things.

2 . 2 W at e r

Hydrogen bonds and the properties of water Use theories to explain natural phenomena: the theory that hydrogen bonds form between water molecules explains waters properties. There is strong experimental evidence or hydrogen bonds, but it remains a theory that they orm between water molecules. Scientists cannot prove without doubt that they exist as they are not directly visible. However, hydrogen bonds are a very useul way o explaining the properties o water. They explain the cohesive, adhesive, thermal and solvent properties o water. It is these distinctive properties that make water so useul to living organisms.

It might seem unwise to base our understanding o the natural world on something that has not been proven to exist. However this is the way that science works  we can assume that a theory is correct i there is evidence or it, i it helps to predict behaviour, i it has not been alsifed and i it helps to explain natural phenomena.

Properties of water Hydrogen bonding and dipolarity explain the cohesive, adhesive, thermal and solvent properties of water. Cohesive properties C ohesion reers to the binding together o two molecules o the same type, or instance two water molecules. Water molecules are cohesive  they cohere, which means they stick to each other, due to hydrogen bonding, described in the previous section. This property is useul or water transport in plants. Water is sucked through xylem vessels at low pressure. The method can only work i the water molecules are not separated by the suction orces. D ue to hydrogen bonding this rarely happens and water can be pulled up to the top o the tallest trees  over a hundred metres.

Adhesive properties Hydrogen bonds can orm between water and other polar molecules, causing water to stick to them. This is called adhesion. This property is useul in leaves, where water adheres to cellulose molecules in cell walls. I water evaporates rom the cell walls and is lost rom the lea via the network o air spaces, adhesive orces cause water to be drawn out o the nearest xylem vessel. This keeps the walls moist so they can absorb carbon dioxide needed or photosynthesis.

Thermal properties Water has several thermal properties that are useul to living organisms: 

High specifc heat capacity. Hydrogen bonds restrict the motion o water molecules and increases in the temperature o water require hydrogen bonds to be broken. Energy is needed to do this. As a result, the amount o energy needed to raise the temperature o water is relatively large. To cool down, water must lose relatively large amounts o energy. Waters temperature remains relatively stable in comparison to air or land, so it is a thermally stable habitat or aquatic organisms.



High latent heat o vap orization. When a molecule evaporates it separates rom other molecules in a liquid and becomes a vapour molecule. The heat needed to do this is called the latent heat o

69

2

M O L E C U L AR B I O LO G Y vaporization. Evaporation therefore has a cooling effect. C onsiderable amounts of heat are needed to evaporate water, because hydrogen bonds have to be broken. This makes it a good evaporative coolant. S weating is an example of the use of water as a coolant. 

High boiling point. The boiling point of a substance is the highest temperature that it can reach in a liquid state. For the same reasons that water has a high latent heat of vaporization, its boiling point is high. Water is therefore liquid over a broad range of temperatures  from 0 C to 1 00 C. This is the temperature range found in most habitats on Earth.

Solvent properties Water has important solvent properties. The polar nature of the water molecule means that it forms shells around charged and polar molecules, preventing them from clumping together and keeping them in solution. Water forms hydrogen bonds with polar molecules. Its partially negative oxygen pole is attracted to positively charged ions and its partially positive hydrogen pole is attracted to negatively charged ions, so both dissolve. C ytoplasm is a complex mixture of dissolved substances in which the chemical reactions of metabolism occurs.

toK

Hydrophilic and hydrophobic

How do scientic explanations difer rom pseudo-scientic explanations?

Substances can be hydrophilic or hydrophobic.

Homeopathy is a practice where remedies are prepared by dissolving things like charcoal, spider venom or deadly nightshade. This mother tincture o harmul substance is diluted again and again to the point where a sample rom the solution is unlikely to contain a single molecule o the solute. It is this ultra-dilute solution that is claimed to have medicinal properties. The properties are reerred to as the memory o water. Despite the large number o practitioners o this practice, no homeopathic remedy has ever been shown to work in a large randomized placebo-controlled clinical trial.

The literal meaning of the word hydrophilic is water-loving. It is used to describe substances that are chemically attracted to water. All substances that dissolve in water are hydrophilic, including polar molecules such as glucose, and particles with positive or negative charges such as sodium and chloride ions. S ubstances that water adheres to, cellulose for example, are also hydrophilic. S ome substances are insoluble in water although they dissolve in other solvents such as propanone ( acetone) . The term hydrophobic is used to describe them, though they are not actually water-fearing. Molecules are hydrophobic if they do not have negative or positive charges and are nonpolar. All lipids are hydrophobic, including fats and oils

 Figure 3

70

When two nonpolar molecules in water come into contact, weak interactions form between them and more hydrogen bonds form between water molecules

2 . 2 W at e r I a nonpolar molecule is surrounded by water molecules, hydrogen bonds orm between the water molecules, but not between the nonpolar molecule and the water molecules. I two nonpolar molecules are surrounded by water molecules and random movements bring them together, they behave as though they are attracted to each other. There is a slight attraction between nonpolar molecules, but more signifcantly, i they are in contact with each other, more hydrogen bonds can orm between water molecules. This is not because they are water-earing: it is simply because water molecules are more attracted to each other than to the nonpolar molecules. As a result, nonpolar molecules tend to join together in water to orm larger and larger groups. The orces that cause nonpolar molecules to join together into groups in water are known as hydrophobic interactions.

comparing water and methane Comparison o the thermal properties o water with those o methane. The properties o water have already been described. Methane is a waste product o anaerobic respiration in certain prokaryotes that live in habitats where oxygen is lacking. Methanogenic prokaryotes live in swamps and other wetlands and in the guts o animals, including termites, cattle and sheep. They also live in waste dumps and are deliberately encouraged to produce methane in anaerobic digesters. Methane can be used as a uel but i allowed to escape into the atmosphere it contributes to the greenhouse eect. Water and methane are both small molecules with atoms linked by single covalent bonds. However water molecules are polar and can orm hydrogen bonds, whereas methane molecules are nonpolar and do not orm hydrogen bonds. As a result their physical properties are very dierent. The data in table 1 shows some o the physical properties o methane and water. The density and specifc heat capacity are given or methane and water in a liquid state. The data shows that water has a higher specifc heat capacity, higher latent heat o vaporization, higher melting point and higher boiling point. Whereas methane is liquid over a range o only 2 2 C , water is liquid over 1 00 C .

Popy

Mhn

W

Formula

CH 4

H 2O

Molecular mass

16

Density Specifc heat capacity

0.46g per cm

18 3

1g per cm 3

2.2 J per g per C

4.2 J per g per C

Latent heat o vaporization

760 J/g

2,257 J/g

Melting point

182 C

0 C

Boiling point

160 C

100 C

 Table 1

Comparing methane and water

 Figure 4 Bubbles of methane gas, produced

by prokaryotes decomposing organic matter at the bottom of a pond have been trapped in ice when the pond froze

71

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cooling the body with sweat Use of water as a coolant in sweat. Sweat is secreted by glands in the skin. The sweat is carried along narrow ducts to the surace o the skin where it spreads out. The heat needed or the evaporation o water in sweat is taken rom the tissues o the skin, reducing their temperature. B lood fowing through the skin is thereore cooled. This is an eective method o cooling the body because water has a high latent heat o vaporization. S olutes in the sweat, especially ions such as sodium, are let on the skin surace and can sometimes be detected by their salty taste.

There are methods o cooling other than sweating, though many o these also rely on heat loss due to evaporation o water. Panting in dogs and birds is an example. Transpiration is evaporative loss o water rom plant leaves; it has a cooling eect which is useul in hot environments.

Sweat secretion is controlled by the hypothalamus o the brain. It has receptors that monitor blood temperature and also receives sensory inputs rom temperature receptors in the skin. I the body is overheated the hypothalamus stimulates the sweat glands to secrete up to two litres o sweat per hour. Usually no sweat is secreted i the body is below the target temperature, though when adrenalin is secreted we sweat even i we are already cold. This is because adrenalin is secreted when our brain anticipates a period o intense activity that will tend to cause the body to overheat.

Transport in blood plasma Methods of transport of glucose, amino acids, cholesterol, fats, oxygen and sodium chloride in blood in relation to their solubility in water. B lood transports a wide variety o substances, using several methods to avoid possible problems and ensure that each substance is carried in large enough quantities or the bodys needs. S odium chloride is an ionic compound that is reely soluble in water, dissolving to orm sodium ions ( Na + ) and chloride ions ( C l - ) , which are carried in blood plasma. Amino acids have both negative and positive charges. B ecause o this they are soluble in water but their solubility varies depending on the R group, some o which are hydrophilic while others are hydrophobic. All amino acids are soluble enough to be carried dissolved in blood plasma.

72

Glucose is a polar molecule. It is reely soluble in water and is carried dissolved in blood plasma. O xygen is a nonpolar molecule. B ecause o the small size o the molecule it dissolves in water but only sparingly and water becomes saturated with oxygen at relatively low concentrations. Also, as the temperature o water rises, the solubility o oxygen decreases, so blood plasma at 3 7 C can hold much less dissolved oxygen than water at 2 0 C or lower. The amount o oxygen that blood plasma can transport around the body is ar too little to provide or aerobic cell respiration. This problem is overcome by the use o hemoglobin in red blood cells. Hemoglobin has binding sites or oxygen and greatly increases the capacity o the blood or oxygen transport.

2 . 3 c a r b o h y d r at e s a n d l i P i d s

Fats molecules are entirely nonpolar, are larger than oxygen and are insoluble in water. They are carried in blood inside lipoprotein complexes. These are groups of molecules with a single layer of phospholipid on the outside and fats inside. The hydrophilic phosphate heads of the phospholipids face outwards and are in contact with water in the blood plasma. The hydrophobic hydrocarbon tails face inwards and are in contact with the fats. There are also proteins in the phospholipid monolayer, hence the name lipoprotein. Cholesterol molecules are hydrophobic, apart from a small hydrophilic region at one end. This is not enough to make cholesterol dissolve in water and instead it is transported with fats in lipoprotein complexes. The cholesterol molecules are positioned in the phospholipid monolayers, with the hydrophilic region facing outwards in the region with the phosphate heads of the phospholipids.

phospholipid protein cholesterol triglyceride

 Figure 5 Arrangement of molecules in

a lipoprotein complex

2.3 c  p understnding  Monosaccharide monomers are linked

together by condensation reactions to orm disaccharides and polysaccharide polymers.  Fatty acids can be saturated, monounsaturated or polyunsaturated.  Unsaturated atty acids can be cis or trans isomers.  Triglycerides are ormed by condensation rom three atty acids and one glycerol.

Ntre of science  Evaluating claims: health claims made about

lipids need to be assessed.

applictions  Structure and unction o cellulose and starch

in plants and glycogen in humans.  Scientifc evidence or health risks o trans-ats and saturated ats.  Lipids are more suitable or long-term energy storage in humans than carbohydrates.  Evaluation o evidence and the methods used to obtain evidence or health claims made about lipids.

Skills  Use o molecular visualization sotware to

compare cellulose, starch and glycogen.  Determination o body mass index by calculation or use o a nomogram.

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toK

carbohydrates

i w cmpeng paradgms gve dferen explanans  a phenmenn, hw can we decde whch s crrec?

Monosaccharide monomers are linked together by condensation reactions to orm disaccharides and polysaccharide polymers.

Thomas Kuhn, in his book The Structure o Scientifc Revolutions adopted the word paradigm to reer to the rameworks that dominate the interpretation o inormation in a scientifc discipline at a particular point in time. The paradigm impacts the kinds o questions that are supposed to be asked. Nutritionism is the reductionist paradigm that the presence o indicator nutrients are the key determinant o healthy ood. Even highly processed ood may be advertised as healthy depending on the degree to which it contains healthy nutrients. Words like carbs, vitamins and polyunsaturated at have entered the everyday lexicon. Some argue that this aligns consumer anxiety with the commercial interests o ood manuacturers. An alternative paradigm or determining the healthiness o ood is argued or by Michael Pollan in his book In Deense o Food. It argues that ood quality should be determined by cultural tradition which tended to look at ood more holistically:

Glucose, ructose and ribose are all examples o monosaccharides. The structure o glucose and ribose molecules was shown in sub-topic 2 .1 . Monosaccharides can be linked together to make larger molecules. 

Monosaccharides are single sugar units.



D isaccharides consist o two monosaccharides linked together. For example, maltose is made by linking two glucose molecules together. S ucrose is made by linking a glucose and a ructose.



Polysaccharides consist o many monosaccharides linked together. S tarch, glycogen and cellulose are polysaccharides. They are all made by linking together glucose molecules. The dierences between them are described later in this sub-topic.

When monosaccharides combine, they do so by a process called condensation ( fgure 1 ) . This involves the loss o an O H rom one molecule and an H rom another molecule, which together orm H 2 O . Thus, condensation involves the combination o subunits and yields water. Linking together monosaccharides to orm disaccharides and polysaccharides is an anabolic process and energy has to be used to do it. ATP supplies energy to the monosaccharides and this energy is then used when the condensation reaction occurs. H

H

HO

The sheer novelty and glamor o the Western diet, with its seventeen thousand new ood products every year and the marketing power  thirty-two billion dollars a year  used to sell us those products, has overwhelmed the orce otradition and let us where we now fnd ourselves: relying on science and journalism and government and marketing to help us decide what to eat

H

H OH

HO

OH

Monosaccharides, C 6 H 12 O 6 e.g. glucose, fructose, galactose

H 2O Condensation

Hydrolysis

(water removed)

(water added)

H HO

Michael Pollan, In Deense oFood: An Eater's Maniesto

H O Glycosidic bond

Condensation

 Figure 1

e.g. maltose, sucrose, lactose

OH

Hydrolysis

H

H HO

Disaccharide, C 12 H 22 O 11

O

O

O

OH

Polysaccharide e.g. starch, glycogen

Condensation and hydrolysis reactions between monosaccharides and disaccharides

74

2 . 3 c a r b o h y d r at e s a n d l i P i d s

Imaging carbohydrate molecules Use of molecular visualization software to compare cellulose, starch and glycogen. The most widely used molecular visualization software is JMol, which can be downloaded free of charge. There are also many websites that use JMol, which are easier to use. S uggestions of suitable websites are available with the electronic resources that accompany this book. When JMol software is being used, you should be able to make these changes to the image of a molecule that you see on the screen: 

Use the scroll function on the mouse to make the image larger or smaller.



Left click and move the mouse to rotate the image.



Right click to display a menu that allows you to change the style of molecular model, label the atoms, make the molecule rotate continuously or change the background colour.

S pend some time developing your skill in molecular visualization and then try these questions to test your skill level and learn more about the structure of polysaccharides.

Questions 1

Select glucose with the ball and stick style with a black background. 

2

4

[2 ]

S elect sucrose with sticks style and a blue background. 

3

What colours are used to show carbon, hydrogen and oxygen atoms?

What is the difference between the glucose ring and the fructose ring in the sucrose molecule?

[1 ]

S elect amylose, which is the unbranched form of starch, with the wireframe style and a white background. If possible select a short amylose chain and then a longer one. 

What is the overall shape of an amylose molecule?

[1 ]



How many glucose molecules in the chain are linked to only one other glucose?

[1 ]

S elect amylopectin, with the styles and colours that you prefer. Amylopectin is the branched form of starch. Zoom in to look closely at a position where there is a branch. A glucose molecule must be linked to an extra third glucose to make the branch. 



What is different about this linkage, compared to the linkages between glucose molecules in unbranched parts of the molecule?

[1 ]

How many glucose molecules are linked to only one other glucose in the amylopectin molecule?

[1 ]



Figure 2 Images of sugars using molecular visualization software  (a) fructose, (b) maltose, (c) lactose

75

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M O L E C U L AR B I O LO G Y

5

Select glycogen. It is similar but not identical to the amylopectin orm o starch. 

6

Select cellulose. 

7

What is the dierence between glycogen and amylopectin? [1 ]

How is it dierent in shape rom the other polysaccharides? [1 ]

Look at the oxygen atom that orms part o the ring in each glucose molecule in the chain. 

What pattern do you notice in the position o these oxygen atoms along the chain?

Polysaccharides Structure and function of cellulose and starch in plants and glycogen in humans. Starch, glycogen and cellulose are all made by linking together glucose molecules, yet their structure and unctions are very dierent. This is due to dierences in the type o glucose used to make them and in the type o linkage between glucose molecules. Glucose has fve O H groups, any o which could be used in condensation reactions, but only three o them are actually used to link to make polysaccharides. The most common link is between the O H on carbon atom 1 ( on the right hand side in molecular diagrams o glucose) and the O H on carbon atom 4 ( shown on the let hand side) . The O H on carbon atom 6 ( shown at the top o molecular diagrams) is used to orm side branches in some polysaccharides.

 Figure 3

Glucose molecule

Glucose can have the OH group on carbon atom 1 pointing either upwards or downwards. In alpha glucose (-glucose) the OH group points downwards but in beta glucose (-glucose) it points upwards. This small dierence has major consequences or polysaccharides made rom glucose. Cellulose is made by linking together -glucose molecules. Condensation reactions link carbon atom 1 to carbon atom 4 on the next -glucose. The OH groups on carbon atom 1 and 4 point in opposite directions: up on carbon 1 and down on carbon 4. To bring these OH groups together and allow a condensation reaction to occur, each -glucose added to the chain has to be positioned at 1 80 to the previous one. The glucose subunits in the chain are oriented alternately upwards and downwards. The consequence o this is that the cellulose molecule is a straight chain, rather than curved.

76

 Figure 4 Cellulose

C ellulose molecules are unbranched chains o - glucose, allowing them to orm bundles with hydrogen bonds linking the cellulose molecules. These bundles are called cellulose microfbrils. They have very high tensile strength and are used as the basis o plant cell walls. The tensile strength o cellulose prevents plant cells rom bursting, even when very high pressures have developed inside the cell due to entry o water by osmosis.

2 . 3 c a r b o h y d r at e s a n d l i P i d s

Starch is made by linking together -glucose molecules. As in cellulose, the links are made by condensation reactions between the OH groups on carbon atom 1 o one glucose and carbon atom 4 o the adjacent glucose. These OH groups both point downwards, so all the glucose molecules in starch can be orientated in the same way. The consequence o this is that the starch molecule is curved, rather than straight. There are two orms o starch. In amylose the chain o -glucose molecules is unbranched and orms a helix. In amylopectin the chain is branched, so has a more globular shape. Starch is only made by plant cells. Molecules o both types o starch are hydrophilic but they are too large to be soluble in water. They are thereore useul in cells where large amounts o glucose need to be stored, but a concentrated glucose solution would cause too much water to enter a cell by osmosis. Starch is used as a store o glucose and thereore o energy in seeds and storage organs such as potato cells. Starch is made as a temporary store in lea cells when glucose is being made aster by photosynthesis than it can be exported to other parts o the plant.

 Figure 5 Starch

glycogen it is easy to add extra glucose molecules or remove them. This can be done at both ends o an unbranched molecule or at any o the ends in a branched molecule. S tarch and glycogen molecules do not have a fxed size and the number o glucose molecules that they contain can be increased or decreased.

Glycogen is very similar to the branched orm o starch, but there is more branching, making the molecule more compact. Glycogen is made by animals and also some ungi. It is stored in the liver and some muscles in humans. Glycogen has the same unction as starch in plants: it acts as a store o energy in the orm o glucose, in cells where large stores o dissolved glucose would cause osmotic problems. With both starch and

 Figure 6 Glycogen

lipids Triglycerides are formed by condensation from three fatty acids and one glycerol. Lipids are a diverse group o carbon compounds that share the property o being insoluble in water. Triglycerides are one o the principal groups o lipid. Examples o triglycerides are the at in adipose tissue in humans

77

2

M O L E C U L AR B I O LO G Y and the oil in sunfower seeds. Fats are liquid at body temperature ( 3 7 C ) but solid at room temperature ( 2 0 C ) whereas oils are liquid at both body temperature and room temperature. A triglyceride is made by combining three atty acids with one glycerol ( see gure 7) . Each o the atty acids is linked to the glycerol by a condensation reaction, so three water molecules are produced. The linkage ormed between each atty acid and the glycerol is an ester bond. This type o bond is ormed when an acid reacts with the O H group in an alcohol. In this case the reaction is between the C O O H group on a atty acid and an O H on the glycerol. Triglycerides are used as energy stores. The energy rom them can be released by aerobic cell respiration. B ecause they do not conduct heat well, they are used as heat insulators, or example in the blubber o Arctic marine mammals.

Glycerol

Fatty acids

H

C

O

H

HO

C (CH 2 ) n CH 3

Triglyceride (fat)

H

H H

C

O

Condensation C (CH 2 ) n CH 3 (water removed) H

C

O

O H

C

O

H

H

C

O

H

HO

O

O HO

H

 Figure 7

C (CH 2 ) n CH 3 O

C (CH 2 ) n CH 3 O 3H 2 O

C (CH 2 ) n CH 3

H

C H

O

C (CH 2 ) n CH 3 O

Ester bond

Formation of a triglyceride from glycerol and three fatty acids

enrgy storag Lipids are more suitable for long term energy storage in humans than carbohydrates. Lipids and carbohydrates are both used or energy storage in humans, but lipids are normally used or long- term energy storage. The lipids that are used are ats. They are stored in specialized groups o cells called adipose tissue. Adipose tissue is located immediately beneath the skin and also around some organs including the kidneys.

greater because ats orm pure droplets in cells with no water associated, whereas each gram o glycogen is associated with about two grams o water, so lipids are actually six times more ecient in the amount o energy that can be stored per gram o body mass. This is important, because we have to carry our energy stores around with us wherever we go. It is even more important or animals such as birds and bats that fy.

There are several reasons or using lipids rather than carbohydrates or long- term energy storage: 

78

The amount o energy released in cell respiration per gram o lipids is double the amount released rom a gram o carbohydrates. The same amount o energy stored as lipid rather than carbohydrate thereore adds hal as much to body mass. In act the mass advantage o lipids is even



S tored lipids have some secondary roles that could not be perormed as well by carbohydrates. B ecause lipids are poor conductors o heat, they can be used as heat insulators. This is the reason or much o our stored at being in sub- cutaneous adipose tissue next to the skin. B ecause at

2 . 3 c a r b o h y d r at e s a n d l i P i d s

is liquid at body temperature, it can also act as a shock absorber. This is the reason or adipose tissue around the kidneys and some other organs. Glycogen is the carbohydrate that is used or energy storage, in the liver and in some muscles. Although lipids are ideal or longterm storage o energy, glycogen is used or short- term storage. This is because glycogen

can be broken down to glucose rapidly and then transported easily by the blood to where it is needed. Fats in adipose tissue cannot be mobilized as rapidly. Glucose can be used either in anaerobic or aerobic cell respiration whereas ats and atty acids can only be used in aerobic respiration. The liver stores up to 1 5 0 grams o glycogen and some muscles store up to 2 % glycogen by mass.

d- qu: Emperor penguins 0.4 D uring the Antarctic winter emale E mperor 8.0 penguins live and eed at sea, but males have to stay on the ice to incubate the single egg the emale has laid. Throughout this time the males eat no ood. Ater 1 6 weeks the eggs hatch and the emales return. While the males are 12.0 incubating the eggs they stand in tightly packed groups o about 3 , 0 0 0 birds. To investigate the captive before reasons or standing in groups, 1 0 male birds were taken rom a colony at Pointe Geologie in 0.4 Antarctica. They had already survived 4 weeks 7.7 without ood. They were kept or 1 4 more weeks without ood in enced enclosures where they could not orm groups. All other conditions were kept the same as in the wild 11.8 colony. The mean air temperature was 1 6 . 4 C . The composition o the captive and the wild birds bodies was measured beore and ater the wild before 1 4- week period o the experiment. The results in kilograms are shown in fgure 8 .

a) C alculate the total mass loss or each group o birds.

[2 ]

i) wild

0.5 6.8 18.2

14.3

0.8 captive after 0.4 6.9 14.4

17.3

2.2 wild after Key water lipid protein other substances

 Figure 8

ii) captive b) C ompare the changes in lipid content o the captive birds with those o the birds living ree in the colony. [2 ] c) B esides being used as an energy source, state another unction o lipid which might be important or penguin survival. [1 ]

79

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M O L E C U L AR B I O LO G Y

Body mass index Determination of body mass index by calculation or use of a nomogram. The body mass index, usually abbreviated to B MI, was developed by a B elgian statistician, Adolphe Quetelet. Two measurements are needed to calculate it: the mass o the person in kilograms and their height in metres. B MI is calculated using this ormula: mass in kilograms B MI = __2 ( height in metres) Units or B MI are kg m - 2 B MI can also be ound using a type o chart called a nomogram. A straight line between the height on the let hand scale and the mass on the right hand scale intersects the B MI on the central scale. The data based questions on page 81 include a B MI nomogram. B MI is used to assess whether a persons body mass is at a healthy level, or is too high or too low. Table 1 shows how this is done:

bMi below 18.5

sttu underweight

18.524.9

normal weight

25.029.9

overweight

30.0 or more

obese

actvty etmtng ody ft prcntg To estimate body fat percentage, measure the thickness of a skinfold in millimetres using calipers in these four places: Front of upper arm Back of upper arm Below scapula Side of waist The measurements are added and then analysis tools available on the internet can be used to calculate the estimate.

 Figure 9

Measuring body fat with skinfold callipers

80



Table 1

In some parts o the world ood supplies are insufcient or are unevenly distributed and many people as a result are underweight. In other parts o the world a likelier cause o being underweight is anorexia nervosa. This is a psychological condition that involves voluntary starvation and loss o body mass. Obesity is an increasing problem in some countries. Excessive ood intake and insufcient exercise cause an accumulation o at in adipose tissue. The amount o body at can be estimated using skinold calipers (fgure 9) . Obesity increases the risk o conditions such as coronary heart disease and type 2 diabetes. It reduces lie expectancy signifcantly and is increasing the overall costs o health care in countries where rates o obesity are rising.



Measuring body mass. What was this persons body mass index if their height was 1.80 metres?

2 . 3 c a r b o h y d r at e s a n d l i P i d s

d  qu: Nomograms and BMI b) S uggest two ways in which the woman could reduce her body mass. [2 ]

Use fgure 1 1 to answer these questions. 1

a) S tate the body mass index o a man who has a mass o 75 kg and a height o 1 .45  metres.

[1 ]

4. O utline the relationship between height and B MI or a fxed body mass.

[1 ]

b) Deduce the body mass status o this man. [1 ] 2

a) State the body mass o the person standing on the scales on the previous page. [1 ] b) The person has a height o 1 .8 metres. D educe their body mass status. [1 ]

3

a) A woman has a height o 1 5 0 cm and a B MI o 40. C alculate the minimum amount o body mass she must lose to reach normal body mass status. S how all o your working. [3 ]

body mass/kg

height/cm

150 140 130 120

125

body mass index

130 135

110 50 100 95 90 85 80 75 70 65 60

140 40

30

145 150 155 160

20

55

165 170

50

175

45

180 40 10 35

185 190 195

30

200 205

25  Figure 10

Jogger

210

 Figure 11

Fatty acids Fatty acids can be saturated, monounsaturated or polyunsaturated. The basic structure o atty acids was described in sub- topic 2 .1 . There is a chain o carbon atoms, with hydrogen atoms linked to them by single covalent bonds. It is thereore a hydrocarbon chain. At one end o the chain is the acid part o the molecule. This is a carboxyl group, which can be represented as C O O H. The length o the hydrocarbon chain is variable but most o the atty acids used by living organisms have between 1 4 and 20 carbon atoms. Another variable eature is the bonding between the carbon atoms. In some atty

81

2

M O L E C U L AR B I O LO G Y OH

O C

H C H

OH

O C

H C H H C H H C H

H C H H C H H C H H C H

OH

O C

H C H

H C H

H C H H C H H C H

H C H

H C H

H C H

H C H

C H

H C H

H C H

C H H C H

H C H

H C H H C H H C H

C H C H

C H H C H

H C H H C H

H C H C H

H C H

H C H H C H

C H H C H H C H

H C H H C H

H C H

H C H H

H

palmitic acid  saturated  non-essential

 Figure 12

linolenic acid  polyunsaturated  all cis  essential  omega 3

C H

H C H

H C H H palmitoleic acid  monounsaturated  cis  non-essential  omega 7

Examples of fatty acids

acids all o the carbon atoms are linked by single covalent bonds, but in other atty acids there are one or more positions in the chain where carbon atoms are linked by double covalent bonds. I a carbon atom is linked to adj acent carbons in the chain by single bonds, it can also bond to two hydrogen atoms. I a carbon atom is linked by a double bond to an adj acent carbon in the chain, it can only bond to one hydrogen atom. A atty acid with single bonds between all o its carbon atoms thereore contains as much hydrogen as it possibly could and is called a saturated fatty acid. Fatty acids that have one or more double bonds are unsaturated because they contain less hydrogen than they could. I there is one double bond, the atty acid is monounsaturated and i it has more than one double bond it is p olyunsaturated. Figure 1 2 shows one saturated atty acid, one monounsaturated and one polyunsaturated atty acid. It is not necessary to remember names o specifc atty acids in IB B iology.

unsatrated fatty acids Unsaturated fatty acids can be cis or trans isomers. In unsaturated atty acids in living organisms, the hydrogen atoms are nearly always on the same side o the two carbon atoms that are double bonded  these are called cis- atty acids. The alternative is or the hydrogens to be on opposite sides  called trans- atty acids. These two conormations are shown in fgure 1 4. In cis-atty acids, there is a bend in the hydrocarbon chain at the double bond. This makes triglycerides containing cis- unsaturated atty acids less good at packing together in regular arrays than saturated atty acids, so it lowers the melting point. Triglycerides with cis- unsaturated atty acids are thereore usually liquid at room temperature  they are oils. Trans-atty acids do not have a bend in the hydrocarbon chain at the double bond, so they have a higher melting point and are solid at room temperature. Trans-atty acids are produced artifcially by partial hydrogenation o vegetable or fsh oils. This is done to produce solid ats or use in margarine and some other processed oods.

H H

H

C C cis

C C H trans

 Figure 13

Double bonds in fatty acids

82

 Figure 14 Fatty acid stereochemistry  (a)

trans (b) cis

2 . 3 c a r b o h y d r at e s a n d l i P i d s

Health risks of fats Scientifc evidence or health risks o trans-ats and saturated ats. There have been many claims about the eects o dierent types o at on human health. The main concern is coronary heart disease ( C HD ) . In this disease the coronary arteries become partially blocked by atty deposits, leading to blood clot ormation and heart attacks. A positive correlation has been ound between saturated atty acid intake and rates o C HD in many research programs. However, fnding a correlation does not prove that saturated ats cause the disease. It could be another actor correlated with saturated at intake, such as low amounts o dietary fbre, that actually causes C HD . There are populations that do not ft the correlation. The Maasai o Kenya or example have a diet that is rich in meat, at, blood and milk. They thereore have a high consumption o saturated ats, yet C HD is almost unknown among the Maasai. Figure 1 7 shows members o another Kenyan tribe that show this trend.

 Figure 15 Triglycerides in

olive oil contain cis-unsaturated fatty acids

D iets rich in olive oil, which contains cis- monounsaturated atty acids, are traditionally eaten in countries around the Mediterranean. The populations o these countries typically have low rates o C HD and it has been claimed that this is due to the intake o cis- monounsaturated atty acids. However, genetic actors in these populations, or other aspects o the diet such as the use o tomatoes in many dishes could explain the C HD rates. There is also a positive correlation between amounts o trans-at consumed and rates o C HD . Other risk actors have been tested, to see i they can account or the correlation, but none did. Trans-ats thereore probably do cause C HD . In patients who had died rom C HD , atty deposits in the diseased arteries have been ound to contain high concentrations o trans-ats, which gives more evidence o a causal link.

narrowed lumen of artery

fatty plaque causing thickening of the artery lining

layer of muscle and elastic bres  Figure 16 Artery

outer coat of artery

showing fatty plaque

 Figure 17

Samburu people of Northern Kenya. Like the Maasai, the Samburu have a diet rich in animal products but rates of heart disease are extremely low

83

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M O L E C U L AR B I O LO G Y

evaluating th halth risks of foods Evaluating claims: health claims made about lipids need to be assessed. Many health claims about oods are made. In some cases the claim is that the ood has a health benet and in other cases it is that the ood is harmul. Many claims have been ound to be alse when they are tested scientically. It is relatively easy to test claims about the eects o diet on health using laboratory animals. Large numbers o genetically uniorm animals can be bred and groups o them with the same age, sex and state o health can be selected or use in experiments. Variables other than diet, such as temperature and amount o exercise, can be controlled so that they do not infuence the results o the experiment. Diets can be designed so that only one dietary actor varies and strong evidence can thus be obtained about the eect o this actor on the animal. Results o animal experiments are oten interesting, but they do not tell us with certainty what the health eects are on humans o a actor in the diet. It would be very dicult to carry out

similar controlled experiments with humans. It might be possible to select matched groups o experimental subj ects in terms o age, sex and health, but unless identical twins were used they would be genetically dierent. It would also be almost impossible to control other variables such as exercise and ew humans would be willing to eat a very strictly controlled diet or a long enough period. Researchers into the health risks o ood must thereore use a dierent approach. Evidence is obtained by epidemiological studies. These involve nding a large cohort o people, measuring their ood intake and ollowing their health over a period o years. S tatistical procedures can then be used to nd out whether actors in the diet are associated with an increased requency o a particular disease. The analysis has to eliminate the eects o other actors that could be causing the disease.

Nature of science question: using volunteers in experiments. D uring the S econd World War, experiments were conducted both in England and in the US using conscientious obj ectors to military service as volunteers. The volunteers were willing to sacrice their health to help extend medical knowledge. A vitamin C trial in E ngland involved 2 0 volunteers. For six weeks they were all given a diet containing 70 mg o vitamin C . Then, or the next eight months, three volunteers were kept on the diet with 70 mg, seven had their dose reduced to 1 0 mg and ten were given no vitamin C . All o these ten volunteers developed scurvy. Three- centimetre cuts were made in their thighs, with the wounds closed up with ve stitches. These wounds ailed to heal. There was also bleeding rom hair ollicles and rom the gums. S ome o the volunteers developed more serious heart problems. The groups given 1 0 mg or 70 mg o vitamin C ared equally well and did not develop scurvy. Experiments on requirements or vitamin C have also been done using real guinea- pigs, which ironically are suitable because guinea-pigs, like

84

humans, cannot synthesize ascorbic acid. D uring trial periods with various intakes o vitamin C , concentrations in blood plasma and urine were monitored. The guinea- pigs were then killed and collagen in bone and skin was tested. The collagen in guinea- pigs with restricted vitamin C had less cross- linking between the protein bres and thereore lower strength. 1

Is it ethically acceptable or doctors or scientists to perorm experiments on volunteers, where there is a risk that the health o the volunteers will be harmed?

2

S ometimes people are paid to participate in medical experiments, such as drug trials. Is this more or less acceptable than using unpaid volunteers?

3

Is it better to use animals or experiments or are the ethical objections the same as with humans?

4

Is it acceptable to kill animals, so that an experiment can be done?

2 . 3 c a r b o h y d r at e s a n d l i P i d s

anlysis of dt on helth risks of lipids Evaluation of evidence and the methods used to obtain the evidence for health claims made about lipids. An evaluation is defned in IB as an assessment o implications and limitations. Evidence or health claims comes rom scientifc research. There are two questions to ask about this research: 1

2



Implications  do the results o the research support the health claim strongly, moderately or not at all?

How widely spread is the data? This is shown by the spread o data points on a scattergraph or the size o error bars on a bar chart. The more widely spread the data, the less likely it is that mean dierences are signifcant.



I statistical tests have been done on the data, do they show signifcant dierences?

Limitations  were the research methods used rigorous, or are there uncertainties about the conclusions because o weaknesses in methodology?

The second question is answered by assessing the methods used. The points below reer to surveys and slightly dierent questions should be asked to assess controlled experiments.

The frst question is answered by analysing the results o the research  either experimental results or results o a survey. Analysis is usually easiest i the results are presented as a graph or other type o visual display. 



Is there a correlation between intake o the lipid being investigated and rate o the disease or the health beneft? This might be either a positive or negative correlation. How large is the dierence between mean ( average) rates o the disease with dierent levels o lipid intake? Small dierences may not be signifcant.



How large was the sample size? In surveys it is usually necessary to have thousands o people in a survey to get reliable results.



How even was the sample in sex, age, state o health and lie style? The more even the sample, the less other actors can aect the results.



I the sample was uneven, were the results adjusted to eliminate the eects o other actors?



Were the measurements o lipid intake and disease rates reliable? S ometimes people in a survey do not report their intake accurately and diseases are sometimes misdiagnosed.

d- qu: Evaluating evidence from a health survey The Nurses Health S urvey is a highly respected survey into the health consequences o many actors. It began in 1 976 with 1 2 1 , 700 emale nurses in the US A and C anada, who completed a lengthy questionnaire about their liestyle actors and medical history. Follow- up questionnaires have been completed every two years since then. D etails o the methods used to assess diet and diagnose coronary heart disease can be ound by reading a research paper in the American Journal o Epidemiology, which is reely available on the internet: O h, K, Hu, FB , Manson, JE, S tamper, MJ and Willett, WC . ( 2 005 ) D ietary Fat Intake and Risk o C oronary Heart D isease in Women: 2 0 Years o Follow-up o the Nurses

Health Study. American Journal of Epidemiology, 1 61 :672 679. doi:1 0.1 093 /aj e/kwi085 To asse ss the eects o trans- ats on rates o C HD , the participants in the survey were divide d into ive groups according to the ir trans- at intake. Q uintile 1 was the 2 0 % o participants with the lowest intake and quintile 5 was the 2 0 % with the highe st intake. The ave rage intake o trans- ats or each quintile was calculated, as a percentage o dietary energy intake. The re lative risk o C HD was o und or each quintile, with Q uintile 1 assigned a risk o 1 . The risk was adj usted or die rences b etween the quintiles in age , body mass index, smoking, alcohol intake , parental

85

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M O L E C U L AR B I O LO G Y

history o C HD , intake o other oods that aect C HD rate s and various othe r actors. Figure 1 8 is a graph showing the percentage o ene rgy rom trans- ats or e ach o the ive quintiles and the adj uste d relative risk o C HD . The e e ct o trans- at intake on relative risk o C HD is statistically signiicant with a conidence level o  9 9 % . 1

S tate the trend shown in the graph.

3

The mean age o nurses in the fve quintiles was not the same. E xplain the reasons or adj usting the results to compensate or the eects o age dierences. [2 ]

5

relative risk of CHD

1.4 1.2 1.0 0.8 0.6 0.4 0.2

S uggest reasons or using only emale nurses in this survey. [3 ]

2

4

1.6

0 1

1.5 2.0 2.5 percentage of energy from trans-fats

[1 ]

3.0

Data for graph

C alculate the chance, based on the statistical tests, o the dierences in C HD risk being due to actors other than trans- at intake. [2 ]

% of energy from trans-fat

1.3

1.6

1.9

2.2

2.8

Relative risk of CHD

1.0

1.08

1.29

1.19

1.33

 Figure 18

D iscuss evidence rom the graph that other actors were having some eect on rates o C HD . [2 ]

Zutphen

USA

Slavonia

Belgrade

Crevalcor

Zrenjanin

Dalmatia

Crete

Montegiorgio

Velika

Rome

Corfu

Ushibuka

Tanushimaru

% Calories as saturated fat

W. Finland

Populations ranked by % calories as saturated fat

E. Finland

data-base questions: Saturated fats and coronary heart disease

22

19

19

18

14

12

10

10

9

9

9

9

8

7

3

3

Death CHD 992 351 420 574 214 288 248 152 86 rate/ 100,000 All yr 1 causes 1727 1318 1175 1088 1477 509 1241 1101 758

9

150

80

290

144

66

88

543 1080 1078 1027 764 1248 1006

 Table 2

1

2

3

86

a) Plot a scattergraph o the data in table 2 .

[5 ]

b) O utline the trend shown by the scattergraph.

[2 ]

C ompare the results or: a) E ast and West Finland;

[2 ]

b) C rete and Montegiorgio.

[2 ]

Evaluate the evidence rom this survey or saturated ats as a cause o coronary heart disease.

[4]

2 .4 Protein s

2.4 P understnding

applictions

 Amino acids are linked together by

 Rubisco, insulin, immunoglobulins, rhodopsin,

condensation to orm polypeptides. There are twenty diferent amino acids in polypeptides synthesized on ribosomes. Amino acids can be linked together in any sequence giving a huge range o possible polypeptides. The amino acid sequence o polypeptides is coded or by genes. A protein may consist o a single polypeptide or more than one polypeptide linked together. The amino acid sequence determines the threedimensional conormation o a protein. Living organisms synthesize many diferent proteins with a wide range o unctions. Every individual has a unique proteome.

 

    

collagen and spider silk as examples o the range o protein unctions.  Denaturation o proteins by heat or deviation o pH rom the optimum.

Skills  Draw molecular diagrams to show the ormation

o a peptide bond.

Ntre of science  Patterns, trends and discrepancies: most but

not all organisms assemble polypeptides rom the same amino acids.

amino cids nd polypeptides Amino acids are linked together by condensation to orm polypeptides. Polypeptides are chains of amino acids that are made by linking together amino acids by condensation reactions. This happens on ribosomes by a process called translation, which will be described in sub- topic 2 .7. Polypeptides are the main component of proteins and in many proteins they are the only component. S ome proteins contain one polypeptide and other proteins contain two or more. The condensation reaction involves the amine group (- NH 2 ) of one amino acid and the carboxyl group (- C OOH) of another. Water is eliminated, as carboxyl group

H

H O

H N

C

peptide bond

amino group

1

C OH

H

O

H N

C

condensation (water removed)

N

C

H

OH

R

R

amino acid

amino acid

H

O

H

H

C

C

N

C

O

H C

OH

H R

R

H2O  Figure 1

Condensation joins two amino acids with a peptide bond

87

2

M O L E C U L AR B I O LO G Y in all condensation reactions, and a new bond is ormed between the two amino acids, called a peptide bond. A dipeptide is a molecule consisting o two amino acids linked by a peptide bond. A polypeptide is a molecule consisting o many amino acids linked by peptide bonds. Polypeptides can contain any number o amino acids, though chains o ewer than 2 0 amino acids are usually reerred to as oligopeptides rather than polypeptides. Insulin is a small protein that contains two polypeptides, one with 2 1 amino acids and the other with 3 0. The largest polypeptide discovered so ar is titin, which is part o the structure o muscle. In humans titin is a chain o 3 4, 3 5 0 amino acids, but in mice it is even longer with 3 5 , 2 1 3 amino acids.

Drawing peptide bonds Draw molecular diagrams to show the ormation o a peptide bond. To orm a dipeptide, two amino acids are linked by a condensation reaction between the amine group o one amino acid and the carboxyl group o the other. This is shown in fgure 1 . The peptide bond is the same, whatever R group the amino acid carries. To test your skill at showing how peptide bonds are ormed, try showing the ormation o a peptide bond between two o the amino acids in fgure 2 . There are sixteen possible dipeptides that can be produced rom these our amino acids. You could also try to draw an oligopeptide o our amino acids, linked by three peptide bonds. I you do this correctly, you should see these eatures:



There is chain o atoms linked by single covalent bonds orming the backbone o the oligopeptide, with a repeating sequence o - N- C- C-



A hydrogen atom is linked by a single bond to each nitrogen atom in the backbone and an oxygen atom is linked by a double bond to one o the two carbon atoms.



The amine ( - NH 2 ) and carboxyl ( - C O O H) groups are used up in orming the peptide bond and only remain at the ends o the chain. These are called the amino and carboxyl terminals o the chain.



The R groups o each amino acid remain and proj ect outwards rom the backbone.

COOH OH H

C H

H

C H

H

H C H

H C H

H 2 N C COOH

H 2 N C COOH

H 2 N C COOH

H

H glutamic acid

H alanine

serine 

H H 2N

C COOH H glycine

Figure 2 Some common amino acids

The diversity of amino acids There are twenty diferent amino acids in polypeptides synthesized on ribosomes. The amino acids that are linked together by ribosomes to make polypeptides all have some identical structural eatures: a carbon atom in the centre o the molecule is bonded to an amine group, a carboxyl group and a hydrogen atom. The carbon atom is also bonded to an R group, which is dierent in each amino acid.

88

2 .4 Protein s Twenty dierent amino acids are used by ribosomes to make polypeptides. The amine groups and the carboxyl groups are used up in orming the peptide bond, so it is the R groups o the amino acids that give a polypeptide its character. The repertoire o R groups allows living organisms to make and use an amazingly wide range o proteins. Some o the dierences are shown in table 1 . It is not necessary to try to learn these specifc dierences but it is important to remember that because o the dierences between their R groups, the twenty amino acids are chemically very diverse. S ome proteins contain amino acids that are not in the basic repertoire o twenty. In most cases this is due to one o the twenty being modifed ater a polypeptide has been synthesized. There is an example o modifcation o amino acids in collagen, a structural protein used to provide tensile strength in tendons, ligaments, skin and blood vessel walls. C ollagen polypeptides made by ribosomes contain proline at many positions, but at some o these positions it is converted to hydroxyproline, which makes the collagen more stable. Nine R groups are hydrophobic with between zero and nine carbon atoms

Eleven R groups are hydrophilic

Seven R groups can become charged Four hydrophilic Four R groups act as Three R groups act as Three R Six R groups R groups are an acid by giving up a a base by accepting a groups contain do not contain polar but never proton and becoming proton and becoming charged rings rings negatively charged positively charged  Table 1

acvy scuvy Ascorbic acid (vitamin C) is needed to convert proline into hydroxyproline, so ascorbic acid deciency leads to abnormal collagen production. From your knowledge o the role o collagen, what efects do you expect this to have? Test your predictions by researching the symptoms o ascorbic acid deciency (scurvy) .

Classifcation o amino acids

amino cids nd origins Patterns, trends and discrepancies: most but not all organisms assemble polypeptides rom the same amino acids. It is a remarkable act that most organisms make proteins using the same 2 0 amino acids. In some cases amino acids are modifed ater a polypeptide has been synthesized, but the initial process o linking together amino acids on ribosomes with peptide bonds usually involves the same 2 0 amino acids. We can exclude the possibility that this trend is due to chance. There must be one or more reasons or it. S everal hypotheses have been proposed: 

These 20 amino acids were the ones produced by chemical processes on Earth beore the origin o lie, so all organisms used them and have continued to use them. Other amino acids might have been used, i they had been available.



They are the ideal 2 0 amino acids or making a wide range o proteins, so natural selection

will always avour organisms that use them and do not use other amino acids. 

All lie has evolved rom a single ancestral species, which used these 2 0 amino acids. B ecause o the way that polypeptides are made by ribosomes, it is difcult or any organism to change the repertoire o amino acids, either by removing existing ones or adding new ones.

B iology is a complicated science and discrepancies are commonly encountered. Some species have been ound that use one o the three codons that normally signal the end o polypeptide synthesis ( stop codons) to encode an extra non- standard amino acid. For example, some species use UGA to code or selenocysteine and some use UAG to code or pyrrolysine.

89

2

M O L E C U L AR B I O LO G Y

dt-bse questios: Commonality of amino acids 1

a) D iscuss which o the three hypotheses or use o the same 2 0 amino acids by most organisms is supported by the evidence. [3 ] b) S uggest ways o testing one o the hypotheses.

2

 Figure 3

C ell walls o bacteria contain peptidoglycan, a complex carbon compound that contains sugars and short chains o amino acids. Some o these amino acids are dierent rom the usual repertoire o 2 0. Also, some o them are right-handed orms o amino acids, whereas the 2 0 amino acids made into polypeptides are always the let-handed orms. D iscuss whether this is a signifcant discrepancy that alsifes the theory that living organisms all make polypeptides using the same 2 0 amino acids. [5 ]

Kohoutek Comet  26 diferent amino acids were ound in an articial comet produced by researchers at the Institut dAstrophysique Spatiale (CNRS/France) , which suggests that amino acids used by the rst living organisms on Earth may have come rom space

Polypeptide diversity

ativity

Amino acids can be linked together in any sequence giving a huge range of possible polypeptides.

clultig polypeptie iversity number of mio is

number of possible mio i sequees

1

20 1

2

20 2

3

400 8,000

4 20 6

64 million 10.24 trillion

 Table 2

Calculate the missing values

[2 ]

Ribosomes link amino acids together one at a time, until a polypeptide is ully ormed. The ribosome can make peptide bonds between any pair o amino acids, so any sequence o amino acids is possible. The number o possible amino acid sequences can be calculated starting with dipeptides ( table 2 ) . B oth amino acids in a dipeptide can be any o the twenty so there are twenty times twenty possible sequences ( 2 0 2 ) . There are 2 0  2 0  2 0 possible tripeptide sequences ( 2 0 3 ) . For a polypeptide o n amino acids there are 2 0 n possible sequences. The number o amino acids in a polypeptide can be anything rom 2 0 to tens o thousands. Taking one example, i a polypeptide has 400 amino acids, there are 2 0 400 possible amino acid sequences. This is a mindbogglingly large number and some online calculators simply express it as infnity. I we add all the possible sequences or other numbers o amino acids, the number is eectively infnite.

Genes and polypeptides The amino acid sequence of polypeptides is coded for by genes. The number o amino acid sequences that could be produced is immense, but living organisms only actually produce a small raction o these. Even so, a typical cell produces polypeptides with thousands o dierent sequences and must store the inormation needed to do this. The amino acid sequence o each polypeptide is stored in a coded orm in the base sequence o a gene.

 Figure 4 Lysozyme with

nitrogen o amine groups shown blue, oxygen red and sulphur yellow. The active site is the clet upper let

90

S ome genes have other roles, but most genes in a cell store the amino acid sequence o a polypeptide. They use the genetic code to do this. Three bases o the gene are needed to code or each amino acid in the polypeptide. In theory a polypeptide with 400 amino acids should require a gene with a sequence o 1 , 2 00 bases. In practice genes are

2 .4 Protein s always longer, with extra base sequences at both ends and sometimes also at certain points in the middle. The base sequence that actually codes for a polypeptide is known to molecular biologists as the open reading frame. O ne puzzle is that open reading frames only occupy a small proportion of the total D NA of a species.

Proteins and polypeptides A protein may consist o a single polypeptide or more than one polypeptide linked together. S ome proteins are single polypeptides, but others are composed of two or more polypeptides linked together. Integrin is a membrane protein with two polypeptides, each of which has a hydrophobic portion embedded in the membrane. Rather like the blade and handle of a folding knife the two polypeptides can either be adj acent to each other or can unfold and move apart when it is working. C ollagen consists of three long polypeptides wound together to form a rope- like molecule. This structure has greater tensile strength than the three polypeptides would if they were separate. The winding allows a small amount of stretching, reducing the chance of the molecule breaking. Hemoglobin consists of four polypeptides with associated non-polypeptide structures. The four parts of hemoglobin interact to transport oxygen more effectively to tissues that need it than if they were separate.

num f plyppd

exmpl

bckgud

1

lysozyme

Enzyme in secretions such as nasal mucus and tears; it kills some bacteria by digesting the peptidoglycan in their cell walls.

2

integrin

Membrane protein used to make connections between structures inside and outside a cell.

collagen

Structural protein in tendons, ligaments, skin and blood vessel walls; it provides high tensile strength, with limited stretching.

hemoglobin

Transport protein in red blood cells; it binds oxygen in the lungs and releases it in tissues with a reduced oxygen concentration.

3

4  Table 3

Example o proteins with diferent numbers o polypeptides

Protein conformations The amino acid sequence determines the three-dimensional conormation o a protein. The conformation of a protein is its three-dimensional structure. The conformation is determined by the amino acid sequence of a protein and its constituent polypeptides. Fibrous proteins such as collagen

 Figure 5 Integrin

embedded in a membrane (grey) shown olded and inactive and open with binding sites inside and outside the cell indicated (red and purple)

acvy Molecular biologists are investigating the numbers o open reading rames in selected species or each o the major groups o living organism. It is still ar rom certain how many genes in each species code or a polypeptide that the organism actually uses, but we can compare current best estimates: 

Drosophila melanogaster, the ruit fy, has base sequences or about 14,000 polypeptides.



Caenorhabditis elegans, a nematode worm with less than a thousand cells, has about 19,000.



Homo sapiens has base sequences or about 23,000 dierent polypeptides.



Arabidopsis thaliana, a small plant widely used in research, has about 27,000.

Can you nd any species with greater or lesser numbers o open reading rames than these?

91

2

M O L E C U L AR B I O LO G Y are elongated, usually with a repeating structure. Many proteins are globular, with an intricate shape that oten includes parts that are helical or sheet-like. Amino acids are added one by one, to orm a polypeptide. They are always added in the same sequence to make a particular polypeptide. In globular proteins the polypeptides gradually old up as they are made, to develop the fnal conormation. This is stabilized by bonds between the R groups o the amino acids that have been brought together by the olding.

 Figure 6 Lysozyme, showing how a polypeptide

can be folded up to form a globular protein. Three sections that are wound to form a helix are shown red and a section that forms a sheet is shown yellow. Other parts of the polypeptide including both of its ends are green

In globular proteins that are soluble in water, there are hydrophilic R groups on the outside o the molecule and there are usually hydrophobic groups on the inside. In globular membrane proteins there are regions with hydrophobic R groups on the outside o the molecule, which are attracted to the hydrophobic centre o the membrane. In fbrous proteins the amino acid sequence prevents olding up and ensures that the chain o amino acids remains in an elongated orm.

Denaturation of proteins Denaturation of proteins by heat or pH extremes. The three- dimensional conormation o proteins is stabilized by bonds or interactions between R groups o amino acids within the molecule. Most o these bonds and interactions are relatively weak and they can be disrupted or broken. This results in a change to the conormation o the protein, which is called denaturation. A denatured protein does not normally return to its ormer structure  the denaturation is permanent. S oluble proteins oten become insoluble and orm a precipitate. This is due to the hydrophobic R groups in the centre o the molecule becoming exposed to the water around by the change in conormation. Heat can cause denaturation because it causes vibrations within the molecule that can break intermolecular bonds or interactions. Proteins vary in their heat tolerance. S ome microorganisms that live in volcanic springs or in hot water near geothermal vents have proteins that are not denatured by temperatures o 80 C or higher. The best known example is D NA polymerase rom Thermus aquaticus, a prokaryote that was discovered in hot springs in Yellowstone National Park. It works best at 80 C and because o this it is widely used in biotechnology. Nevertheless, heat causes denaturation o most proteins at much lower temperatures.

92

E xtremes o pH, both acidic and alkaline, can cause denaturation. This is because charges on R groups are changed, breaking ionic bonds within the protein or causing new ionic bonds to orm. As with heat, the three-dimensional structure o the protein is altered and proteins that have been dissolved in water oten become insoluble. There are exceptions: the contents o the stomach are normally acidic, with a pH as low as 1 .5 , but this is the optimum pH or the protein-digesting enzyme pepsin that works in the stomach.

 Figure 7

When eggs are heated, proteins that were dissolved in both the white and the yolk are denatured. They become insoluble so both yolk and white solidify

2 .4 Protein s

Protein functions Living organisms synthesize many diferent proteins with a wide range o unctions. O ther groups o carbon compounds have important roles in the cell, but none can compare with the versatility o proteins. They can be compared to the worker bees that perorm almost all the tasks in a hive. All o the unctions listed here are carried out by proteins.

acvy du xpm A solution o egg albumen in a test tube can be heated in a water bath to nd the temperature at which it denatures. The efects o pH can be investigated by adding acids and alkalis to test tubes o egg albumen solution. To quantiy the extent o denaturation, a colorimeter can be used as denatured albumen absorbs more light than dissolved albumen.



C atalysis  there are thousands o dierent enzymes to catalyse specifc chemical reactions within the cell or outside it.



Muscle contraction  actin and myosin together cause the muscle contractions used in locomotion and transport around the body.



C ytoskeletons  tubulin is the subunit o microtubules that give animals cells their shape and pull on chromosomes during mitosis.



Tensile strengthening  fbrous proteins give tensile strength needed in skin, tendons, ligaments and blood vessel walls.



B lood clotting  plasma proteins act as clotting actors that cause blood to turn rom a liquid to a gel in wounds.

bx



Transp ort of nutrients and gases  proteins in blood help transport oxygen, carbon dioxide, iron and lipids.

Botox is a neurotoxin obtained rom Clostridium botulinum bacteria.



C ell adhesion  membrane proteins cause adj acent animal cells to stick to each other within tissues.

1



Membrane transp ort  membrane proteins are used or acilitated diusion and active transport, and also or electron transport during cell respiration and photosynthesis.

What are the reasons or injecting it into humans?

2

What is the reason or Clostridium botulinum producing it?

3

What are the reasons or injecting it rather than taking it orally?



Hormones  some such as insulin, FS H and LH are proteins, but hormones are chemically very diverse.



Recep tors  binding sites in membranes and cytoplasm or hormones, neurotransmitters, tastes and smells, and also receptors or light in the eye and in plants.



Packing of D NA  histones are associated with D NA in eukaryotes and help chromosomes to condense during mitosis.



Immunity  this is the most diverse group o proteins, as cells can make huge numbers o dierent antibodies.

acvy

There are many biotechnological uses or proteins including enzymes or removing stains, monoclonal antibodies or pregnancy tests or insulin or treating diabetics. Pharmaceutical companies now produce many dierent proteins or treating diseases. These tend to be very expensive, as it is still not easy to synthesize proteins artifcially. Increasingly, genetically modifed organisms are being used as microscopic protein actories.

93

2

M O L E C U L AR B I O LO G Y

exampls of protins Rubisco, insulin, immunoglobulins, rhodopsin, collagen and spider silk as examples o the range o protein unctions. Six proteins which illustrate some o the unctions o proteins are described in table 4.

rubo

inuln

This name is an abbreviation or ribulose bisphosphate carboxylase, which is arguably the most important enzyme in the world. The shape and chemical properties o its active site allow it to catalyse the reaction that xes carbon dioxide rom the atmosphere, which provides the source o carbon rom which all carbon compounds needed by living organisms can be produced. It is present at high concentrations in leaves and so is probably the most abundant o all proteins on Earth.

This hormone is produced as a signal to many cells in the body to absorb glucose and help reduce the glucose concentration o the blood. These cells have a receptor or insulin in their cell membrane to which the hormone binds reversibly. The shape and chemical properties o the insulin molecule correspond precisely to the binding site on the receptor, so insulin binds to it, but not other molecules. Insulin is secreted by  cells in the pancreas and is transported by the blood.

immunoglobuln

rhodopn

These proteins are also known as antibodies. They have sites at the tips o their two arms that bind to antigens on bacteria or other pathogens. The other parts o the immunoglobulin cause a response, such as acting as a marker to phagocytes that can engul the pathogen. The binding sites are hypervariable. The body can produce a huge range o immunoglobulins, each with a diferent type o binding site. This is the basis o specic immunity to disease.

Vision depends on pigments that absorb light. One o these pigments is rhodopsin, a membrane protein o rod cells o the retina. Rhodopsin consists o a light sensitive retinal molecule, not made o amino acids, surrounded by an opsin polypeptide. When the retinal molecule absorbs a single photon o light, it changes shape. This causes a change to the opsin, which leads to the rod cell sending a nerve impulse to the brain. Even very low light intensities can be detected.

collagen

spde lk

There are a number o diferent orms o collagen but all are rope-like proteins made o three polypeptides wound together. About a quarter o all protein in the human body is collagen  it is more abundant than any other protein. It orms a mesh o bres in skin and in blood vessel walls that resists tearing. Bundles o parallel collagen molecules give ligaments and blood vessel walls their immense strength. It orms part o the structure o teeth and bones, helping to prevent cracks and ractures.

Diferent types o silk with diferent unctions are produced by spiders. Dragline silk is stronger than steel and tougher than Kevlar. It is used to make the spokes o spiders webs and the lielines on which spiders suspend themselves. When rst made it contains regions where the polypeptide orms parallel arrays. Other regions seem like a disordered tangle, but when the silk is stretched they gradually extend, making the silk extensible and very resistant to breaking.

Protoms Every individual has a unique proteome. A proteome is all o the proteins produced by a cell, a tissue or an organism. B y contrast, the genome is all o the genes o a cell, a tissue or an organism. To fnd out how many dierent proteins are being produced, mixtures o proteins are extracted rom a sample and are then separated

94

2 .4 Protein s by gel electrophoresis. To identiy whether or not a particular protein is present, antibodies to the protein that have been linked to a fuorescent marker can be used. I the cell fuoresces, the protein is present. Whereas the genome o an organism is xed, the proteome is variable because dierent cells in an organism make dierent proteins. Even in a single cell the proteins that are made vary over time depending on the cells activities. The proteome thereore reveals what is actually happening in an organism, not what potentially could happen. Within a species there are strong similarities in the proteome o all individuals, but also dierences. The proteome o each individual is unique, partly because o dierences o activity but also because o small dierences in the amino acid sequence o proteins. With the possible exception o identical twins, none o us have identical proteins, so each o us has a unique proteome. E ven the proteome o identical twins can become dierent with age.

 Figure 8

Proteins rom a nematode worm have been separated by gel electrophoresis. Each spot on the gel is a diferent protein

acvy acv cc: gm d pm We might expect the proteome of an organism to be smaller than its genome, as some genes do not code for polypeptides. In fact the proteome is larger. How could an organism produce more proteins than the number of genes that its genome contains?

95

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M O L E C U L AR B I O LO G Y

2.5 enzyms understnding  Enzymes have an active site to which specic 

  

substrates bind. Enzyme catalysis involves molecular motion and the collision o substrates with the active site. Temperature, pH and substrate concentration afect the rate o activity o enzymes. Enzymes can be denatured. Immobilized enzymes are widely used in industry.

Ntre of science

applictions  Methods o production o lactose-ree milk and

its advantages.

Skills

 Experimental design: accurate quantitative

measurements in enzyme experiments require replicates to ensure reliability.

 Design o experiments to test the efect o

temperature, pH and substrate concentration on the activity o enzymes.  Experimental investigation o a actor afecting enzyme activity. (Practical 3)

active sites nd enzymes Enzymes have an active site to which specic substrates bind . Enzymes are globular proteins that work as catalysts  they speed up chemical reactions without being altered themselves. Enzymes are oten called biological catalysts because they are made by living cells and speed up biochemical reactions. The substances that enzymes convert into products in these reactions are called substrates. A general equation or an enzyme- catalysed reaction is: e nzym e  product substrate _______

 Figure 1

Computer-generated image of the enzyme hexokinase, with a molecule of its substrate glucose bound to the active site. The enzyme bonds a second substrate, phosphate, to the glucose, to make glucose phosphate

96

Enzymes are ound in all living cells and are also secreted by some cells to work outside. Living organisms produce many dierent enzymes  literally thousands o them. Many dierent enzymes are needed, as enzymes only catalyse one biochemical reaction and thousands o reactions take place in cells, nearly all o which need to be catalysed. This property is called enzymesubstrate sp ecifcity. It is a signifcant dierence between enzymes and non- biological catalysts such as the metals that are used in catalytic converters o vehicles. To be able to explain enzymesubstrate specifcity, we must look at the mechanism by which enzymes speed up reactions. This involves the

2 . 5 en z yM e s substrate, or substrates binding to a special region on the surace o the enzyme called the active site (see fgure 1 ) . The shape and chemical properties o the active site and the substrate match each other. This allows the substrate to bind, but not other substances. Substrates are converted into products while they are bound to the active site and the products are then released, reeing the active site to catalyse another reaction.

data-ba qutio: Biosynthesis of glycogen The Nobel Prize or Medicine was won in 1 947 by Gerty C ori and her husband C arl. They isolated two enzymes that convert glucose phosphate into

glycogen. Glycogen is a polysaccharide, composed o glucose molecules bonded together in two ways, called 1 , 4 and 1 , 6 bonds ( see fgure 2 ) . 4

4 bonding

 Figure 2

1

2

3

1 1

4 bonding plus a 6 bond forming a side-branch

a) D escribe the shape o C urve B .

[2 ]

b) Explain the shape o C urve B .

[2 ]

% conversion

1

C urve B was obtained using enzymes that had not been heat- treated.

Bonding in glycogen

Explain why two dierent enzymes are needed or the synthesis o glycogen rom glucose phosphate.

80 60

[2 ]

40

The ormation o side-branches increases the rate at which glucose phosphate molecules can be linked on to a growing glycogen molecule. Explain the reason or this. [2 ]

20

C urve A was obtained using heat- treated enzymes. Explain the shape o curve A.

[2 ]

B

A 10

20

30

40

50 min

 Figure 3

shows the percentage conversion of glucose phosphate to glycogen by the two enzymes, over a 50-minute period

enzym activity Enzyme catalysis involves molecular motion and the collision of substrates with the active site. E nzyme activity is the catalysis o a reaction by an enzyme. There are three stages: 

The substrate binds to the active site o the enzyme. S ome enzymes have two substrates that bind to dierent parts o the active site.



While the substrates are bound to the active site they change into dierent chemical substances, which are the products o the reaction.



The products separate rom the active site, leaving it vacant or substrates to bind again.

A substrate molecule can only bind to the active site i it moves very close to it. The coming together o a substrate molecule and an active site is known as a collision. This might suggest a high velocity impact between two vehicles on a road, but that would be a misleading image and we need to think about molecular motion in liquids to understand how substrateactive site collisions occur. With most reactions the substrates are dissolved in water around the enzyme. B ecause water is in a liquid state, its molecules and all

97

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toK Why hs he lck nd key mdel n been lly superseded by he induced-f mdel? The lock and key model and the induced-t model were both developed to help to explain enzyme activity. Models like these are simplied descriptions, which can be used to make predictions. Scientists test these predictions, usually by perorming experiments. I the results agree with the predictions, then the model is retained; i not then the model is modied or replaced. The German scientist Emil Fischer introduced the lock and key model in 1890. Daniel Koshland suggested the induced-t model in 1959 in the United States. The conormational changes predicted by Koshland's model were subsequently observed using high-resolution X-ray analysis o enzymes and other newly developed techniques. Although much experimental evidence has accumulated conrming predictions based on the induced-t model, it is still just viewed as a model o enzyme activity.

the particles dissolved in it are in contact with each other and are in continual motion. E ach particle can move separately. The direction of movement repeatedly changes and is random, which is the basis of diffusion in liquids. B oth substrates and enzymes with active sites are able to move, though most substrate molecules are smaller than the enzyme so their movement is faster. S o, collisions between substrate molecules and the active site occur because of random movements of both substrate and enzyme. The substrate may be at any angle to the active site when the collision occurs. Successful collisions are ones in which the substrate and active site are correctly aligned to allow binding to take place.

water molecules

substrates

active site part of enzyme  Figure 4 Enzyme-substrate collisions. If random

movements bring any of the substrate molecules close to the active site with the correct orientation, the substrate can bind to the active site

Factors afecting enzyme activity aciviy Mking  hyphesis Bacillus licheniformis lives in soil and on decomposing eathers. What is the reason or it producing a protease that works best at alkaline pH? Make a hypothesis to explain the observations. How could you test your hypothesis?

98

Temperature, pH and substrate concentration afect the rate o activity o enzymes. Enzyme activity is afected by temperature in two ways 

In liquids, the particles are in continual random motion. When a liquid is heated, the particles in it are given more kinetic energy. Both enzyme and substrate molecules therefore move around faster at higher temperatures and the chance of a substrate molecule colliding with the active site of the enzyme is increased. Enzyme activity therefore increases.



When enzymes are heated, bonds in the enzyme vibrate more and the chance of the bonds breaking is increased. When bonds in the enzyme break, the structure of the enzyme changes, including the active site. This change is permanent and is called denaturation. When an enzyme molecule has been denatured, it is no longer able to catalyse reactions. As more and more enzyme molecules in a solution become denatured, enzyme activity falls. Eventually it stops altogether, when the enzyme has been completely denatured. So, as temperature rises there are reasons for both increases and decreases in enzyme activity. Figure 5 shows the effects of temperature on a typical enzyme.

2 . 5 en z yM e s

Enzymes are sensitive to pH

Most enzymes have an optimum pH at which their activity is highest. I the pH is increased or decreased rom the optimum, enzyme activity decreases and eventually stops altogether. When the hydrogen ion concentration is higher or lower than the level at which the enzyme naturally works, the structure o the enzyme is altered, including the active site. B eyond a certain pH the structure o the enzyme is irreversibly altered. This is another example o denaturation. E nzyme s do no t all have the same p H o p timu m  in act, the re is a wide range . This re le cts the wide range o  p H e nviro nme nts in which e nzyme s wo rk. Fo r e xamp le , the p ro te ase se cre te d b y Bacillus lichen iform is has a p H o p timum b e twe e n 9 and 1 0 . This b acte rium is cu lture d to p ro duce its alkaline - to le rant p ro te ase o r u se in b io lo gical lau ndry de te rge nts, which are alkaline . Figure 6 sho ws the p H range o  so me o  the p lace s whe re e nzyme s wo rk. Figu re  7 sho ws the e e cts o  p H o n an e nzyme that is adap te d to wo rk at ne u tral p H.

rate of reaction

The pH scale is used to measure the acidity or alkalinity o a solution. The lower the pH, the more acid or the less alkaline a solution is. Acidity is due to the presence o hydrogen ions, so the lower the pH, the higher the hydrogen ion concentration. The pH scale is logarithmic. This means that reducing the pH by one unit makes a solution ten times more acidic. A solution at pH 7 is neutral. A solution at pH 6 is slightly acidic; pH 5 is ten times more acidic than pH 6, pH 4 is one hundred times more acidic than pH 6, and so on.

rate at which reaction decreases owing to denaturation of enzyme molecules

0

20

optimum temperature

actual rate of reaction

30 40 temperature/C

50

60

enzyme activity

Key stomach acidic hot springs decaying plant matter large intestine small intestine alkaline lakes

1 2 3 4 5 6

E nzymes cannot catalyse reactions until the substrate binds to the active site. This happens because o the random movements o molecules in liquids that result in collisions between substrates and active sites. I the concentration o substrates is increased, substrateactive site collisions will take place more requently and the rate at which the enzyme catalyses its reaction increases.

7 8 9 10  Figure 6

Optimum pH at which enzyme activity is fastest (pH 7 is optimum for most enzymes) .

As pH increases or decreases from the optimum, enzyme activity is reduced. This is because the shape of the active site is altered so the substrate does not t so well. Most enzymes are denatured by very high or low pH, so the enzyme no longer catalyses the reaction.

enzyme activity

I the relationship between substrate concentration and enzyme activity is plotted on a graph, a distinctive curve is seen ( fgure 8) , rising less and less steeply, but never quite reaching a maximum.

10

 Figure 5 Temperature and

Enzyme activity is afected by substrate concentration

However, there is another trend that needs to be considered. Ater the binding o a substrate to an active site, the active site is occupied and unavailable to other substrate molecules until products have been ormed and released rom the active site. As the substrate concentration rises, more and more o the active sites are occupied at any moment. A greater and greater proportion o substrateactive site collisions are thereore blocked. For this reason, the increases in the rate at which enzymes catalyse reactions get smaller and smaller as substrate concentration rises.

rate at which reaction increases owing to increased kinetic energy of substrate and enzyme molecules

pH  Figure 7

pH and enzyme activity

99

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M O L E C U L AR B I O LO G Y

Denaturation Enzymes can be denatured. enzyme activity

Enzymes are proteins, and like other proteins their structure can be irreversibly altered by certain conditions. This process is denaturation and both high temperatures and either high or low pH can cause it.

substrate concentration

When an enzyme has been denatured, the active site is altered so the substrate can no longer bind, or i its binds, the reaction that the enzyme normally catalyses does not occur. In many cases denaturation causes enzymes that were dissolved in water to become insoluble and orm a precipitate.

 Figure 8

The efect o substrate concentration on enzyme activity

Quantitative experiments Experimental design: accurate quantitative measurements in enzyme experiments require replicates to ensure reliability. O ur understanding o enzyme activity is based on evidence rom experiments. To obtain strong evidence these experiments must be careully designed and ollow some basic principles: 



measurements should be accurate, which in science means close to the true value; and



the experiment should be repeated, so that the replicate results can be compared to assess how reliable they are.

the results o the experiment should be quantitative, not j ust descriptive;

data-base questions: Digesting jello cubes a) describing whether the solution around the cubes is colourless or a shade o pink or red

Figure 9 shows apparatus that can be used to investigate protein digestion. tube

b) taking a sample o the solution and measuring its absorbance in a colorimeter

tight-tting lid

c) nding the mass o the cubes using an electronic balance. [3 ]

protease in a solution with known pH  Figure 9

100

I method ( c) was chosen, discuss whether it would be better to nd the mass o all o the cubes o j ello together, or nd the mass o each one separately. [2 ]

3

I the j ello cubes have a mass o 0.5 grams, state whether it is accurate enough to measure their mass to:

gelatine cubes

Tube used to investigate the rate o digestion o gelatine

I the cubes are made rom sugar- ree j ello ( j elly) , the colouring that they contain will gradually be released as the protein is digested by the protease. The questions below assume that strawberryfavoured j ello with red colouring has been used! 1

2

Explain whether these methods o assessing the rate o protein digestion are acceptable:

a) the nearest gram ( g) b) the nearest milligram ( mg) c) the nearest microgram ( g) .

[3 ]

2 . 5 en z yM e s

4

To obtain accurate mass measurements o the j ello cubes, it is necessary to remove them rom the tube and dry their surace to ensure that there are no drips o solution rom the tube adhering. Explain the reason or drying the surace o the blocks. [2 ]

7

D raw a graph o the results in the table.

8

D escribe the relationship between pH and papain activity. [3 ]

9

D iscuss the conclusions that can be drawn rom this data about the precise optimum pH o papain. [2 ]

Table 1 gives the results that were obtained using sugar-ree jello cubes and a protease called papain, extracted rom the fesh o resh pineapples. 5 6

D iscuss whether the results in table 1 are reliable.

[2 ]

Most o the results were obtained using an extract o protease rom one pineapple, but ater this ran out, a second pineapple was used to obtain more protease or use in the experiment. a) Deduce which results were obtained using the second extract.

[1 ]

b) S uggest how the use o a second extract could have aected the results. [2 ]

ph

Ma dcra (mg)

2

80

87

77

3

122

127

131

4

163

166

164

5

171

182

177

6

215

210

213

7

167

163

84

8

157

157

77

9

142

146

73

[5 ]

 Table 1

Designing enzyme experiments Design o experiments to test the efect o temperature, pH and substrate concentration on the activity o enzymes. 1

2

The actor that you are going to investigate is the independent variable. You need to decide:

clock could be used to measure the time taken or a colour change;



how you are going to vary it, or example with substrate concentration you would obtain a solution with the highest concentration and dilute it to get lower concentrations;



what units should be used or measuring the dependent variable, or example seconds rather than minutes or hours would be used or measuring a rapid colour change;



what units should be used or measuring the independent variable, or example temperature is measured in degrees C elsius;



how many repeats you need to get reliable enough results.



what range you need or the independent variable, including the highest and lowest levels and the number o intermediate levels.

The variable that you measure to nd out how ast the enzyme is catalysing the reaction is the dependent variable. You need to decide: 

how you are going to measure it, including the choice o meter or other measuring device, or example an electronic stop

3

Other actors that could aect the dependent are control variables. You need to decide: 

what all the control variables are;



how each o them can be kept constant;



what level they should be kept at, or example temperature should be kept at the optimum or the enzyme i pH is being investigated, but actors that might inhibit enzymes should be kept at a minimum level.

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M O L E C U L AR B I O LO G Y

enzym xprimnts Experimental investigation o a actor afecting enzyme activity. There are many worthwhile enzyme experiments. The method that ollows can be used to investigate the eect o substrate concentration on the activity o catalase. C atalase is one o the most widespread enzymes. It catalyses the conversion o hydrogen peroxide, a toxic by- product o metabolism, into water and oxygen. The apparatus shown in fgure 1 0 can be used to investigate the activity o catalase in yeast. The experiment could be repeated using the same concentration o yeast, but dierent hydrogen peroxide concentrations. Another possible investigation would be to assess the catalase concentrations in other cell types, such as liver, kidney or germinating seeds. These tissues would have to be macerated and then mixed with water at the same concentration as the yeast. 1

D escribe how the activity o the enzyme catalase could be measured using the apparatus shown in fgure 1 0. [2 ]

2

Explain why a yeast suspension must always be thoroughly stirred beore a sample o it is taken or use in an experiment. [2]

3

S tate two actors, apart rom enzyme concentration, that should be kept

 Figure 11

102

Enzyme experiment

constant i investigating the eect o substrate concentration.

[2 ]

4

Predict whether the enzyme activity will change more i substrate concentration is increased by 0. 2 mol dm - 3 or i it is decreased by the same amount. [2 ]

5

Explain why tissues such as liver must be macerated beore investigating catalase activity in them.

[2 ]

Safety goggles must be worn if this experiment is performed. Care should be taken not to get hydrogen peroxide on the skin. oxygen

yeast three-way tap

measuring cylinder water

0.8 mol dm 2 3 hydrogen peroxide  Figure 10

Apparatus for measuring catalase activity

water

2 . 5 en z yM e s

data-ba qutio: Designing an experiment to fnd the eect o temperature on lipase. Lipase converts fats into fatty acids and glycerol. It therefore causes a decrease in pH. This pH change can be used to measure the activity of lipase. Figure 1 2 shows suitable apparatus.

2

tube contents mixed when both have reached target temperature

thermometer 3

4 thermostatically controlled water bath  Figure 12

lipase

[2 ]

b) S tate the units for measuring the dependent variable.

[1 ]

c) Explain the need for at least three replicate results for each temperature in this experiment.

[2 ]

a) List the control factors that must be kept constant in this experiment.

[3 ]

b) Explain how these control factors can be kept constant.

[2 ]

c) S uggest a suitable level for each control factor.

[3 ]

S uggest reasons for: a) milk being used to provide a source of lipids in this experiment rather than vegetable oil. [1 ]

milk mixed with sodium carbonate (an alkali) and phenolphthalein (a pH indicator)

b) the thermometer being placed in the tube containing the larger, rather than the smaller, volume of liquid [1 ]

Apparatus for investigating the activity of lipase

Phenolphthalein is pink in alkaline conditions, but becomes colourless when the pH drops to 7. The time taken for this colour change can be used to measure the activity of lipase at different temperatures. Alternatively, pH changes could be followed using a pH probe and data- logging software. 1

a) Explain how you would measure the dependent variable accurately.

c) the substrate being added to the enzyme, rather than the enzyme to the substrate. 5

S ketch the shape of graph that you would expect from this experiment, with a temperature range from 0 C to 80 C on the x- axis and time taken for the indicator to change colour on the y- axis. [2 ]

6

Explain whether lipase from human pancreas or from germinating castor oil seeds would be expected to have the higher optimum temperature. [2 ]

a) State the independent variable in this experiment and how you would vary it. [2 ] b) S tate the units for measuring the independent variable.

[1 ]

c) State an appropriate range for the independent variable.

[2 ]

[1 ]

Immobilized enzymes Immobilized enzymes are widely used in industry. In 1 897 the B uchner brothers, Hans and E duard, showed that an extract of yeast, containing no yeast cells, would convert sucrose into alcohol. The door was opened to the use of enzymes to catalyse chemical processes outside living cells. Louis Pasteur had claimed that fermentation of sugars to alcohol could only occur if living cells were present. This was part of the theory of

103

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M O L E C U L AR B I O LO G Y

toK Wha is he diference beween dgma and hery? Ater the discovery in the 19th century o the conversion o sugar into alcohol by yeast, a dispute developed between two scientists, Justus von Liebig and Louis Pasteur. In 1860 Pasteur argued that this process, called ermentation, could not occur unless live yeast cells were present. Liebig claimed that the process was chemical and that living cells were not needed. Pasteurs view refected the vitalistic dogma  that the substances in animals and plants could only be made under the infuence o a vital spirit or vital orce. These contrasting views were as much infuenced by political and religious actors as by scientic evidence. The dispute was only resolved ater the death o both men. In 1897 the Buchner brothers, Hans and Eduard, showed that an extract o yeast, containing no yeast cells, did indeed convert sucrose into alcohol. The vitalistic dogma was overthrown and the door was opened to the use o enzymes to catalyse chemical processes outside living cells.

vitalism, which stated that substances in animals and plants can only be made under the infuence o a vital spirit or vital orce. The articial synthesis o urea, described in sub- topic 2 . 1 , had provided evidence against vitalism, but the B uchners research provided a clearer alsication o the theory. More than 5 00 enzymes now have commercial uses. Figure 1 3 shows a classication o commercially useul enzymes. Some enzymes are used in more than one type o industry. other industries 5% agriculture 11%

miscellaneous 4%

medical 21% biosensor 16% food & nutrition 23%

biotechnology 46% environment 13%

energy 3%  Figure 13

The enzymes used in industry are usually immobilized. This is attachment o the enzymes to another material or into aggregations, so that movement o the enzyme is restricted. There are many ways o doing this, including attaching the enzymes to a glass surace, trapping them in an alginate gel, or bonding them together to orm enzyme aggregates o up to 0. 1 mm diameter. Enzyme immobilization has several advantages.

104



The enzyme can easily be separated rom the products o the reaction, stopping the reaction at the ideal time and preventing contamination o the products.



Ater being retrieved rom the reaction mixture the enzyme may be recycled, giving useul cost savings, especially as many enzymes are very expensive.



Immobilization increases the stability o enzymes to changes in temperature and pH, reducing the rate at which they are degraded and have to be replaced.



S ubstrates can be exposed to higher enzyme concentrations than with dissolved enzymes, speeding up reaction rates.

2 . 6 s tru ctu r e o f d n a an d r n a

lctose-free mik Methods o production o lactose-ree milk and its advantages. Lactose is the sugar that is naturally present in milk. It can be converted into glucose and galactose by the enzyme lactase: lactose  glucose + galactose. Lactase is obtained rom Kluveromyces lactis, a type o yeast that grows naturally in milk. B iotechnology companies culture the yeast, extract the lactase rom the yeast and puriy it or sale to ood manuacturing companies. There are several reasons or using lactase in ood processing: 



S ome people are lactose-intolerant and cannot drink more than about 2 5 0 ml o milk per day, unless it is lactose- reduced ( see fgure 1 4) . Galactose and glucose are sweeter than lactose, so less sugar needs to be added to sweet oods containing milk, such as milk shakes or ruit yoghurt.



Lactose tends to crystallize during the production o ice cream, giving a gritty texture. B ecause glucose and galactose are more soluble than lactose they remain dissolved, giving a smoother texture.



B acteria erment glucose and galactose more quickly than lactose, so the production o yoghurt and cottage cheese is aster. Thailand South India Crete France Finland Sweden 0%

50% 100% lactose intolerance

 Figure 14 Rates of lactose intolerance

2.6 s  dna  rna understnding  The nucleic acids DNA and RNA are polymers o

nucleotides.  DNA difers rom RNA in the number o strands normally present, the base composition and the type o pentose.  DNA is a double helix made o two antiparallel strands o nucleotides linked by hydrogen bonding between complementary base pairs.

Ntre of science  Using models as representation o the real

world: Crick and Watson used model-making to discover the structure o DNA.

appictions  Crick and Watsons elucidation o the structure

o DNA using model-making.

Skis  Drawing simple diagrams o the structure o

single nucleotides and o DNA and RNA, using circles, pentagons and rectangles to represent phosphates, pentoses and bases.

105

2

M O L E C U L AR B I O LO G Y

Nucleic cids nd nucleotides The nucleic acids DNA and RNA are polymers o nucleotides. phosphate

sugar

base

O O

P

O 5

CH 2 O

O

1

C

C

N

Nucleic acids were frst discovered in material extracted rom the nuclei o cells, hence their name. There are two types o nucleic acid: D NA and RNA. Nucleic acids are very large molecules that are constructed by linking together nucleotides to orm a polymer. Nucleotides consist o three parts:

4

C3

2

OH  Figure 1

C



a sugar, which has fve carbon atoms, so is a pentose sugar;

OH



a p hosp hate group, which is the acidic, negatively- charged part o nucleic acids; and



a base that contains nitrogen and has either one or two rings o atoms in its structure.

The parts of a nucleotide

Figure 1 shows these parts and how they are linked together. The base and the phosphate are both linked by covalent bonds to the pentose sugar. Figure 2 shows a nucleotide in symbolic orm. To link nucleotides together into a chain or polymer, covalent bonds are ormed between the phosphate o one nucleotide and the pentose sugar o the next nucleotide. This creates a strong backbone or the molecule o alternating sugar and phosphate groups, with a base linked to each sugar.

 Figure 2

A simpler representation of a nucleotide

There are our dierent bases in both D NA and RNA, so there are our dierent nucleotides. The our dierent nucleotides can be linked together in any sequence, because the phosphate and sugar used to link them are the same in every nucleotide. Any base sequence is thereore possible along a D NA or RNA molecule. This is the key to nucleic acids acting as a store o genetic inormation  the base sequence is the store o inormation and the sugar phosphate backbone ensures that the store is stable and secure.

Difeences between DNa nd rNa DNA difers rom RNA in the number o strands normally present, the base composition and the type o pentose. HOH 2 C

OH

O H

H

H

H OH

HOH 2 C

H

2

There are usually two polymers o nucleotides in D NA but only one in RNA. The polymers are oten reerred to as strands, so D NA is double- stranded and RNA is single-stranded.

3

The our bases in D NA are adenine, cytosine, guanine and thymine. The our bases in RNA are adenine, cytosine, guanine and uracil, so the dierence is that uracil is present instead o thymine in RNA.

H OH

OH

The sugar within DNA is deoxyribose (top) and the sugar in RNA is ribose (bottom)

106

The sugar within D NA is deoxyribose and the sugar in RNA is ribose. Figure 3 shows that deoxyribose has one ewer oxygen atom than ribose. The ull names o D NA and RNA are based on the type o sugar in them  deoxyribonucleic acid and ribonucleic acid.

OH

H

 Figure 3

1

H O

H

There are three important dierences between the two types o nucleic acid:

2 . 6 s tru ctu r e o f d n a an d r n a

d-b qi: Chargafs data D NA samples from a range of species were analysed in terms of their nucleotide composition by Edwin C hargaff, an Austrian biochemist, and by others. The data is presented in table 1 . 1

2

C ompare the base composition of Mycobacterium tuberculosis ( a prokaryote) with the base composition of the eukaryotes shown in the table. [2 ] C alculate the base ratio A+ G/T + C , for humans and for Mycobacterium tuberculosis. S how your working. [2 ]

s  dna

Gp

3

4

5

E valuate the claim that in the D NA of eukaryotes and prokaryotes the amount of adenine and thymine are equal and the amounts of guanine and cytosine are equal.

[2 ]

E xplain the ratios between the amounts of bases in eukaryotes and prokaryotes in terms of the structure of D NA.

[2 ]

S uggest reasons for the difference in the base composition of bacteriophage T2 and the polio virus. [2 ]

ai

Gi

cyi

thymi

Human

Mammal

31.0

19.1

18.4

31.5

Cattle

Mammal

28.7

22.2

22.0

27.2

Salmon

Fish

29.7

20.8

20.4

29.1

Sea urchin

Invertebrate

32.8

17.7

17.4

32.1

Wheat

Plant

27.3

22.7

22.8

27.1

Yeast

Fungus

31.3

18.7

17.1

32.9

Mycobacterium tuberculosis Bacteriophage T2 Polio virus

Bacterium Virus Virus

15.1 32.6 30.4

34.9 18.2 25.4

35.4 16.6 19.5

14.6 32.6 0.0

 Table 1

Dwing DNa nd rNa molecules Drawing simple diagrams of the structure of single nucleotides and of DNA and RNA, using circles, pentagons and rectangles to represent phosphates, pentoses and bases. The structure of D NA and RNA molecules can be shown in diagrams using simple symbols for the subunits: 

circles for phosphates;



pentagons for pentose sugar;



rectangles for bases.

Figure 2 shows the structure of a nucleotide, using these symbols. The base and the phosphate are linked to the pentose sugar. The base is linked to C 1  the carbon atom on the right hand side of the pentose sugar. The phosphate is linked to C 5  the carbon atom on the side  Figure 4 Simplifed

diagram o RNA

107

2

M O L E C U L AR B I O LO G Y covalent bond

P S

A

P

chain on the upper let side o the pentose sugar. The positions o these carbon atoms are shown in fgure 1 .

S

T

To show the structure o RNA, draw a polymer o nucleotides, with a line to show the covalent bond linking the phosphate group o each nucleotide to the pentose in the next nucleotide. The phosphate is linked to C 3 o the pentose  the carbon atom that is on the lower let.

P

P S

C

S

G

P S P S

G

I you have drawn the structure o RNA correctly, the two ends o the polymer will be dierent. They are reerred to as the 3  and the 5  terminals.

P



The phosphate o another nucleotide could be linked to the C 3 atom o the 3  terminal.



The pentose o another nucleotide could be linked to the phosphate o the 5  terminal.

S

A

T

P

S

C

P

P Hydrogen bonds are formed between two bases

Key:

S  sugar A

P  phosphate

C T

 nitrogenous bases

G

 Figure 5 Simplifed

diagram o DNA

Structure of DNa

5 end 3 end complementary base pairs

S P S

P A

T

S

G

S

C

P

hydrogen bonds

S P

P

C

S

S

G

P A

T P

S

S S P S P T

S

S

P G

P C

S

G

Each strand consists o a chain o nucleotides linked by covalent bonds.

P



The two strands are parallel but run in opposite directions so they are said to be antiparallel. O ne strand is oriented in the direction 5  to 3  and the other is oriented in the direction 3  to 5 .



The two strands are wound together to orm a double helix.



The strands are held together by hydrogen bonds between the nitrogenous bases. Adenine ( A) is always paired with thymine ( T) and guanine ( G) with cytosine ( C ) . This is reerred to as comp lementary base p airing, meaning that A and T complement each other by orming base pairs and similarly G and C complement each other by orming base pairs.

G

S S P

S P

S sugarphosphate backbone S C

S 3 end

P 5 end  Figure 6 The double helix

108

D rawings o the structure o D NA on paper cannot show all eatures o the three-dimensional structure o the molecule. Figure 6 represents some o these eatures. 

P S

DNA is a double helix made of two antiparallel strands of nucleotides linked by hydrogen bonding between complementary base pairs.

P

A C

A P

P

P C

T

G

S

S

To show the structure o DNA, draw a strand o nucleotides, as with RNA, then a second strand alongside the frst. The second strand should be run in the opposite direction, so that at each end o the DNA molecule, one strand has a C 3 terminal and the other a C 5 terminal. The two strands are linked by hydrogen bonds between the bases. Add letters or names to indicate the bases. Adenine (A) only pairs with thymine (T) and cytosine (C ) only pairs with guanine (G) .

2 . 6 s tru ctu r e o f d n a an d r n a

d-b qi: The bases in DNA Look at the molecular models in fgure 7 and answer the ollowing questions. 1

2

3

Identiy three similarities between adenine and guanine. [3 ]

S tate one dierence between adenine and the other bases. [1 ]

4

C ompare the structure o cytosine and thymine.

Each o the bases in D NA has a nitrogen atom bonded to a hydrogen atom in a similar position, which appears in the lower let in each case in fgure 7. D educe how this nitrogen is used when a nucleotide is being assembled rom its subunits. [2 ]

5

Guanine

Adenine

[4]

Although the bases have some shared eatures, each one has a distinctive chemical structure and shape. Remembering the unction o D NA, explain the importance or the bases each to be distinctive. [5 ]

Cytosine

Thymine

 Figure 7

Molecular models Using models as representation of the real world: Crick and Watson used model-making to discover the structure of DNA. The word model in English is derived rom the Latin word modus, meaning manner or method. Models were originally architects plans, showing how a new building might be constructed. Threedimensional models were then developed to give a more realistic impression o what a proposed building would be like. Molecular models also show a possible structure in three dimensions, but whereas architects models are used to decide whether a building should become reality in the uture, molecular models help us to discover what the structure o a molecule actually is. Models in science are not always three- dimensional and do not always propose structures. They can be theoretical concepts and they can represent systems or processes. The common eature o models is that they are proposals, which are made to be tested. As with architecture, models in science are oten rej ected and replaced. Model- making played a critical part in C rick and Watsons discovery o the structure o D NA, but it took two attempts beore they were successul.

109

2

M O L E C U L AR B I O LO G Y

toK

crik nd Wtsons models of DNa struture

Wha is he relaive rle  cmpeiin and cperain in scienifc research?

Crick and Watsons discovery o the structure o DNA using model-making.

Three prominent research groups openly competed to elucidate the structure o DNA: Watson and Crick were working at Cambridge; Maurice Wilkins and Rosalind Franklin were working at Kings College o the University o London; and Linus Pauling's research group was operating out o Caltech in the United States.

C rick and Watsons success in discovering the structure o D NA was based on using the evidence to develop possible structures or D NA and testing them by model- building. Their rst model consisted o a triple helix, with bases on the outside o the molecule and magnesium holding the two strands together with ionic bonds to the phosphate groups on each strand. The helical structure and the spacing between subunits in the helix tted the X- ray diraction pattern obtained by Rosalind Franklin.

A stereotype o scientists is that they take a dispassionate approach to investigation. The truth is that science is a social endeavour involving a number o emotion-infuenced interactions between science. In addition to the joy o discovery, scientists seek the esteem o their community. Within research groups, collaboration is important, but outside o their research group competition oten restricts open communication that might accelerate the pace o scientic discovery. On the other hand, competition may motivate ambitious scientists to work tirelessly.

It was dicult to get all parts o this model to t together satisactorily and it was rej ected when Franklin pointed out that there would not be enough magnesium available to orm the cross links between the strands. Another deciency o this rst model was that is that it did not take account o C hargas nding that the amount o adenine equals the thymine and the amount o cytosine equals the amount o guanine. To investigate the relationship between the bases in D NA pieces o cardboard were cut out to represent their shapes. These showed that A- T and C - G base pairs could be ormed, with hydrogen bonds linking the bases. The base pairs were equal in length so would t between two outer sugar-phosphate backbones. Another fash o insight was needed to make the parts o the molecule t together: the two strands in the helix had to run in opposite directions  they must be antiparallel. C rick and Watson were then able to build their second model o the structure o D NA. They used metal rods and sheeting cut to shape and held together with small clamps. B ond lengths were all to scale and bond angles correct. Figure 8 shows C rick and Watson with the newly constructed model. The model convinced all those who saw it. A typical comment was It j ust looked right. The structure immediately suggested a mechanism or copying D NA. It also led quickly to the realization that the genetic code must consist o triplets o bases. In many ways the discovery o D NA structure started the great molecular biology revolution, with eects that are still reverberating in science and in society.

 Figure 8 Crick and

110

Watson and their DNA model

2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n

2.7 dna p, p   understnding  The replication o DNA is semi-conservative and 





 

 

depends on complementary base pairing. Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds. DNA polymerase links nucleotides together to orm a new strand, using the pre-existing strand as a template. Transcription is the synthesis o mRNA copied rom the DNA base sequences by RNA polymerase. Translation is synthesis o polypeptides on ribosomes. The amino acid sequence o polypeptides is determined by mRNA according to the genetic code. Codons o three bases on mRNA correspond to one amino acid in a polypeptide. Translation depends on complementary base pairing between codons on mRNA and anticodons on tRNA.

applictions  Use o Taq DNA polymerase to produce multiple

copies o DNA rapidly by the polymerase chain reaction (PCR) .  Production o human insulin in bacteria as an example o the universality o the genetic code allowing gene transer between species.

Skills  Use a table o the genetic code to deduce which

codon(s) corresponds to which amino acid.  Analysis o Meselson and Stahls results to obtain support or the theory o semiconservative replication o DNA.  Use a table o mRNA codons and their corresponding amino acids to deduce the sequence o amino acids coded by a short mRNA strand o known base sequence.  Deducing the DNA base sequence or the mRNA strand.

Ntre of science  Obtaining evidence or scientifc theories:

Meselson and Stahl obtained evidence or the semi-conservative replication o DNA.

Semi-conservtive repliction of DNa The replication o DNA is semi-conservative and depends on complementary base pairing. When a cell prepares to divide, the two strands o the double helix separate ( see fgure 2 ) . Each o these original strands serves as a guide, or template, or the creation o a new strand. The new strands are ormed by adding nucleotides, one by one, and linking them together. The result is two D NA molecules, both composed o an original strand and a newly synthesized strand. For this reason, D NA replication is reerred to as being semi-conservative.

111

2

M O L E C U L AR B I O LO G Y

adenine

thymine

cytosine

guanine

guanine

cytosine

thymine

The base sequence on the template strand determines the base sequence on the new strand. Only a nucleotide carrying a base that is complementary to the next base on the template strand can successully be added to the new strand (fgure 1 ) . This is because complementary bases orm hydrogen bonds with each other, stabilizing the structure. I a nucleotide with the wrong base started to be inserted, hydrogen bonding between bases would not occur and the nucleotide would not be added to the chain. The rule that one base always pairs with another is called complementary base pairing. It ensures that the two D NA molecules that result rom DNA replication are identical in their base sequences to the parent molecule that was replicated.

obtaining evidence fr the thery f semicnservative replicatin

adenine

Obtaining evidence or scientifc theories: Meselson and Stahl obtained evidence or the semi-conservative replication o DNA.

 Figure 1

S emi- conservative replication is an example o a scientifc theory that seemed intuitively right, but nonetheless needed to be backed up with evidence. Laboratories around the world attempted to confrm experimentally that replication o D NA is semi- conservative and soon convincing evidence had been obtained.

Parental DNA G C C G C G A T

G C T A T A C G

Replication fork A T G

C

A

T

G C T A

C T A T A C G

T A C C G A A T A T

C G T A A T A T

G C A T T A G

Parental strand  Figure 2

G C A T T A G C

New strand

New Parental strand strand

Semi-conservative replication

In 1 95 8 Matthew Meselson and Franklin S tahl published the results o exceedingly elegant experiments that provided very strong evidence or semi- conservative replication. They used 1 5 N, a rare isotope o nitrogen that has one more neutron than the normal 14 N isotope, so is denser. In the 1 93 0s Harold Urey had developed methods o puriying stable isotopes that could be used as tracers in biochemical pathways. 1 5 N was one o these. Meselson and S tahl devised a new method o separating D NA containing 1 5 N in its bases rom D NA with 1 4N. The technique is called caesium chloride density gradient centriugation. A solution o caesium chloride is spun in an ultracentriuge at nearly 45 , 000 revolutions per minute or 2 0 hours. The dense caesium ions tend to move towards the bottom o the tube but do not sediment ully because o diusion. A gradient is established, with the greatest caesium concentration, and thereore density, at the bottom and the lowest at the top o the tube. Any substance centriuged with the caesium chloride solution becomes concentrated at a level corresponding with its density. Meselson and S tahl cultured the bacterium E. coli or ourteen generations in a medium where the only nitrogen source was 1 5 N. Almost all nitrogen atoms in the bases o the D NA in the bacteria were thereore 1 5 N. They then transerred the bacteria abruptly to a medium in which all the nitrogen was 1 4 N. At the temperature used to culture them, the generation time was 5 0 minutes  the bacteria divided and thereore replicated their D NA once every 5 0 minutes.

112

2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n

Meselson and S tahl collected samples o D NA rom the bacterial culture or several hours rom the time when it was transerred to the 1 4 N medium. They extracted the D NA and measured its density by caesium chloride density gradient centriugation. The D NA could be detected because it absorbs ultraviolet light, and so created a dark band when the tubes were illuminated with ultraviolet. Figure 3 shows the results. In the next part o this sub- topic there is guidance in how to analyse the changes in position o the dark bands.

avy nw xpm hqu Meselson and Stahl used three techniques in their experiments that that were relatively new. Identiy a technique used by them that was developed: a) by Urey in the 1930s b) by Pickels in the 1 940s c) by M eselson and Stahl them selves in the 1 950s.

avy 0

0.3

0.7

1.0

1.5

2.0

2.5

3.0

4.0

generations  Figure 3

Meselson nd Sthls DNa repliction experiments Analysis o Meselson and Stahls results to obtain support or the theory o semi-conservative replication o DNA. The data- based question below will guide you through the analysis o Meselson and S tahls results and help to build your skills in this aspect o science.

Mg h vy To model helicase activity you could use some two-stranded rope or string and a split key ring. The strands in the rope are helical and represent the two strands in DNA. Open the key ring and put one strand o the rope inside it. Close the ring so that the other strand is outside. Slide the ring along the string to separate the strands. What problems are revealed by this model o the activity o helicase? Use the internet to fnd the solution used by living organisms.

d-b qu: The Meselson and Stahl experiment In order or cell division to occur, DNA must be duplicated to ensure that progeny cells have the same genetic inormation as the parent cells. The process o duplicating DNA is termed replication. The MeselsonStahl experiment sought to understand the mechanism o replication. Did it occur in a conservative ashion, a semi-conservative ashion or in a dispersive ashion (see fgure 4) ? Meselson and Stahl grew E. coli in a medium containing heavy nitrogen ( 1 5 N) or a number o generations. They then transerred the bacteria

to a 1 4N medium. S amples o the bacteria were taken over a period o time and separated by density gradient centriugation, a method in which heavier molecules settle urther down in acentriuge tube than lighter ones. 1

The single band o D NA at the start ( 0 generations) had a density o 1 . 72 4 g cm -3 . The main band o D NA ater our generations had a density o 1 . 71 0 g cm -3 . Explain how D NA with a lower density had been produced by the bacteria. [2 ]

113

2

M O L E C U L AR B I O LO G Y

2

a) Estimate the density o the D NA ater one generation. [2 ] b) Explain whether the density o D NA ater one generation alsifes any o the three possible mechanisms or D NA replication shown in fgure 4. [3 ]

3

4 5

6

Predict the results o centriuging a mixture o D NA rom 0 generations and 2  generations.

[2 ]

a) D escribe the results ater two generations, including the density o the D NA. [3 ] b) E xplain whether the results ater two generations alsiy any o the three possible mechanisms or D NA replication.

[3 ]

Explain the results ater three and our generations.

[2 ]

Figure 4 shows D NA rom E. coli at the start ( 0 generations) and ater one generation, with strands o D NA containing 1 5 N shown red and strands containing 1 4N shown green. Redraw either ( a) , ( b) or ( c) , choosing the mechanism that is supported by Meselson and S tahls experiment. Each D NA molecule can be shown as two parallel lines rather than a helix and the colours do not have to be red and green. D raw the D NA or two more generations o replication in a medium containing 1 4N. [3 ]

Dispersive

Conservative Semi-conservative

Newly synthesized strand Original template strand  Figure 4 Three possible mechanisms for

DNA replication

Helicase Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds. B eore D NA replication can occur, the two strands o the molecule must separate so that they can each act as a template or the ormation o a new strand. The separation is carried out by helicases, a group o enzymes that use energy rom ATP. The energy is required or breaking hydrogen bonds between complementary bases. One well-studied helicase consists o six globular polypeptides arranged in a donut shape. The polypeptides assemble with one strand o the D NA molecule passing through the centre o the donut and the other outside it. Energy rom ATP is used to move the helicase along the DNA molecule, breaking the hydrogen bonds between bases and parting the two stands. D ouble- stranded D NA cannot be split into two strands while it is still helical. Helicase thereore causes the unwinding o the helix at the same time as it separates the strands.

114

2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n

DNa polymese DNA polymerase links nucleotides together to form a new strand, using the pre-existing strand as a template. O nce helicase has unwound the double helix and split the D NA into two strands, replication can begin. Each o the two strands acts as a template or the ormation o a new strand. The assembly o the new strands is carried out by the enzyme D NA polymerase. D NA polymerase always moves along the template strand in the same direction, adding one nucleotide at a time. Free nucleotides with each o the our possible bases are available in the area where D NA is being replicated. Each time a nucleotide is added to the new strand, only one o the our types o nucleotide has the base that can pair with the base at the position reached on the template strand. D NA polymerase brings nucleotides into the position where hydrogen bonds could orm, but unless this happens and a complementary base pair is ormed, the nucleotide breaks away again. O nce a nucleotide with the correct base has been brought into position and hydrogen bonds have been ormed between the two bases, D NA polymerase links it to the end o the new strand. This is done by making a covalent bond between the phosphate group o the ree nucleotide and the sugar o the nucleotide at the existing end o the new strand. The pentose sugar is the 3  terminal and the phosphate group is the 5  terminal, so D NA polymerase adds on the 5  terminal o the ree nucleotide to the 3  terminal o the existing strand. D NA polymerase gradually moves along the template strand, assembling the new strand with a base sequence complementary to the template strand. It does this with a very high degree o fdelity  very ew mistakes are made during D NA replication.

Pcr  the polymese hin etion Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR) . The polymerase chain reaction ( PC R) is a technique used to make many copies o a selected D NA sequence. O nly a very small quantity o the D NA is needed at the start. The D NA is loaded into a PC R machine in which a cycle o steps repeatedly doubles the quantity o the selected D NA. This involves double- stranded D NA being separated into two single strands at one stage o the cycle and single strands combining to orm double-stranded D NA at another stage. The two strands in D NA are held together by hydrogen bonds. These are weak interactions, but in a D NA molecule there are large numbers

o them so they hold the two strands together successully at the temperatures normally encountered by most cells. I D NA is heated to a high temperature, the hydrogen bonds eventually break and the two strands separate. I the D NA is then cooled hydrogen bonds can orm, so the strands pair up again. This is called re- annealing. The PC R machine separates DNA strands by heating them to 95 C or fteen seconds. It then cools the DNA quickly to 5 4 C . This would allow reannealing o parent strands to orm double-stranded DNA. However, a large excess o short sections o single-stranded DNA called primers is present. The

115

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primers bind rapidly to target sequences and as a large excess o primers is present, they prevent the re-annealing o the parent strands. C opying o the single parent strands then starts rom the primers. The next stage in PCR is synthesis o doublestranded DNA, using the single strands with primers as templates. The enzyme Taq DNA polymerase is used to do this. It was obtained rom a bacterium, Thermus aquaticus, ound in hot springs, including those o Yellowstone National Park. The temperatures o these springs range rom 50 C to 80 C. Enzymes in most organisms would rapidly denature at such high temperatures, but those o Thermus aquaticus, including its DNA polymerase, are adapted to be very heat-stable to resist denaturation. Taq DNA polymerase is used because it can resist the brie period at 95 C used to separate the DNA

strands. It would work at the lower temperature o 5 4 C that is used to attach the primers, but its optimum temperature is 72 C . The reaction mixture is thereore heated to this temperature or the period when Taq DNA polymerase is working. At this temperature it adds about 1 ,000 nucleotides per minute, a very rapid rate o DNA replication. When enough time has elapsed or replication o the selected base sequence to be complete, the next cycle is started by heating to 95 C . A cycle o PC R can be completed in less than two minutes. Thirty cycles, which ampliy the D NA by a actor o a billion, take less than an hour. With the help o Taq D NA polymerase, PC R allows the production o huge numbers o copies o a selected base sequence in a very short time.

Select the DNA sequence to be copied

Twice as many DNA molecules can be copied in the next cycle

Raise temperature 15 seconds to 95C to separate the two strands

80 seconds Raise temperature to 72C to allow rapid DNA replication by Taq DNA polymerase  Figure 5

Lower temperature abruptly to 54C to allow binding of primers to DNA

25 seconds

 Figure 6

Transcription Transcription is the synthesis of mRNA copied from the DNA base sequences by RNA polymerase. This sequence o bases in a gene does not, in itsel, give any observable characteristic in an organism. The unction o most genes is to speciy the sequence o amino acids in a particular polypeptide. It is proteins that oten directly or indirectly determine the observable characteristics o an individual. Two processes are needed to produce a specifc polypeptide, using the base sequence o a gene. The frst o these is transcrip tion. Transcription is the synthesis o RNA, using D NA as a template. B ecause RNA is single- stranded, transcription only occurs along one o the two strands o D NA. What ollows is an outline o transcription: 

116

The enzyme RNA polymerase binds to a site on the D NA at the start o a gene.

2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n



RNA polymerase moves along the gene separating D NA into single strands and pairing up RNA nucleotides with complementary bases on one strand o the D NA. There is no thymine in RNA, so uracil pairs in a complementary ashion with adenine.



RNA polymerase orms covalent bonds between the RNA nucleotides.



The RNA separates rom the D NA and the double helix reorms.



Transcription stops at the end o the gene and the completed RNA molecule is released.

The product o transcription is a molecule o RNA with a base sequence that is complementary to the template strand o D NA. This RNA has a base sequence that is identical to the other strand, with one exception  there is uracil in place o thymine. So, to make an RNA copy o the base sequence o one strand o a D NA molecule, the other strand is transcribed. The D NA strand with the same base sequence as the RNA is called the sense strand. The other strand that acts as the template and has a complementary base sequence to both the RNA and the sense strand is called the antisense strand. RNA polymerase free RNA nucleotides direction of transcription

antisense strand of DNA

3

5

5

3

sense strand of DNA

RNA molecule

 Figure 7

The second o the two processes needed to produce a specifc polypeptide is translation. Translation is the synthesis o a polypeptide, with an amino acid sequence determined by the base sequence o a molecule o RNA. The production o RNA by transcription and how its base sequence is determined by a gene was described in the previous part o this sub- topic. Translation takes place on cell structures in the cytoplasm known as ribosomes. Ribosomes are complex structures that consist o a small and a large subunit, with binding sites or each o the molecules that take part in the translation. Figure 9 shows the two subunits o a ribosome. Each is composed o RNA molecules (pink and yellow) and proteins (purple) . Part o the large subunit (green) is the site that makes peptide bonds between amino acids, to link them together into a polypeptide.

TRANSCRIPTION

Translation is synthesis of polypeptides on ribosomes.

DNA

RNA TRANSLATION

Translation

POLYPEPTIDE  Figure 8

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 Figure 9

Large and small subunits of the ribosome with proteins shown in purple, ribosomal RNA in pink and yellow and the site that catalyses the formation of peptide bonds green

Messenge rNa nd the genetic code The amino acid sequence of polypeptides is determined by mRNA according to the genetic code. RNA that carries the inormation needed to synthesize a polypeptide is called messenger RNA, usually abbreviated to mRNA. The length o mRNA molecules varies depending on the number o amino acids in the polypeptide but an average length or mammals is about 2,000 nucleotides. In the genome there are many dierent genes that carry the inormation needed to make a polypeptide with a specifc amino acid sequence. At any time a cell will only need to make some o these polypeptides. O nly certain genes are thereore transcribed and only certain types o mRNA will be available or translation in the cytoplasm. C ells that need or secrete large amounts o a particular polypeptide make many copies o the mRNA or that polypeptide. For example, insulin- secreting cells in the pancreas make many copies o the mRNA needed to make insulin. Although most RNA is mRNA, there are other types; or example, transer RNA is involved in decoding the base sequence o mRNA into an amino acid sequence during translation and ribosomal RNA is part o the structure o the ribosome. They are usually reerred to as tRNA and rRNA.

data-base questions: Interpreting electron micrographs The electron micrographs in fgure 1 0 show transcription, translation and D NA replication.

show up more clearly. Identiy each o these structures:

1

a) the red structure in the central micrograph

2

118

D educe, with reasons, which process is occurring in each electron micrograph. The colour in the electron micrographs has been added to make the dierent structures

[5 ]

b) the thin blue molecule near the lower edge o the right- hand micrograph

2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n

c) the blue molecules o variable length attached to this thin blue molecule

e) the green molecules in the let- hand micrograph.

[5]

d) the red molecule in the let-hand micrograph

 Figure 10

codons Codons of three bases on mRNA correspond to one amino acid in a polypeptide. The translation dictionary that enables the cellular machinery to convert the base sequence on the mRNA into an amino acid sequence is called the genetic code. There are our dierent bases and twenty amino acids, so one base cannot code or one amino acid. There are sixteen combinations o two bases, which is still too ew to code or all o the twenty amino acids. Living organisms thereore use a triplet code, with groups o three bases coding or an amino acid. A sequence o three bases on the mRNA is called a codon. E ach codon codes or a specifc amino acid to be added to the polypeptide. Table 1 lists all o the 64 possible codons. The three bases o an mRNA codon are designated in the table as frst, second and third positions. Note that dierent codons can code or the same amino acid. For example the codons GUU and GUC both code or the amino acid valine. For this reason, the code is said to be degenerate. Note also that three codons are stop codons that code or the end o translation. Amino acids are carried on another kind o RNA, called tRNA. Each amino acid is carried by a specifc tRNA, which has a three- base anticodon complementary to the mRNA codon or that particular amino acid.

f p (5 ) U

C

A

G

s p u Phe Phe Leu Leu Leu Leu Leu Leu IIe IIe IIe Met Val Val Val Val

c Ser Ser Ser Ser Pro Pro Pro Pro Thr Thr Thr Thr Ala Ala Ala Ala

a Tyr Tyr Stop Stop His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu

G Cys Cys Stop Trp Arg Arg Arg Arg Ser Ser Arg Arg Gly Gly Gly Gly

th p (3 ) U C A G U C A G U C A G U C A G

 Table 1

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Deoding base sequenes Use of a table of the genetic code to deduce which codon(s) corresponds to which amino acid; use of a table of mRNA codons and their corresponding amino acids to deduce the sequence of amino acids coded by a short mRNA strand of known base sequence; deducing the DNA base sequence for the mRNA strand. There is no need to try to memorize the genetic code, but i a table showing it is available, you should be able to make various deductions. 1

Which codons correspond to an amino acid?

Three letters are used to indicate each amino acid in the table o the genetic code. Each o the 20 amino acids has between one and six codons. Read o the three letters o each codon or the amino acid. For example, the amino acid methionine, shown as Met on the table, has one codon which is AUG. 2

Questions 1

What base sequence in D NA would be transcribed to give the base sequence of a strand of mRNA?

A strand o mRNA is produced by transcribing the anti- sense strand o the D NA. This thereore has a

D educe the codons or a) Tryptophan ( Trp)

What amino acid sequence would be translated from a sequence of codons in a strand of mRNA?

The frst three bases in the mRNA sequence are the codon or the frst amino acid, the next three bases are the codon or the second base and so on. Look down the let hand side o the table to fnd the frst base o a codon, across the top o the table to fnd the second base and down the right hand side to fnd the third base. For example, GCA codes or the amino acid alanine, which is abbreviated to Ala in the table. 3

base sequence complementary to the mRNA. For example, the codon AUG in mRNA is transcribed rom the base sequence TAC on the antisense strand o the D NA. A longer example is that the base sequence GUAC GUAC G is transcribed rom C ATGC ATGC . Note that adenine pairs with thymine in D NA but with uracil in RNA.

b) Tyrosine ( Tyr)

2

c) Arginine ( Arg)

[3 ]

D educe the amino acid sequences that correspond to these mRNA sequences:

[3 ]

a) AC G 3

b) C AC GGG

c) C GC GC GAGG [3 ]

I mRNA contains the base sequence C UC AUC GAAUAAC C C a) deduce the amino acid sequence o the polypeptide translated rom the mRNA

[2 ]

b) deduce the base sequence o the antisense strand transcribed to produce the mRNA. [2 ]

codons and antiodons Translation depends on complementary base pairing between codons on mRNA and anticodons on tRNA. Three components work together to synthesize polypeptides by translation:

120



mRNA has a sequence o codons that specifes the amino acid sequence o the polypeptide;



tRNA molecules have an anticodon o three bases that binds to a complementary codon on mRNA and they carry the amino acid corresponding to that codon;



ribosomes act as the binding site or mRNA and tRNAs and also catalyse the assembly o the polypeptide.

2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n A summary o the main events o translation ollows: 1

An mRNA binds to the small subunit o the ribosome.

2

A molecule o tRNA with an anticodon complementary to the frst codon to be translated on the mRNA binds to the ribosome.

3

A second tRNA with an anticodon complementary to the second codon on the mRNA then binds. A maximum o two tRNAs can be bound at the same time.

4

The ribosome transers the amino acid carried by the frst tRNA to the amino acid on the second tRNA, by making a new peptide bond. The second tRNA is then carrying a chain o two amino acids  a dipeptide.

5

The ribosome moves along the mRNA so the frst tRNA is released, the second becomes the frst.

6

Another tRNA binds with an anticodon complementary to the next codon on the mRNA.

7

The ribosome transers the chain o amino acids carried by the frst tRNA to the amino acid on the second tRNA, by making a new peptide bond.

S tages 4, 5 and 6 are repeated again and again, with one amino acid added to the chain each time the cycle is repeated. The process continues along the mRNA until a stop codon is reached, when the completed polypeptide is released. The accuracy o translation depends on complementary base pairing between the anticodon on each tRNA and the codon on mRNA. Mistakes are very rare, so polypeptides with a sequence o hundreds o amino acids are regularly made with every amino acid correct. amino acid

growing polypeptide chain large sub unit of ribosome tRNA

tRNA mRNA

anticodon  Figure 11

Production of human insulin in bacteria Production of human insulin in bacteria as an example of the universality of the genetic code allowing gene transfer between species. D iabetes in some individuals is due to destruction o cells in the pancreas that secrete the hormone insulin. It can be treated by inj ecting insulin into the blood. Porcine and bovine insulin, extracted rom the pancreases o pigs and cattle, have both

been widely used. Porcine insulin has only one dierence in amino acid sequence rom human insulin and bovine insulin has three dierences. S hark insulin, which has been used or treating diabetics in Japan, has seventeen dierences.

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D espite the dierences in the amino acid sequence between animal and human insulin, they all bind to the human insulin receptor and cause lowering o blood glucose concentration. However, some diabetics develop an allergy to animal insulins, so it is preerable to use human insulin. In 1 982 human insulin became commercially available or the rst time. It was produced using genetically modied E. coli bacteria. S ince then methods o production have been developed using yeast cells and more recently safower plants. Each o these species has been genetically modied by transerring the gene or making human insulin to it. This is done in such a way that the gene is transcribed to produce mRNA and the mRNA is translated to produce harvestable quantities o insulin. The insulin produced has exactly the same amino acid sequence as i the gene was being transcribed and translated in human cells.

This may seem obvious, but it depends on each tRNA with a particular anticodon having the same amino acid attached to it as in humans. In other words, E. coli, yeast and safower ( a prokaryote, a ungus and a plant) all use the same genetic code as humans ( an animal) . It is ortunate or  Figure 12 genetic engineers that all organisms, with very ew exceptions, use the same genetic code as it makes gene transer possible between widely diering species.

2.8 cell respiration  Figure 12 Text to be added. understnding

 Cell respiration is the controlled release o

energy rom organic compounds to produce ATP.  ATP rom cell respiration is immediately available as a source o energy in the cell.  Anaerobic cell respiration gives a small yield o ATP rom glucose.  Aerobic cell respiration requires oxygen and gives a large yield o ATP rom glucose.

Ntre of science  Assessing the ethics o scientifc research:

the use o invertebrates in respirometer experiments has ethical implications.

122

applictions  Use o anaerobic cell respiration in yeasts to

produce ethanol and carbon dioxide in baking.  Lactate production in humans when anaerobic respiration is used to maximize the power o muscle contractions.

Skills  Analysis o results rom experiments involving

measurement o respiration rates in germinating seeds or invertebrates using a respirometer.

2 . 8 c e l l r e s P i r at i o n

relese of enegy by cell espition Cell respiration is the controlled release of energy from organic compounds to produce ATP. C ell respiration is one o the unctions o lie that all living cells perorm. O rganic compounds are broken down to release energy, which can then be used in the cell. For example, energy is released in muscle fbres by breaking down glucose into carbon dioxide and water. The energy can then be used or muscle contraction. In humans the source o the organic compounds broken down in cell respiration is the ood that we eat. C arbohydrates and lipids are oten used, but amino acids rom proteins may be used i we eat more protein than needed. Plants use carbohydrates or lipids previously made by photosynthesis.

 Figure 1

Breaking down 8 grams of glucose in cell respiration provides enough energy to sprint 100 metres

C ell respiration is carried out using enzymes in a careul and controlled way, so that as much as possible o the energy released is retained in a usable orm. This orm is a chemical substance called adenosine triphosphate, almost always abbreviated to ATP. To make ATP, a phosphate group is linked to adenosine diphosphate, or AD P. E nergy is required to carry out this reaction. The energy comes rom the breakdown o organic compounds. ATP is not transerred rom cell to cell and all cells require a continuous supply. This is the reason or cell respiration being an essential unction o lie in all cells.

aTP is  souce of enegy ATP from cell respiration is immediately available as a source of energy in the cell. C ells require energy or three main types o activity. 

S ynthesizing large molecules like D NA, RNA and proteins.



Pumping molecules or ions across membranes by active transport.



Moving things around inside the cell, such as chromosomes, vesicles, or in muscle cells the protein fbres that cause muscle contraction.

cell respiration

ADP 1 phosphate

ATP

active cell processes  Figure 2

The energy or all o these processes is supplied by ATP. The advantage o ATP as an energy supply is that the energy is immediately available. It is released simply by splitting ATP into AD P and phosphate. The AD P and phosphate can then be reconverted to ATP by cell respiration. When energy rom ATP is used in cells, it is ultimately all converted to heat. Although heat energy may be useul to keep an organism warm, it cannot be reused or cell activities and is eventually lost to the environment. This is the reason or cells requiring a continual source o ATP or cell activities.

 Figure 3

Infra red photo of toucan showing that it is warmer than its surroundings due to heat generated by respiration. Excess heat is dissipated by sending warm blood to the beak

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M O L E C U L AR B I O LO G Y

anerobic respirtion Anaerobic cell respiration gives a small yield of ATP from glucose. Glucose is broken down in anaerobic cell respiration without using any oxygen. The yield o ATP is relatively small, but the ATP can be produced quickly. Anaerobic cell respiration is thereore useul in three situations:

 Figure 4 The mud

in mangrove swamps is defcient in oxygen. Mangrove trees have evolved vertical roots called pneumatophores which they use to obtain oxygen rom the air



when a short but rapid burst o ATP production is needed;



when oxygen supplies run out in respiring cells;



in environments that are decient in oxygen, or example waterlogged soils.

The products o anaerobic respiration are not the same in all organisms. In humans, glucose is converted to lactic acid, which is usually in a dissolved orm known as lactate. In yeast and plants glucose is converted to ethanol and carbon dioxide. B oth lactate and ethanol are toxic in excess, so must be removed rom the cells that produce them, or be produced in strictly limited quantities.

activity

S ummary equations

does bioethnol solve or mke more problems?

glucose

There has been much debate about bioethanol production. A renewable fuel that cuts down on carbon emissions is obviously desirable. What are the arguments against bioethanol production?

lactate

AD P ATP This occurs in animals including humans. glucose

ethanol + carbon dioxide

AD P ATP This occurs in yeasts and plants.

Yest nd its uses Use of anaerobic cell respiration in yeasts to produce ethanol and carbon dioxide in baking. Yeast is a unicellular ungus that occurs naturally in habitats where glucose or other sugars are available, such as the surace o ruits. It can respire either aerobically or anaerobically. Anaerobic cell respiration in yeast is the basis or production o oods, drinks and renewable energy. B read is made by adding water to four, kneading the mixture to make dough and then baking it. Usually an ingredient is added to the dough to create bubbles o gas, so that the baked bread has a lighter texture. Yeast is oten this ingredient. Ater kneading, the dough is kept warm to encourage the yeast to respire. Any oxygen in the dough is soon used up so the yeast carries out anaerobic cell respiration. The carbon dioxide produced by anaerobic cell respiration cannot escape rom the dough and orms bubbles. The swelling o the dough due to  Figure 5

124

2 . 8 c e l l r e s P i r at i o n

the production o bubbles o carbon dioxide is called rising. Ethanol is also produced by anaerobic cell respiration, but it evaporates during baking. B ioethanol is ethanol produced by living organisms, or use as a renewable energy source. Although any plant matter can be utilized as a eed stock and various living organisms can be used to convert the plant matter into ethanol, most bioethanol is produced rom sugar cane and corn ( maize) , using yeast. Yeast converts sugars into ethanol in large ermenters by anaerobic respiration. O nly sugars can be converted, so starch and cellulose must rst be broken down into sugars. This is done using enzymes. The ethanol produced by the yeasts is puried by distillation and various methods are then used to remove water rom it to improve its combustion. Most bioethanol is used as a uel in vehicles, sometimes in a pure state and sometimes mixed with gasoline ( petrol) .  Figure 6

d-b qu: Monitoring anaerobic cell respiration in yeast The apparatus in gure 7 was used to monitor mass changes during the brewing o wine. The fask was placed on an electronic balance, which was connected to a computer or data-logging. The results are shown in gure 8. C alculate the total loss o mass during the experiment and the mean daily loss. airlock to prevent entry of oxygen

electronic balance connected to a datalogging computer

yeast in a solution of sugar and nutrients

E xplain the loss o mass.

3

S uggest two reasons or the increasing rate o mass loss rom the start o the experiment until day 6. [2 ]

4

S uggest two reasons or the mass remaining constant rom day 1 1 onwards. [2 ]

[3 ]

555 550 545

555.00  Figure 7

[3 ]

560

mass / g

1

2

Yeast data-logging apparatus

0

1

2

3

4

5

6 7 8 9 time / days

 Figure 8 Monitoring anaerobic cell

10 11 12 13

respiration in yeast

anerobic respirtion in humns Lactate production in humans when anaerobic respiration is used to maximize the power of muscle contractions. The lungs and blood system supply oxygen to most organs o the body rapidly enough or aerobic respiration to be used, but sometimes we

resort to anaerobic cell respiration in muscles. The reason is that anaerobic respiration can supply ATP very rapidly or a short period o time. It is

125

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M O L E C U L AR B I O LO G Y

thereore used when we need to maximize the power o muscle contractions. In our ancestors maximally powerul muscle contractions will have been needed or survival by allowing escape rom a predator or catching o prey during times o ood shortage. These events rarely occur in our lives today. Instead anaerobic respiration is more likely to be used during training or sport. These are examples: 

weight liters during the lit;



short- distance runners in races up to 400 metres;



long- distance runners, cyclists and rowers during a sprint fnish.

Anaerobic cell respiration involves the production o lactate, so when it is being used to supply ATP, the concentration o lactate in a muscle increases. There is a limit to the concentration that the body can tolerate and this limits how much anaerobic respiration can be done. This is the reason or the short timescale over which the power o muscle contractions can be maximized. We can only sprint or a short distance  not more than 400 metres.

Ater vigorous muscle contractions, the lactate must be broken down. This involves the use o oxygen. It can take several minutes or enough oxygen to be absorbed or all lactate to be broken down. The demand or oxygen that builds up during a period o anaerobic respiration is called the oxygen debt.

 Figure 9

Short bursts of intense exercise are fuelled by ATP from anaerobic cell respiration

aerobic respirtion Aerobic cell respiration requires oxygen and gives a large yield of ATP from glucose. I oxygen is available to a cell, glucose can be more ully broken down to release a greater quantity o energy than in anaerobic cell respiration. Whereas the yield o ATP is only two molecules per glucose with anaerobic cell respiration, it is more than thirty per glucose with aerobic cell respiration. Aerobic cell respiration involves a series o chemical reactions. C arbon dioxide and water are produced. In most organisms carbon dioxide is a waste product that has to be excreted, but the water is oten useul. In humans about hal a litre is produced per day. glucose + oxygen

carbon dioxide + water

AD P to ATP  Figure 10

The desert rat never needs to drink despite only eating dry foods, because aerobic cell respiration supplies its water needs

126

In eukaryotic cells most o the reactions o aerobic cell respiration, including all o the reactions that produce carbon dioxide, happen inside the mitochondrion.

2 . 8 c e l l r e s P i r at i o n

respiometes Analysis of results from experiments involving measurement of respiration rates in germinating seeds or invertebrates using a respirometer. A respirometer is any device that is used to measure respiration rate. There are many possible designs. Most involve these parts:

in volume. I possible the temperature inside the respirometer should be controlled using a thermostatically controlled water bath.



A sealed glass or plastic container in which the organism or tissue is placed.

Respirometers can be used to perorm various experiments:



An alkali, such as potassium hydroxide, to absorb carbon dioxide.



the respiration rate o dierent organisms could be compared;



A capillary tube containing fuid, connected to the container.



the eect o temperature on respiration rate could be investigated;



respiration rates could be compared in active and inactive organisms.

O ne possible design o respirometer is shown in gure 1 1 , but it is possible to design simpler versions that require only a syringe with a capillary tube attached to it. I the respirometer is working correctly and the organisms inside are carrying out aerobic cell respiration, the volume o air inside the respirometer will reduce and the fuid in the capillary tube will move towards the container with the organisms. This is because oxygen is used up and carbon dioxide produced by aerobic cell respiration is absorbed by the alkali. The position o the fuid should be recorded several times. I the rate o movement o the fuid is relatively even, the results are reliable. I the temperature inside the respirometer fuctuates, the results will not be reliable because an increase in air temperature causes an increase

graduated 1 cm 3 syringe

wire basket containing animal tissue lter paper rolled to form a wick potassium hydroxide solution

capillary tube  Figure 11

Diagram of a respirometer

The table below shows the results o an experiment in which the eect o temperature on respiration in germinating pea seeds was investigated. To analyse these results you should rst check to see i the repeats at each temperature are close enough or you to decide that the results are reliable. You should then calculate mean results or each temperature. The next stage is to plot a graph o the mean results, with temperature on the horizontal x-axis and the rate o movement o fuid on the vertical y-axis. Range bars can be added to the graph by plotting the lowest and highest result at each temperature and joining them with a ruled line. The graph will allow you to conclude what the relationship is between the temperature and the respiration rate o the germinating peas.

tmpu (c)

Mvm  fud  pm (mm m - 1 ) 1 dg

2d dg

3d dg

5

2.0

1.5

2.0

10

2.5

2.5

3.0

15

3.5

4.0

4.0

20

5.5

5.0

6.0

25

6.5

8.0

7.5

30

11.5

11.0

9.5

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M O L E C U L AR B I O LO G Y

data-bas qustions: Oxygen consumption in tobacco hornworms

1

a) Predict, using the data in the graphs, how the respiration rate o a larva will change as it grows rom moulting until it reaches the critical weight. [1 ] b) Explain the change in respiration rate that you have described. [2 ]

2

a) D iscuss the trends in respiration rate in larvae above the critical weight. [2 ]

3

S uggest a reason or earlier moulting in larvae reared in air with reduced oxygen content. [2 ] before critical weight 5th instar

0.12 0.10 0.08 0.06 0.04 0.02

after critical weight 0.16 0.14 0.12 0.10 0.08

1 0.025

2

3

4

5

6

4th instar

0.020 0.015 0.010 0.005

7 8 9 10 11 12 13 0.032 0.030 0.028 0.026 0.024 0.022 0.020 0.018

0.20.30.40.50.60.70.80.9 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0.000

3rd instar

4 6 0.0 0.0 weight (g)

1.0 1.1 1.2 1.3 1.4 0.009 0.008 0.007 0.006 0.005 0.004 0.003 6 18 0.1 0 . weight (g)

0.2 0 0.2 2 0.2 4 0.2 6

Each data point on the graphs shows the body mass and respiration rate o one larva. For each instar the results have been divided into younger larvae with low to intermediate body mass and older larvae with intermediate to high body mass. The results are plotted on separate graphs. The intermediate body mass is reerred to as the critical weight.

The researchers reared some tobacco hornworms in air with reduced oxygen content. They ound that the instar larvae moulted at a lower body mass than larvae reared in normal air with 2 0% oxygen.

0.0 8 0.1 0 0.1 2 0.1 4

The graphs below (fgure 1 2 ) show measurements made using a simple respirometer o the respiration rate o 3rd, 4th and 5 th instar larvae. D etails o the methods are given in the paper published by the biologists who carried out the research. The reerence to the research is C allier V and Nijhout H F (2 01 1 ) C ontrol o body size by oxygen supply reveals size-dependent and size-independent mechanisms o molting and metamorphosis. PNAS;1 08:1 46641 4669. This paper is reely available on the internet at http://www.pnas.org/ content/1 08/35 /1 4664.ull.pd+ html.

b) S uggest reasons or the dierence in the trends between the periods below and above the critical weight. [2 ]

respiration rate (ml O 2 /min)

Tobacco hornworms are the larvae o Manduca sexta. Adults o this species are moths. Larvae emerge rom the eggs laid by the adult emale moths. There are a series o larval stages called instars. Each instar grows and then changes into the next one by shedding its exoskeleton and developing a new larger one. The exoskeleton includes the tracheal tubes that supply oxygen to the tissues.

 Figure 12

Respiration rates of tobacco hornworms (after Callier and Nijhout, 2011)

ethics of animal us in rspiromtrs Assessing the ethics o scientifc research: the use o invertebrates in respirometer experiments has ethical implications. It is important or all scientists to assess the ethics o their research. There has been intense debate about the ethics o using animals in experiments. When discussing ethical issues, do

128

we consider the consequences such as benefts to students who are learning science? D o we consider intentions? For example, i the animals are harmed unintentionally does that change

2 . 9 Ph o to s yn th e s i s

whether the experiment was ethical or not? Are there absolute principles o right and wrong: or example, can we say that animals should never be subj ect to conditions that are outside what they would encounter in their natural habitat? B eore carrying out respirometer experiments involving animals these questions should be answered to help to decide whether the experiments are ethically acceptable: 1

Is it acceptable to remove animals rom their natural habitat or use in an experiment and can they be saely returned to their habitat?

2

Will the animals suer pain or any other harm during the experiment?

3

C an the risk o accidents that cause pain or suering to the animals be minimized during the experiment? In particular, can contact with the alkali be prevented?

4

Is the use o animals in the experiment essential or is there an alternative method that avoids using animals?

It is particularly important to consider the ethics o animal use in respirometer experiments because the International B accalaureate Organization has issued a directive that laboratory or eld experiments and investigations need to be undertaken in an ethical way. An important aspect o this is that experiments should not be undertaken in schools that infict pain or harm on humans or other living animals.

2.9 P understnding  Photosynthesis is the production o carbon 



  

compounds in cells using light energy. Visible light has a range o wavelengths with violet the shortest wavelength and red the longest. Chlorophyll absorbs red and blue light most eectively and refects green light more than other colours. Oxygen is produced in photosynthesis rom photolysis o water. Energy is needed to produce carbohydrates and other carbon compounds rom carbon dioxide. Temperature, light intensity and carbon dioxide concentration are possible limiting actors on the rate o photosynthesis.

applictions  Changes to the Earths atmosphere, oceans and

rock deposition due to photosynthesis.

Skills  Design o experiments to investigate limiting

actors on photosynthesis.  Separation o photosynthetic pigments by chromatography.  Drawing an absorption spectrum or chlorophyll and an action spectrum or photosynthesis.

Ntre of science  Experimental design: controlling relevant

variables in photosynthesis experiments is essential.

129

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M O L E C U L AR B I O LO G Y

What is photosynthesis? Photosynthesis is the production of carbon compounds in cells using light energy. Living organisms require complex carbon compounds to build the structure of their cells and to carry out life processes. S ome organisms are able to make all the carbon compounds that they need using only light energy and simple inorganic substances such as carbon dioxide and water. The process that does this is called photosynthesis. Photosynthesis is an example of energy conversion, as light energy is converted into chemical energy in carbon compounds. The carbon compounds produced include carbohydrates, proteins and lipids.

 Figure 2

The trees in one hectare of redwood forest in California can have a biomass of more than 4,000 tonnes, mostly carbon compounds produced by photosynthesis  Figure 1

Leaves absorb carbon dioxide and light and use them in photosynthesis

Separating photosynthetic pigments by chromatography Separation of photosynthetic pigments by chromatography. (Practical 4) C hloroplasts contain several types of chlorophyll and other pigments called accessory pigments. B ecause these pigments absorb different ranges of wavelength of light, they look a different colour to us. Pigments can be separated by chromatography. You may be familiar with paper chromatography but thin layer chromatography gives better results. This is done with a plastic strip that has been coated with a thin layer of a porous material. A spot containing pigments extracted from leaf tissue is placed near one end of the strip. A solvent is allowed to run up the strip, to separate the different types of pigment.

130

1

Tear up a leaf into small pieces and put them in a mortar.

2

Add a small amount of sand for grinding.

 Figure 3

Thin layer chromatography

2 . 9 Ph o to s yn th e s i s

3

Add a small volume o propanone ( acetone) .

4

Use the pestle to grind the lea tissue and dissolve out the pigments.

Carotene

orange

0.98

5

I the propanone all evaporates, add a little more.

Chlorophyll a

blue green

0.59

6

When the propanone has turned dark green, allow the sand and other solids to settle, then pour the propanone o into a watch glass.

Chlorophyll b

yellow green

0.42

Phaeophytin

olive green

0.81

Xanthophyll 1

yellow

0.28

Xanthophyll 2

yellow

0.15

7

8

9

Use a hair drier to evaporate o all the propanone and water rom the cells cytoplasm. When you have just a smear o dry pigments in the watch glass, add 34 drops o propanone and use a paint brush to dissolve the pigments. Use the paint brush to transer a very small amount o the pigment solution to the TLC strip. Your aim is to make a very small spot o pigment in the middle o the strip, 1 0 millimetres rom one end. It should be very dark. This is achieved by repeatedly putting a small drop onto the strip and then allowing it to dry beore adding another amount. You can speed up drying by blowing on the spot or by using the hair drier.

1 0 When the spot is dark enough, slide the other end o the strip into the slot in a cork or bung that fts into a tube that is wider than the TLC strip. The slot should hold the strip frmly. 1 1 Insert the cork and strip into a specimen tube. The TLC strip should extend nearly to the bottom o the tube, but not quite touch.

sp umb

clu

da mv (mm)

1 2 3 4 5

rf

nam f pgm

Pgm

clu f pgm

rf

1 2 Mark the outside o the tube j ust below the level o the spot on the TLC strip. 1 3 Take the strip and cork out o the tube. 1 4 Pour running solvent into the specimen tube up to the level that you marked. 1 5 Place the specimen tube on a lab bench where it will not be disturbed. C areully lower the TLC strip and cork into the tube, so that the tube is sealed and the TLC strip is j ust dipping into the running solvent.  The solvent must NO T touch the pigment spot. 1 6 Leave the tube completely alone or about fve minutes, to allow the solvent to run up through the TLC strip. You can watch the pigments separate, but D O NO T TO UC H THE  TUB E . 1 7 When the solvent has nearly reached the top o the strip, remove it rom the tube and separate it rom the cork. 1 8 Rule two pencil lines across the strip, one at the level reached by the solvent and one at the level o the initial pigment spot. 1 9 D raw a circle around each o the separated pigment spots and a cross in the centre o the circle.

6 7 8  Figure 4 Chromatogram

of leaf pigments

Table o standard R  values

131

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M O L E C U L AR B I O LO G Y

2 0 Using a ruler with millimetre markings, measure the distance moved by the running solvent ( the distance between the two lines) and the distance moved by each pigment ( the distance between the lower line and the cross in the centre o the circle) .

2 1 C alculate the R  or each pigment, where R  is the distance run by the pigment divided by the distance run by the solvent. 22 Show all your results in the table above, starting with the pigment that had moved least ar.

Waveengths of ight Visible light has a range o wavelengths with violet the shortest wavelength and red the longest. Sunlight or simply light is made up o all the wavelengths o electromagnetic radiation that our eyes can detect. It is thereore visible to us and other wavelengths are invisible. There is a spectrum o electromagnetic radiation rom very short to very long wavelengths. Shorter wavelengths such as X-rays and ultraviolet radiation have high energy; longer wavelengths such as inrared radiation and radio waves have lower energy. Visible light has wavelengths longer than ultraviolet and shorter than inrared. The range o wavelengths o visible light is 400 to 700 nanometres. When droplets o water in the sky split sunlight up and a rainbow is ormed, dierent colours o light are visible. This is because sunlight is a mixture o dierent wavelengths, which we see as dierent colours, including violet, blue, green and red. Violet and blue are the shorter wavelengths and red is the longest wavelength. The wavelengths o light that are detected by the eye are also those used by plants in photosynthesis. A reason or this is that they are emitted by the sun and penetrate the E arths atmosphere in larger quantities than other wavelengths, so are particularly abundant.

 Figure 5 In

a rainbow the wavelengths of visible light are separated

solar radiation reaching the Earths surface/W m 2 2

1.5

blue 5 4502 500 nm green 5 5252 575 nm red

5 6502 700 nm

1.0

0.5

0 500

1000

1500

2000

2500

3000

wavelength /nm  Figure 6 The spectrum

of electromagnetic radiation reaching the Earths surface

light absorption by chorophy Chlorophyll absorbs red and blue light most eectively and refects green light more than other colours. 132

The frst stage in photosynthesis is the absorption o sunlight. This involves chemical substances called pigments. A white or transparent substance does not absorb visible light. Pigments are substances that do

2 . 9 Ph o to s yn th e s i s

absorb light and thereore appear coloured to us. Pigments that absorb all o the colours appear black, because they emit no light. There are pigments that absorb some wavelengths o visible light but not others. For example, the pigment in a gentian fower absorbs all colours except blue. It appears blue to us, because this part o the sunlight is refected and can pass into our eye, to be detected by cells in the retina. Photosynthesizing organisms use a range o pigments, but the main photosynthetic pigment is chlorophyll. There are various orms o chlorophyll but they all appear green to us. This is because they absorb red and blue light very eectively, but the intermediate green light much less eectively. Wavelengths o green light thereore are refected. This is the reason or the main colour in ecosystems dominated by plants being green.

 Figure 7

Gentian fowers contain the pigment delphinidin, which refects blue light and absorbs all other wavelengths.

absorption nd ction spectr Drawing an absorption spectrum for chlorophyll and an action spectrum for photosynthesis.



When drawing both action and absorption spectra, the horizontal x-axis should have the legend wavelength, with nanometres shown as the units. The scale should extend rom 400 to 700 nanometres.



O n an action spectrum the y-axis should be used or a measure o the relative amount o photosynthesis. This is oten given as a percentage o the maximum rate, with a scale rom 0 to 1 00% .

It is not dicult to explain why action and absorption spectra are very similar: photosynthesis can only occur in wavelengths o light that chlorophyll or the other photosynthetic pigments can absorb. 100

chlorophyll a chlorophyll b carotenoids

% absorption

An action spectrum is a graph showing the rate o photosynthesis at each wavelength o light. An absorption spectrum is a graph showing the percentage o light absorbed at each wavelength by a pigment or a group o pigments.

400  Figure 8

500

600 wavelength (nm)

700

Absorption spectra o plant pigments

O n an absorption spectrum the y- axis should have the legend % absorption, with a scale rom 0 to 1 00% .



Ideally data points or specic wavelengths should be plotted and then a smooth curve be drawn through them. I this is not possible, the curve rom a published spectrum could be copied.

photosynthesis (% of max rate)

100 

400  Figure 9

500

600 wavelength (nm)

700

Action spectrum o a plant pigment

133

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M O L E C U L AR B I O LO G Y

data-bas qustions: Growth of tomato seedlings in red, green and blue light Tomato seeds were germinated and grown or 3 0 days in light produced by red, orange, green and blue light emitting diodes. Four dierent colours o LE D were tested and two combinations o colours. In every treatment the tomato plants received the same intensity o photons o light. The peak wavelength o light emitted by each wavelength is shown in the table below, together with the mean lea area and height o the seedlings. Plants oten grow tall, with weak stems and small leaves when they are receiving insufcient light or photosynthesis.

1

Plot a graph to show the relationship between wavelength, lea area and height. Hint: i you need two dierent scales on the y-axis you can put one on the let hand side o the graph and the other on the right hand side. D o not attempt to plot the results or combinations o LE D s. [6]

2

Using your graph, deduce the relationship between the lea area o the seedlings and their height. [1 ]

3

E valuate the data in the table or a grower o tomato crops in greenhouses who is considering using LED s to provide light. [3 ]

Pak wavngt o igt mitt by led (nm)

la ara o sings (m 2 )

higt o sings (mm)

Red

630

5.26

192

Orange

600

4.87

172

Green

510

5.13

161

Blue

450

7.26

128

Red and Blue



5.62

99

Red, Green and Blue



5.92

85

coours o leds

Source: Xiaoying, Shirong, Taotao, Zhigang and Tezuka (2012) . Regulation o the growth and photosynthesis o cherry tomato seedlings by diferent light irradiations o light emitting diodes (LED) . African Journal of Biotechnology Vol. 11(22) , pp. 6169-6177

oxygen prductin in phtsynthesis Oxygen is produced in photosynthesis from photolysis of water. O ne o the essential steps in photosynthesis is the splitting o molecules o water to release electrons needed in other stages. H 2 O  4e  + 4H + + O 2 This reaction is called photolysis because it only happens in the light and the word lysis means disintegration. All o the oxygen generated in photosynthesis comes rom photolysis o water. O xygen is a waste product and diuses away.

 Figure 10 Photosynthesizing organisms seem

insignicant in relation to the size o the Earth but over billions o years they have changed it signicantly

134

efts o potosyntsis on t eart Changes to the Earths atmosphere, oceans and rock deposition due to photosynthesis. Prokaryotes were the frst organisms to perorm photosynthesis, starting about 3,500 million years ago. They were joined millions o years later by algae and plants, which have been carrying out photosynthesis ever since.

2 . 9 Ph o to s yn th e s i s One consequence o photosynthesis is the rise in the oxygen concentration o the atmosphere. This began about 2,400 million years ago (mya) , rising to 2% by volume by 2,200 mya. This is known as the Great Oxidation Event. At the same time the E arth experienced its frst glaciation, presumably due to a reduction in the greenhouse eect. This could have been due to the rise in oxygenation causing a decrease in the concentration o methane in the atmosphere and photosynthesis causing a decrease in carbon dioxide concentration. B oth methane and carbon dioxide are potent greenhouse gases. The increase in oxygen concentrations in the oceans between 2 , 400 and 2 , 2 00 mya caused the oxidation o dissolved iron in the water, causing it to precipitate onto the sea bed. A distinctive rock ormation was produced called the banded iron ormation, with layers o iron oxide alternating with other minerals. The reasons or the banding are not yet ully understood. The banded iron ormations are the most important iron ores, so it is thanks to photosynthesis in bacteria billions o years ago that we have abundant supplies o steel today.

av dfr mpr Pl

cmp  mpr (%) CO 2

N2

Ar

O2 H 2O

98

1

1

0

0.04 78

1

21 0.1

Venus Earth Mars

0

96 2.5 1.5 2.5 0.1

What are the main diferences between the composition o the Earth's atmospheres and the atmosphere o the other planets. What is the cause o these diferences?

The oxygen concentration o the atmosphere remained at about 2 % rom 2 , 2 00 mya until about 75 0- 63 5 mya. There was then a signifcant rise to 2 0% or more. This corresponds with the period when many groups o multicellular organisms were evolving.

40

av

30

lg 

20

1500 10

CO 2 uptake/mol h 2 1

oxygen/% of atmosphere

50

1000

0 4.0

3.0

2.0 Millions of years ago ( 1,000)

1.0

0

 Figure 11

Production of carbohydrates

75 150 225 300 light intensity /J dm 2 2 s 2 1

of an experiment in which the rate of photosynthesis was found by measuring the uptake of carbon dioxide

1

What is the reason or a CO 2 uptake rate o  200 in darkness?

2

What can you predict about cell respiration and photosynthesis at the point where the net rate o CO 2 uptake is zero?

carbon dioxide + water  carbohydrate + oxygen To carry out this process, energy is required. A chemical reaction that involves putting in energy is described as endothermic. Reactions involving the production o oxygen are usually endothermic in living systems. Reactions involving combining smaller molecules to make larger ones are also oten endothermic and molecules o carbohydrate such as glucose are much larger than carbon dioxide or water.

0 200

 Figure 12 The graph shows the results

Energy is needed to produce carbohydrates and other carbon compounds rom carbon dioxide. Plants convert carbon dioxide and water into carbohydrates by photosynthesis. The simple equation below summarizes the process:

500

135

2

M O L E C U L AR B I O LO G Y

ativity increase in biomass of grass /kg ha - 1 h - 1

co 2 nentrtin 40 30

limiting fators

20

Temperature, light intensity and carbon dioxide concentration are possible limiting factors on the rate of photosynthesis.

10 0

210

100 200 300 400 CO 2 /cm 3 m - 3 air

 Figure 13

In this graph the rate of photosynthesis was measured indirectly by measuring the change in plant biomass.

1

2

The energy for the conversion of carbon dioxide into carbohydrate is obtained by absorbing light. This is the reason for photosynthesis only occurring in the light. The energy absorbed from light does not disappear  it is converted to chemical energy in the carbohydrates.

The maximum carbon dioxide concentration of the atmosphere is 380 cm 3 m 3 air. Why is the concentration often lower near leaves? In what weather conditions is carbon dioxide concentration likely to be the limiting factor for photosynthesis?

The rate of photosynthesis in a plant can be affected by three external factors: 

temperature;



light intensity;



carbon dioxide concentration.

E ach of these factors can limit the rate if they are below the optimal level. These three factors are therefore called limiting factors. According to the concept of limiting factors, under any combination of light intensity, temperature and carbon dioxide concentration, only one of the factors is actually limiting the rate of photosynthesis. This is the factor that is furthest from its optimum. If the factor is changed to make it closer to the optimum, the rate of photosynthesis increases, but changing the other factors will have no effect, as they are not the limiting factor. O f course, as the limiting factor is moved closer to its optimum, while keeping the other factors constant, a point will be reached where this factor is no longer the one that is furthest from its optimum and another factor becomes the limiting factor. For example, at night, light intensity is presumably the limiting factor for photosynthesis. When the sun rises and light intensity increases, temperature will usually take over as the limiting factor. As the temperature increases during the morning, carbon dioxide concentration might well become the limiting factor.

controed variabes in imiting fator experiments Experimental design: controlling relevant variables in photosynthesis experiments is essential. In any experiment, it is important to control all variables other than the independent and dependent variable that you are investigating. The independent variable is the one that you deliberately vary in the experiment with a range of levels that you choose. The dependent variable is what you measure during the experiment, to see if it is affected by the independent variable.

136

2 . 9 Ph o to s yn th e s i s

It is essential during this type o experiment to be sure that the independent variable is the only actor that could be aecting the dependent variable. All other variables that might aect the independent variable must thereore be controlled. These are questions that you need to answer when you are designing an experiment to investigate a limiting actor on photosynthesis: 

Which limiting actor will you investigate? This will be your independent variable.



How will you measure the rate o photosynthesis? This will be your dependent variable.



How will you keep the other limiting actors at a constant and optimal level? These will be your controlled variables.

Investigating limiting factors Design of experiments to investigate limiting factors on photosynthesis. There are many possible experimental designs. A method that can be used to investigate the eect o carbon dioxide concentration is given below. You could either modiy this to investigate a dierent limiting actor or you could develop an entirely dierent design.

Investigating the efect o carbon dioxide on photosynthesis acv tmprur 100 % of maximum rate

I a stem o pondweed such as Elodea, Cabomba or Myriophyllum is placed upside- down in water and the end o the stem is cut, bubbles o gas may be seen to escape. I these are collected and tested, they are ound to be mostly oxygen, produced by photosynthesis. The rate o oxygen production can be measured by counting the bubbles. Factors that might aect the rate o photosynthesis can be varied to fnd out what eect this has. In the method below carbon dioxide concentration is varied.

50

0

1

Enough water to fll a large beaker is boiled and allowed to cool. This removes carbon dioxide and other dissolved gases.

2

The water is poured repeatedly rom one beaker to another, to oxygenate the water. Very little carbon dioxide will dissolve.

3

A stem o pondweed is placed upside-down in the water and the end o its stem is cut. No bubbles are expected to emerge, as the water contains almost no carbon dioxide. The temperature o the water should be about 25 C and the water should be very brightly illuminated. Suitable apparatus is shown in fgure 1 6.

1

Enough sodium hydrogen carbonate is added to the beaker to raise the carbon dioxide concentration by 0.01 mol dm - 3 . I bubbles emerge, they are counted or 3 0 seconds, repeating the counts until two or three consistent results are obtained.

What was the optimum temperature for photosynthesis in this plant?

2

What was the maximum temperature for photosynthesis?

4

0

10 20 30 40 50 temperature/C

 Figure 14 In

this graph the rate of photosynthesis was measured indirectly by measuring the change in plant biomass

137

2

M O L E C U L AR B I O LO G Y

sodium hydrogen carbonate

5

Enough sodium hydrogen carbonate is added to raise the concentration by another 0.01 mol dm 3 . B ubble counts are done in the same way.

6

The procedure above is repeated again and again until further increases in carbon dioxide do not affect the rate of bubble production.

Questions 1 pondweed

Why are the following procedures necessary? a) B oiling and then cooling the water before the experiment. b) Keeping the water at 2 5 C and brightly illuminating it. c) Repeating bubble counts until several consistent counts have been obtained.

water at 25 C

2

What other factor could be investigated using bubble counts with pondweed and how would you design the experiment?

3

How could you make the measurement of the rate of oxygen production more accurate?

light source

 Figure 15 Apparatus or measuring

photosynthesis rates in diferent concentrations o carbon dioxide

138

Question s

Questions 1

2

Lipase is a digestive enzyme that accelerates the breakdown o triglycerides in the small intestine. In the laboratory, the rate o activity o lipase can be detected by a decline in pH. Explain what causes the pH to decline. [4]

a) ( i)

( ii) S tate the mass units that are shown in the equation. [2 ] b) ( i)

% of protien digested

c) Explain how it is possible to synthesize such large masses o ATP during races. [3 ] d) D uring a 1 00 m race, 80 g o ATP is needed but only 0.5 dm 3 o oxygen is consumed. D educe how ATP is being produced. [3 ]

lgh f Vm f xyg cmd  c rac/m rpra drg h rac/dm 3

immobilized papain

80

dissolved papain

60

C alculate the mass o ATP produced per [2 ] dm 3 o oxygen.

( ii) C alculate the mass o ATP produced per race in table 1 . [4]

Papain is a protease that can be extracted rom pineapple ruits. Figure 1 7 shows the eect o temperature on the activity o papain. The experiment was perormed using papain dissolved in water and then repeated with the same quantity o papain that had been immobilized by attaching it to a solid surace. The results show the percentage o the protein in the reaction mixture that was digested in a fxed time. 100

S tate the volume units that are shown in the equation. [1 ]

40 20

1500

36

10,000

150

42,300

700

 Table 1

0

 Figure 17

a) ( i)

O utline the eects o temperature on the activity o dissolved papain. [2 ]

( ii) E xplain the eects o temperature on the activity o dissolved papain. [2 ] b) ( i)

C ompare the eect o temperature on the activity o immobilized papain with the eect on dissolved papain. [2 ]

( ii) S uggest a reason or the dierence that you have described. [2 ] (iii) In some parts o the human body, enzymes are immobilized in membranes. Suggest one enzyme and a part o the body where it would be useul or it to be immobilized in a membrane. [2]

3

The equation below summarizes the results o metabolic pathways used to produce ATP, using energy rom the oxidation o glucose. glucose + oxygen + (ADP + Pi)  1 80 g 1 34.4 dm 3 1 8.25 kg carbon dioxide + water + ATP 1 34.4 dm3 1 08 g 1 8.25 kg

4

Figure 1 8 shows the eects o varying light intensity on the carbon dioxide absorption by leaves, at dierent, fxed carbon dioxide concentrations and temperatures. a) D educe the limiting actor or photosynthesis at: ( i) W

( ii) X

( iii) Y

( iv) Z.

[4]

b) Explain why curves I and II are the same between 1 and 7 units o light intensity. [3 ] c) Explain the negative values or carbon dioxide absorption when the leaves were in low light intensities. [3 ] 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1

rate of CO 2 absorption / arbitrary units

20 30 40 50 60 70 80 temperature /C

 Figure 18

Z

IV 0.4%CO 2 at 30C

III 0.4%CO 2 at 20C X

Y II 0.13%CO 2 at 30C I 0.13%CO 2 at 20C

W 1 2 3 4 5 6 7 light intensity / arbitrary units

139

2

M O L E C U L AR B I O LO G Y 5

Figure 1 9 shows the results o an experiment in which Chlorella cells were given light o wavelengths rom 660 nm ( red) up to 700 nm ( ar red) . The rate o oxygen production by photosynthesis was measured and the yield o oxygen per photon o light was calculated. This gives a measure o the efciency o photosynthesis at each wavelength. The experiment was then repeated with supplementary light with a wavelength o 65 0 nm at the same time as each o the wavelengths rom 660 to 700 nm, but with the same overall intensity o light as in the frst experiment.

yeild of oxygen molecules per photon of light

with supplementary light without supplementary light

0.10

0.05

680 700 wavelength (nm)

 Figure 19 Photon yield o photosynthesis in diferent light

intensities

140

b) D escribe the eect o the supplementary light. [2 ] c) E xplain how the error bars help in drawing conclusions rom this experiment. [2 ] d) The probable maximum yield o oxygen was 0. 1 2 5 molecules per photon o light. C alculate how many photons are needed to produce one oxygen molecule in photosynthesis. [2 ] e) O xygen production by photolysis involves this reaction: 4H 2 O  O 2 + 2 H 2 O + 4H + + 4e E ach photon o light is used to excite an electron ( raise it to a higher energy level) . C alculate how many times each electron produced by photolysis must be excited during the reactions o photosynthesis. [2 ]

0.15

0 660

a) D escribe the relationship between wavelength o light and oxygen yield, when there was no supplementary light. [2 ]

3C E LGLE Bn IEOtLI CO sG Y Iroducio

E very living organism inherits a blueprint or lie rom its parents. The inheritance o genes ollows patterns. C hromosomes carry genes in a linear sequence that is shared by members o a species. Alleles segregate during meiosis

allowing new combinations to be ormed by the usion o gametes. B iologists have developed techniques or artifcial manipulation o D NA, cells and organisms.

3.1 Genes Uderadig  A gene is a heritable actor that consists o

     

a length o DNA and inuences a specic characteristic. A gene occupies a specic position on one type o chromosome. The various specic orms o a gene are alleles. Alleles difer rom each other by one or a ew bases only. New alleles are ormed by mutation. The genome is the whole o the genetic inormation o an organism. The entire base sequence o human genes was sequenced in the Human Genome Project.

Applicaio  The causes o sickle cell anemia, including a

base substitution mutation, a change to the base sequence o mRNA transcribed rom it and a change to the sequence o a polypeptide in hemoglobin.  Comparison o the number o genes in humans with other species.

skill  Use o a database to determine diferences in

the base sequence o a gene in two species.

naure of ciece  Developments in scientic research ollow

improvements in technology: gene sequencers, essentially lasers and optical detectors, are used or the sequencing o genes.

141

3

G e n e ti cs

What is a gene? A gene is a heritable actor that consists o a length o DNA and infuences a specic characteristic. Genetics is the branch o biology concerned with the storage o inormation in living organisms and how this inormation can be passed rom parents to progeny. The word genetics was used by biologists long beore the method o inormation storage was understood. It came rom the word genesis, meaning origins. B iologists were interested in the origins o eatures such as baldness, blue eyes and much more. S omething must be the cause o these eatures and be passed on to ospring where the eatures would again develop. Experiments in the 1 9th century showed that there were indeed actors in living organisms that infuenced specic characteristics and that these actors were heritable. They could be passed on to ospring by pea plants, ruit fies and all other organisms. There was intense research into genetics rom the early 2 0th century onwards and the word gene was invented or the heritable actors. O ne obvious question was the chemical composition o genes. B y the middle o the 2 0th century there was strong evidence that genes were made o D NA. There are relatively ew D NA molecules in a cell  j ust 46 in a typical human cell or example  yet there are thousands o genes. We can thereore deduce that each gene consists o a much shorter length o D NA than a chromosome and that each chromosome carries many genes.

Comparing numbers of genes Comparison o the number o genes in humans with other species. How many genes does it take to make a bacterium, a banana plant or a bat, and how many are needed to make a human? We see ourselves as more complex in structure, physiology and behaviour so we might expect to

Group Prokaryotes

Brief description

Numbers of genes

Haemophilus infuenzae

Pathogenic bacterium

1,700

Escherichia coli

Gut bacterium

3,200

Protoctista

Trichomonas vaginalis

Unicellular parasite

60,000

Fungi

Saccharomyces cerevisiae (Yeast)

Unicellular ungus

6,000

Plants

Oryza sativa (Rice)

Crop grown or ood

41,000

Arabidopsis thaliana (Thale cress)

Small annual weed

26,000

Populus trichocarpa (Black cottonwood)

Large tree

46,000

Drosophila melanogaster (Fruit fy)

Larvae consume ripe ruit

14,000

Caenorhabditis elegans

Small soil roundworm

19,000

Homo sapiens (Humans)

Large omnivorous biped

23,000

Daphnia pulex (Water fea)

Small pond crustacean

31,000

Animals

142

Name of species

have more genes. The table shows whether this is true. It gives a range o predicted gene numbers. They are based on evidence rom the D NA o these species but are not precise counts o gene numbers as these are not yet known.

3 .1 GEN Es

Where are genes located?

Activity

A gene occupies a specifc position on one type o chromosome.

Etimating the number of human gene

E xperiments in which dierent varieties o plant or animals are crossed show that genes are linked in groups and each group corresponds to one o the types o chromosome in a species. For example, there are our groups o linked genes in ruit fies and our types o chromosome. Maize has ten groups o linked genes and ten types o chromosome and in humans the number o both is 2 3 .

7q22.2

7q15.2

7q21.3 7q21.1

7q14.3

7q14.1

7q12.1 7q12.3

7q11.22

7q21.3 7q21.13 7q21.11

7q31.33 7q31.31 7q31.1 7q22.2

7q33 7q35

7q32.2

7q36.2

Each gene occupies a specic position on the type o chromosome where it is located. This position is called the locus o the gene. Maps showing the sequence o genes along chromosomes in ruit fies and other organisms were produced by crossing experiments, but much more detailed maps can now be produced when the genome o a species is sequenced.

In October 1970 Scientifc American published an estimate that the human genome might consist o as many as 10 million genes. How many times greater than the current predicted number is this? What reasons can you give or such a huge overestimate in 1970?

 Figure 1

Chromosome 7: an example o a human chromosome. It consists o a single DNA molecule with approximately 170 million base pairs  about 5% o the human genome. The pattern o banding, obtained by staining the chromosome, is diferent rom other human chromosomes. Several thousand genes are located on chromosome 7, mostly in the light bands, each o which has a unique identiying code. The locus o a ew o the genes on chromosome 7 is shown

What are alleles? The various specifc orms o a gene are alleles. Gregor Mendel is usually regarded as the ather o genetics. He crossed varieties o pea plants, or example tall pea plants with dwar peas and white- fowered pea plants with purple-fowered. Mendel deduced that the dierences between the varieties that he crossed together were due to dierent heritable actors. We now know that these pairs o heritable actors are alternative orms o the same gene. For example there are two orms o the gene that infuences height, one making pea plants tall and the other making the plants dwar. These dierent orms are called alleles. There can be more than two alleles o a gene. O ne o the rst examples o multiple alleles to be discovered is in mice. A gene that infuences coat colour has three alleles, making the mice yellow, grey and black. There are three alleles o the gene in humans that determines AB O blood groups. In some cases there are large numbers o dierent alleles o a gene, or example the gene that infuences eye colour in ruit fies. As alleles are alternative orms o the same gene, they occupy the same position on one type o chromosome  they have the same locus. O nly one allele can occupy the locus o the gene on a chromosome. Most animal and plant cells have two copies o each type o chromosome, so

 Figure 2

Diferent coat colours in mice

143

3

G e n e ti cs we can expect two copies o a gene to be present. These could be two o the same allele o the gene or two dierent alleles.

Diferences between alleles Alleles difer rom each other by one or a ew bases only. A gene consists o a length o D NA, with a base sequence that can be hundreds or thousands o bases long. The dierent alleles o a gene have slight variations in the base sequence. Usually only one or a very small number o bases are dierent, or example adenine might be present at a particular position in the sequence in one allele and cytosine at that position in another allele. Positions in a gene where more than one base may be present are called single nucleotide polymorphisms, abbreviated to S NPs and pronounced snips. Several snips can be present in a gene, but even then the alleles o the gene dier by only a ew bases.

Comparing genes Use o a database to determine diferences in the base sequence o a gene in two species One outcome o the Human Genome Project is that the techniques that were developed have enabled the sequencing o other genomes. This allows gene sequences to be compared. The results o this comparison can be used to determine evolutionary relationships. Also, the identifcation o conserved sequences allows species to be chosen or exploring the unction o that sequence. 

C hoose Fast A and the sequence should appear. C opy the sequence and paste it into a .txt fle or notepad fle.



Repeat with a number o dierent species that you want to compare and save the fles.



To have the computer align the sequence or you, download the sotware called C lustalX and run it.



In the File menu, choose Load S equences.



C hoose gene rom the search menu.





Enter the name o a gene plus the organism, such as cytochrome oxidase 1 ( C O X1 ) or pan ( chimpanzee) .

S elect your fle. Your sequences should show up in the C lustalX window.



Under the Alignment menu choose D o C omplete Alignment. The example below shows the sequence alignment o 9 dierent organisms.

Move your mouse over the section Genomic regions, transcripts, and products until Nucleotide Links appears.





144

Go to the website called GenB ank ( http://www.ncbi. nlm.nih.gov/pubmed/)



Figure 3

3 .1 GEN Es

Data-baed quetion: COX-2, smoking and stomach cancer C O X- 2 is a gene that codes or the enzyme cyclooxygenase. The gene consists o over 6 , 000 nucleotides. Three single nucleotide polymorphisms have been discovered that are associated with gastric adenocarcinoma, a cancer o the stomach. O ne o these S NPs occurs at nucleotide 1 1 9 5 . The base at this nucleotide can be either adenine or guanine. A large survey in C hina involved sequencing both copies o the C O X- 2 gene in 3 5 7 patients who had developed gastric adenocarcinoma and in 9 85 people who did not have the disease. All o these people were asked whether they had ever smoked cigarettes. Table 1 shows the 3 5 7 patients with gastric adenocarcinoma categorized according to whether they were smokers or non- smokers and whether they had two copies o C O X-2 with G at nucleotide 1 1 95 ( GG) or at least one copy o the gene with A at this position ( AG or AA) . The results are shown as percentages. Table 2 shows the same categorization or the 985 people who did not have this cancer. 1

Predict, using the data, which o bases G or A is more common at nucleotide 1 1 95 in the controls. [2 ]

2

a) Calculate the total percentage o the patients that were smokers and the total percentage o controls that were smokers. [2 ] b) Explain the conclusion that can be drawn rom the dierence in the percentages. [2 ]

3

4

D educe, with a reason, whether G or A at nucleotide 1 1 95 is associated with an increased risk o gastric adenocarcinoma.

[2 ]

D iscuss, using the data, whether the risk o gastric adenocarcinoma is increased equally in all smokers. [2 ] GG

AG or AA

Smokers

9.8%

43.7%

Non-smokers

9.5%

40.0%

 Table 1

Patients with cancer GG

AG or AA

Smokers

9.4%

35.6%

Non-smokers

12.6%

42.4%

 Table 2

Patients without cancer

Mutation

Activity

New alleles are ormed by mutation.

New allele

New alleles are ormed rom other alleles by gene mutation. Mutations are random changes  there is no mechanism or a particular mutation being carried out. The most signifcant type o mutation is a base substitution. One base in the sequence o a gene is replaced by a dierent base. For example, i adenine was present at a particular point in the base sequence it could be substituted by cytosine, guanine or thymine.

Recent research into mutation involved nding the base sequence o all genes in parents and their ofspring. It showed that there was one base mutation per 1.2  10 8 bases. Calculate how many new alleles a child is likely to have as a result o mutations in their parents. Assume that there are 25,000 human genes and these genes are 2,000 bases long on average.

A random change to an allele that has developed by evolution over perhaps millions o years is unlikely to be benefcial. Almost all mutations are thereore either neutral or harmul. S ome mutations are lethal  they cause the death o the cell in which the mutation occurs. Mutations in body cells are eliminated when the individual dies, but mutations in cells that develop into gametes can be passed on to ospring and cause genetic disease.

Source: Campbell, CD, et al. (2012) Estimating the human mutation rate using autozygosity in a founder population. Nature Genetics, 44: 1277-1281. doi: 10.1038/ng.2418

145

3

G e n e ti cs

TOK

sickle cell anemia

What criteria can be used to distinguish between correlation and cause and efect? There is a correlation between high requencies o the sickle-cell allele in human populations and high rates o inection with Falciparum malaria. Where a correlation exists, it may or may not be due to a causal link. Consider the inormation in fgure 4 to decide whether sickle-cell anemia causes inection with malaria. b)

a)

Key Frequency of Hb s allele (%) 1520 1015 510

The causes o sickle cell anemia, including a base substitution mutation, a change to the base sequence o mRNA transcribed rom it and a change to the sequence o a polypeptide in hemoglobin. S ickle- cell anemia is the commonest genetic disease in the world. It is due to a mutation o the gene that codes or the alpha- globin polypeptide in hemoglobin. The symbol or this gene is Hb. Most humans have the allele Hb A . I a base substitution mutation converts the sixth codon o the gene rom GAG to GTG, a new allele is ormed, called Hb S. The mutation is only inherited by ospring i it occurs in a cell o the ovary or testis that develops into an egg or sperm. When the Hb S allele is transcribed, the mRNA produced has GUG as its sixth codon instead o GAG, and when this mRNA is transcribed, the sixth amino acid in the polypeptide is valine instead o glutamic acid. This change causes hemoglobin molecules to stick together in tissues with low oxygen concentrations. The bundles o hemoglobin molecules that are ormed are rigid enough to distort the red blood cells into a sickle shape.

05

Figure 4 Map ( a) shows the requency o the sickle cell allele and map (b) shows malaria afected areas in Arica and Western Asia

These sickle cells cause damage to tissues by becoming trapped in blood capillaries, blocking them and reducing blood fow. When sickle cells return to high oxygen conditions in the lung, the hemoglobin bundles break up and the cells return to their normal shape. These changes occur time ater time, as the red blood cells circulate. Both the hemoglobin and the plasma membrane are damaged and the lie o a red blood cell can be shortened to as little as 4 days. The body cannot replace red blood cells at a rapid enough rate and anemia thereore develops. So, a small change to a gene can have very harmul consequences or individuals that inherit the gene. It is not known how oten this mutation has occurred but in some parts o the world the Hb S allele is remarkably common. In parts o East Arica up to 5 % o newborn babies have two copies o the allele and develop severe anemia. Another 3 5 % have one copy so make both normal hemoglobin and the mutant orm. These individuals only suer mild anemia.

Figure 5 Micrographs o sickle cells and normal red blood cells

146

3 .1 GEN Es

Wha is a genome? The genome is the whole of the genetic information of an organism. Among biologists today the word genome means the whole o the genetic inormation o an organism. Genetic inormation is contained in D NA, so a living organisms genome is the entire base sequence o each o its D NA molecules. 

In humans the genome consists o the 46 molecules that orm the chromosomes in the nucleus plus the D NA molecule in the mitochondrion. This is the pattern in other animals, though the number o chromosomes is usually dierent.



In plant species the genome is the D NA molecules o chromosomes in the nucleus plus the D NA molecules in the mitochondrion and the chloroplast.



The genome o prokaryotes is much smaller and consists o the D NA in the circular chromosome, plus any plasmids that are present.

the Human Genome Projec The entire base sequence of human genes was sequenced in the Human Genome Project. The Human Genome Proj ect began in 1 990. Its aim was to fnd the base sequence o the entire human genome. This proj ect drove rapid improvements in base sequencing techniques, which allowed a drat sequence to be published much sooner than expected in 2 000 and a complete sequence in 2 003 . Although knowledge o the entire base sequence has not given us an immediate and total understanding o human genetics, it has given us what can be regarded as a rich mine o data, which will be worked by researchers or many years to come. For example, it is possible to predict which base sequences are protein- coding genes. There are approximately 2 3 , 000 o these in the human genome. O riginally, estimates or the number o genes were much higher. Another discovery was that most o the genome is not transcribed. O riginally called j unk D NA,  it is being increasingly recognized that within these j unk regions, there are elements that aect gene expression as well as highly repetitive sequences, called satellite D NA. The genome that was sequenced consists o one set o chromosomes  it is a human genome rather than the human genome. Work continues to fnd variations in sequence between dierent individuals. The vast maj ority o base sequences are shared by all humans giving us genetic unity, but there are also many single nucleotide polymorphisms which contribute to human diversity. S ince the publication o the human genome, the base sequence o many other species has been determined. C omparisons between these genomes reveal aspects o the evolutionary history o living organisms that were previously unknown. Research into genomes will be a developing theme o biology in the 2 1 st century.

Activity Ethic of genome reearch Ethical questions about genome research are worth discussing. Is it ethical to take a DNA sample from ethnic groups around the world and sequence it without their permission? Is it ethical for a biotech company to patent the base sequence of a gene to prevent other companies from using it to conduct research freely? Who should have access to this genetic information? Should employers, insurance companies and law enforcement agencies know our genetic makeup?

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techniques used for genome sequencing Developments in scientifc research ollow improvements in technology: gene sequencers, essentially lasers and optical detectors, are used or the sequencing o genes. The idea o sequencing the entire human genome seemed impossibly dicult at one time but improvements in technology towards the end o the 20th century made it possible, though still very ambitious. These improvements continued once the project was underway and drat sequences were thereore completed much sooner than expected. Further advances are allowing the genomes o other species to be sequenced at an ever increasing rate. To sequence a genome, it is rst broken up into small lengths o D NA. Each o these is sequenced separately. To nd the base sequence o a ragment o D NA, single- stranded copies o it are made using D NA polymerase, but the process is stopped beore the whole base sequence has been copied by putting small quantities o a non- standard nucleotide into the reaction mixture. This is done separately with non-standard nucleotides carrying each o the our possible D NA bases. Four samples o D NA copy o varying length are produced, each with one o our D NA bases at the end o each copy. These our samples are separated according to length by gel electrophoresis. For each number o nucleotides in the copy there is a band in j ust one o the our tracks in the gel, rom which the sequence o bases in the D NA can be deduced.

fuorescent marker is used or the copies ending in each o the our bases. 

The samples are mixed together and all the D NA copies are separated in one lane o a gel according to the number o nucleotides.



A laser scans along the lane to make the fuorescent markers fuoresce.



An optical detector is used to detect the colours o fuorescence along the lane. There is a series o peaks o fuorescence, corresponding to each number o nucleotides



A computer deduces the base sequence rom the sequence o colours o fuorescence detected.

The maj or advance in technology that speeded up base sequencing by automating it is this: 

148

C oloured fuorescent markers are used to mark the D NA copies. A dierent colour o

Figure 6 Sequencing read from the DNA of Pinor Noir variety of grape

3 .2 Ch rOmOsOm Es

3.2 Coooe Udertadig  Prokaryotes have one chromosome consisting  





   



o a circular DNA molecule. Some prokaryotes also have plasmids but eukaryotes do not. Eukaryote chromosomes are linear DNA molecules associated with histone proteins. In a eukaryote species there are diferent chromosomes that carry diferent genes. Homologous chromosomes carry the same sequence o genes but not necessarily the same alleles o those genes. Diploid nuclei have pairs o homologous chromosomes. Haploid nuclei have one chromosome o each pair. The number o chromosomes is a characteristic eature o members o a species. A karyogram shows the chromosomes o an organism in homologous pairs o decreasing length. Sex is determined by sex chromosomes and autosomes are chromosomes that do not determine sex.

Applicatio  Cairnss technique or measuring the length

o DNA molecules by autoradiography.  Comparison o genome size in T2 phage, Escherichia coli, Drosophila melanogaster, Homo sapiens and Paris japonica.  Comparison o diploid chromosome numbers o Homo sapiens, Pan troglodytes, Canis familiaris, Oryza sativa, Parascaris equorum.  Use o karyotypes to deduce sex and diagnose Down syndrome in humans.

skill  Use o online databases to identiy the locus o

a human gene and its protein product.

nature of ciece  Developments in scientic research ollow

improvements in techniques: autoradiography was used to establish the length o DNA molecules in chromosomes.

Bacterial chromoome Prokaryotes have one chromosome consisting o a circular DNA molecule. The structure of prokaryotic cells was described in sub- topic 1 . 2 . In most prokaryotes there is one chromosome, consisting of a circular D NA molecule containing all the genes needed for the basic life processes of the cell. The D NA in bacteria is not associated with proteins, so is sometimes described as naked.

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G e n e ti cs B ecause only one chromosome is present in a prokaryotic cell, there is usually only a single copy o each gene. Two identical copies are present briefy ater the chromosome has been replicated, but this is a preparation or cell division. The two genetically identical chromosomes are moved to opposite poles and the cell then splits in two.

Plasmids Some prokaryotes also have plasmids but eukaryotes do not. Plasmids are small extra DNA molecules that are commonly ound in prokaryotes but are very unusual in eukaryotes. They are usually small, circular and naked, containing a ew genes that may be useul to the cell but not those needed or its basic lie processes. For example, genes or antibiotic resistance are oten located in plasmids. These genes are benecial when an antibiotic is present in the environment but are not at other times. Plasmids are not always replicated at the same time as the chromosome o a prokaryotic cell or at the same rate. Hence there may be multiple copies o plasmids in a cell and a plasmid may not be passed to both cells ormed by cell division. C opies o plasmids can be transerred rom one cell to another, allowing spread through a population. It is even possible or plasmids to cross the species barrier. This happens i a plasmid that is released when a prokaryotic cell dies is absorbed by a cell o a dierent species. It is a natural method o gene transer between species. Plasmids are also used by biologists to transer genes between species articially.

Figure 1 (a) Circular DNA molecule from a bacterium (b) Bacterium preparing to divide

trimethoprim resistance

genes to help the plasmid spread

penicillin family resistance

disinfectant resistance

streptomycin family resistance

vancomycin resistance

Figure 2 The pLW1043 plasmid

Usig autoradiography to measure DnA molecules Developments in scientifc research ollow improvements in techniques: autoradiography was used to establish the length o DNA molecules in chromosomes. Quantitative data is usually considered to be the strongest type o evidence or or against a hypothesis, but in biology it is sometimes images that provide the most convincing evidence.

150

D evelopments in microscopy have allowed images to be produced o structures that were previously invisible. These sometimes conrm existing ideas but sometimes also change our understanding.

3 .2 Ch rOmOsOm Es

Autoradiography was used by biologists rom the 1 940s onwards to discover where specic substances were located in cells or tissues. John C airns used the technique in a dierent way in the 1 96 0s. He obtained images o whole D NA molecules rom E. coli bacteria. At the time it was not clear whether the bacterial

chromosome was a single D NA molecule or more than one, but the images produced by C airns answered this question. They also revealed replication orks in D NA or the rst time. C airnss technique was used by others to investigate the structure o eukaryote chromosomes.

Measurig the legth of DnA molecules Cairnss technique for measuring the length of DNA molecules by autoradiography. John C airns produced images o D NA molecules rom E.coli using this technique: 

C ells were grown or two generations in a culture medium containing tritiated thymidine. Thymidine consists o the base thymine linked to deoxyribose and is used by E. coli to make nucleotides that it uses in D NA replication. Tritiated thymidine contains tritium, a radioactive isotope o hydrogen, so radioactively labelled D NA was produced by replication in the E. coli cells.



The cells were then placed onto a dialysis membrane and their cell walls were digested using the enzyme lysozyme. The cells were gently burst to release their D NA onto the surace o the dialysis membrane.



A thin lm o photographic emulsion was applied to the surace o the membrane and let in darkness or two months. D uring that time some o the atoms o tritium in the D NA decayed and emitted high energy electrons, which react with the lm.



At the end o the two-month period the lm was developed and examined with a microscope. At each point where a tritium atom decayed there is a dark grain. These indicate the position o the D NA.

The images produced by C airns showed that the chromosome in E. coli is a single circular D NA molecule with a length o 1 , 1 00 m. This is remarkably long given that the length o the E coli cells is only 2 m. Autoradiography was then used by other researchers to produce images o eukaryotic chromosomes. An image o a chromosome rom the ruit fy Drosophila melanogaster was produced that was 1 2 , 00 0 m long. This corresponded with the total amount o D NA known to be in a D. melanogaster chromosome, so or this species at least a chromosome contains one very long D NA molecule. In contrast to prokaryotes, the molecule was linear rather than circular.

Figure 3

Eukaryote chromosomes Eukaryote chromosomes are linear DNA molecules associated with histone proteins. C hromosomes in eukaryotes are composed o D NA and protein. The D NA is a single immensely long linear D NA molecule. It is associated with histone proteins. Histones are globular in shape and are wider

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G e n e ti cs than the D NA. There are many histone molecules in a chromosome, with the D NA molecule wound around them. Adj acent histones in the chromosome are separated by short stretches o the D NA molecule that are not in contact with histones. This gives a eukaryotic chromosome the appearance o a string o beads during interphase.

Diferences between chromosomes In a eukaryote species there are diferent chromosomes that carry diferent genes. Eukaryote chromosomes are too narrow to be visible with a light microscope during interphase. During mitosis and meiosis the chromosomes become much shorter and atter by supercoiling, so are visible i stains that bind either D NA or proteins are used. In the frst stage o mitosis the chromosomes can be seen to be double. There are two chromatids, with identical DNA molecules produced by replication.

Figure 4 In an electron micrograph the histones give a eukaryotic chromosome the appearance of a string of beads during interphase

7S DNA thr

OH PH phe 16S

cyt b pro

There are at least two dierent types in every eukaryote but in most species there are more than that. In humans or example there are 2 3 types o chromosome.

val 23S leu

PL

glu N6

gln ala control loop asn ribosomal RNA cys transfer RNAs protein coding gene tyr

N5 leu ser his

ser N4 a rg

When the chromosomes are examined during mitosis, dierent types can be seen. They dier both in length and in the position o the centromere where the two chromatids are held together. The centromere can be positioned anywhere rom close to an end to the centre o the chromosome.

lys N 3gly OX3 ATPase

N1 ile f-met N2 trp OL OX1

asp OX2

Figure 5 Gene map of the human mitochondrial chromosome. There are genes on both of the two DNA strands. The chromosomes in the nucleus are much longer, carry far more genes and are linear rather than circular

Every gene in eukaryotes occupies a specifc position on one type o chromosome, called the locus o the gene. Each chromosome type thereore carries a specifc sequence o genes arranged along the linear D NA molecule. In many chromosomes this sequence contains over a thousand genes. C rossing experiments were done in the past to discover the sequence o genes on chromosome types in Drosophila melanogaster and other species. The base sequence o whole chromosomes can now be ound, allowing more accurate and complete gene sequences to be deduced. Having the genes arranged in a standard sequence along a type o chromosome allows parts o chromosomes to be swapped during meiosis.

Homologous chromosomes Homologous chromosomes carry the same sequence o genes but not necessarily the same alleles o those genes. I two chromosomes have the same sequence o genes they are homologous. Homologous chromosomes are not usually identical to each other because, or at least some o the genes on them, the alleles are dierent. I two eukaryotes are members o the same species, we can expect each o the chromosomes in one o them to be homologous with at least one chromosome in the other. This allows members o a species to interbreed.

152

3 .2 Ch rOmOsOm Es

Data-baed quetion: Comparing the chromosomes of mice and humans

Activity

Figure 6 shows all of the types of chromosome in mice and in humans. Numbers and colours are used to indicate sections of mouse chromosomes that are homologous to sections of human chromosomes. Mouse and human genetic similarities Mouse chromosomes 1

2

3 10 9 2 11 15

6 2 18 1

10

10 22 21 19 12

6

Human chromosomes 7

7 2 3 10 12 13 13

2 7

14

15

7 6

3 10 14 8

5

13

8 22

14 17

19

X

16 5

12

21

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

11 15 6 3

18 6 16 21 6 19 18 2

10 18 5

water at 25 C

18 19

20

21

22

X

Y

Y X

10

Figure 6 Chromosomes 1

2

D educe the number of types of chromosomes in mice and in humans.

[2 ]

Identify the two human chromosome types that are most similar to mouse chromosomes.

[2 ]

3

Identify mouse chromosomes which contain sections that are not homologous to human chromosomes. [2 ]

4

S uggest reasons for the many similarities between the mouse and human genomes.

5

polystyrene disc with hole cut through beaker

garlic bulb

1

Y

11 9

1 Garlic has large chromosomes so is an ideal choice for looking at chromosomes. Cells in mitosis are needed. Garlic bulbs grow roots if they are kept for 3 or 4 days with their bases in water, at about 25C. Root tips with cells in mitosis are yellow in colour, not white.

16

17 16 22 3

2

11 19

19 4 19

11 16 10 11

7

1

1

8

11 15

4

4

9 19

19

9 4 3 1

8

7

3

12 22 7 2 16 5

5 8

8

20

11 6

4

micocope invetigation of galic coooe

[2 ]

D educe how chromosomes have mutated during the evolution of animals such as mice and humans. [2 ]

2 Root tips are put in a mixture of a stain that binds to the chromosomes and acid, which loosens the connections between the cell walls. A length of about 5 mm is suitable. Ten parts of acetoorcein to one part of 1.0 mol dm -3 hydrochloric acid gives good results. stainacid mixture

5 mm long garlic root tip

watch glass

3 The roots are heated in the stainacid mixture on a hot plate, to 80C for 5 minutes. One of the root tips is put on a microscope slide, cut in half and the 2.5 mm length furthest from the end of the root is discarded. root tip watch glass

Comparing the genome sizes

8 7

1

5

3

6

2 4

Comparison of genome size in T2 phage, Escherichia coli, Drosophila melanogaster, Homo sapiens and Paris japonica. The genomes of living organisms vary by a huge amount. The smallest genomes are those of viruses, though they are not usually regarded as living organisms. The table on the next page gives the genome size of one virus and four living organisms. O ne of the four living organisms is a prokaryote. It has much the smallest genome. The genome size of eukaryotes depends on the size and number of chromosomes. It is correlated with the complexity of the organism, but is not directly proportional. There are several reasons for this. The proportion of the D NA that acts as functional genes is very variable and also the amount of gene duplication varies.

hot plate set at 80 C

4 A drop of stain and a cover slip is added and the root tip is squashed to spread out the cells to form a layer one cell thick. The chromosomes can then be examined and counted and the various phases of mitosis should also be visible. thumb pressing down to squash root tip cover slip

microscope slide

folded lter paper

153

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G e n e ti cs

Organism

Genome size (million base pairs)

T2 phage

0.18 5

Escherichia coli

Description Virus that attacks Escherichia coli Gut bacterium

140

Fruit fy

Homo sapiens

3,000

Humans

Paris japonica

150,000

Drosophila melanogaster

Woodland plant

Finding the loci of human genes Use o online databases to identiy the locus o a human gene and its protein product. The locus o a gene is its particular position on homologous chromosomes. Online databases can be used to fnd the locus o human genes. There is an example o such a database in the Online Mendelian Inheritance in Man website, maintained by Johns Hopkins University. 

S earch or the abbreviation O MIM to open the home page.



C hoose Search Gene Map.



Enter the name o a gene into the S earch Gene Map box. This should bring up a table with inormation about the gene, including its locus, starting with the chromosome on which the gene is located. S uggestions o human genes are shown on the right.



An alternative to entering the name o a gene is to select a chromosome rom 1 2 2 or one o the sex chromosomes X or Y. A complete sequence o gene loci will be displayed,

together with the total number o gene loci on that chromosome.

Gene name

Description of gene

DRD4

A gene that codes or a dopamine receptor that is implicated in a variety o neurological and psychiatric conditions.

CFTR

A gene that codes or a chloride channel protein. An allele o this gene causes cystic brosis.

HBB

The gene that codes or the beta-globin subunit o hemoglobin. An allele o this gene causes sickle cell anemia.

F8

The gene that codes or Factor VIII, one o the proteins needed or the clotting o blood. The classic orm o hemophilia is caused by an allele o this gene.

TDF

Testis determining actor  the gene that causes a etus to develop as a male.

Haploid nuclei Haploid nuclei have one chromosome o each pair. A haploid nucleus has one chromosome o each type. It has one ull set o the chromosomes that are ound in its species. Haploid nuclei in humans contain 2 3 chromosomes or example. Gametes are the sex cells that use together during sexual reproduction. Gametes have haploid nuclei, so in humans both egg and sperm cells contain 2 3 chromosomes.

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3 .2 Ch rOmOsOm Es

Diploid nuclei Diploid nuclei have pairs of homologous chromosomes. A diploid nucleus has two chromosomes of each type. It has two full sets of the chromosomes that are found in its species. D iploid nuclei in humans contain 46 chromosomes for example. When haploid gametes fuse together during sexual reproduction, a zygote with a diploid nucleus is produced. When this divides by mitosis, more cells with diploid nuclei are produced. Many animals and plants consist entirely of diploid cells, apart from the cells that they are using to produce gametes for sexual reproduction. D iploid nuclei have two copies of every gene, apart from genes on the sex chromosomes. An advantage of this is that the effects of harmful recessive mutations can be avoided if a dominant allele is also present. Also, organisms are often more vigorous if they have two different alleles of genes instead of j ust one. This is known as hybrid vigour and is the reason for strong growth of F 1 hybrid crop plants.

Figure 7 Mosses coat the trunks of the laurel trees in this forest in the Canary Islands. Mosses are unusual because their cells are haploid. In most eukaryotes the gametes are haploid but not the parent that produces them

Chromosome numbers The number of chromosomes is a characteristic feature of members of a species. O ne of the most fundamental characteristics of a species is the number of chromosomes. O rganisms with a different number of chromosomes are unlikely to be able to interbreed so all the interbreeding members of a species need to have the same number of chromosomes. The number of chromosomes can change during the evolution of a species. It can decrease if chromosomes become fused together or increase if splits occur. There are also mechanisms that can cause the chromosome number to double. However, these are rare events and chromosome numbers tend to remain unchanged over millions of years of evolution.

Figure 8 Trillium luteum cell with a diploid number of 12 chromosomes. Two of each type of chromosome are present

Comparing chromosome numbers Comparison of diploid chromosome numbers of Homo sapiens, Pan troglodytes, Canis familiaris, Oryza sativa, Parascaris equorum. The Oxford English D ictionary consists of twenty large volumes, each containing a large amount of information about the origins and meanings of words. This information could have been published in a smaller number of larger volumes or in a larger number of smaller volumes. There is a parallel with the numbers and sizes of chromosomes in

eukaryotes. Some have a few large chromosomes and others have many small ones. All eukaryotes have at least two different types of chromosome, so the diploid chromosome number is at least four. In some cases it is over a hundred. The table on the next page shows the diploid chromosome number of selected species.

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scientifc name o pecie

Figure 9 Who has more chromosomes  a dog or its owner?

Englih name

Diploid chromoome number

Parascaris equorum

horse threadworm

4

Oryza sativa

rice

24

Homo sapiens

humans

46

Pan troglodytes

chimpanzee

48

Canis amiliaris

dog

78

Data-baed quetion: Diferences in chromosome number Plant Haplopappus gracilis Luzula purpurea (woodrush) Crepis capillaris Vicia aba (eld bean) Brassica oleracea (cabbage) Citrullus vulgaris (water melon) Lilium regale (royal lily) Bromus texensis Camellia sinesis (Chinese tea) Magnolia virginiana (sweet bay) Arachis hypogaea (peanut) Cofea arabica (cofee) Stipa spartea (porcupine grass) Chrysoplenum alterniolium (saxirage) Aster laevis (Michaelmas daisy) Glyceria canadensis (manna grass) Carya tomentosa (hickory) Magnolia cordata Rhododendron keysii

Chromoome number 4 6 8 12 18 22 24 28 30 38 40 44 46 48 54 60 64 76 78

Animal Parascaris equorum (horse threadworm) Aedes aegypti (yellow ever mosquito) Drosophila melanogaster (ruity) Musca domestica (house y) Chorthippus parallelus (grasshopper) Cricetulus griseus (Chinese hamster) Schistocerca gregaria (desert locust) Desmodus rotundus (vampire bat) Mustela vison (mink) Felis catus (domestic cat) Mus musculus (mouse) Mesocricetus auratus (golden hamster) Homo sapiens (modern humans) Pan troglodytes (chimpanzee) Ovis aries (domestic sheep) Capra hircus (goat) Dasypus novemcinctus (armadillo) Ursus americanus (American black bear) Canis amiliaris (dog)

Table 1

156

1

There are many different chromosome numbers in the table, but some numbers are missing, for example, 5 , 7, 1 1 , 1 3 . Explain why none of the species has 1 3 chromosomes. [3 ]

2

D iscuss, using the data in the table, the hypothesis that the more complex an organism is, the more chromosomes it has. [4]

3

E xplain why the size of the genome of a species cannot be deduced from the number of chromosomes. [1 ]

4

S uggest, using the data in table 1 , a change in chromosome structure that may have occurred during human evolution. [2 ]

3 .2 Ch rOmOsOm Es

sex determination female

male

XX

XY

Sex is determined by sex chromosomes and autosomes are chromosomes that do not determine sex. There are two chromosomes in humans that determine sex:

X

X



the Y chromosome is much smaller and has its centromere near the end.

B ecause the X and Y chromosomes determine sex they are called the sex chromosomes. All the other chromosomes are autosomes and do not affect whether a fetus develops as a male or female. The X chromosome has many genes that are essential in both males and females. All humans must therefore have at least one X chromosome. The Y chromosome only has a small number of genes. A small part of the Y chromosome has the same sequence of genes as a small part of the X chromosome, but the genes on the remainder of the Y chromosome are not found on the X chromosome and are not needed for female development.

XX X

the X chromosome is relatively large and has its centromere near the middle.

Y



XX

XY XY

1 female : 1 male

Figure 10 Determination of gender

O ne Y chromosome gene in particular causes a fetus to develop as a male. This is called either S RY or TD F. It initiates the development of male features, including testes and testosterone production. B ecause of this gene a fetus with one X and one Y chromosome develops as a male. A fetus that has two X chromosomes and no Y chromosome does not have the TD F gene so ovaries develop instead of testes and female sex hormones are produced, not testosterone. Females have two X chromosomes. Females pass on one of their two X chromosomes in each egg cell, so all offspring inherit an X chromosome from their mother. The gender of a human is determined at the moment of fertilization by one chromosome carried in the sperm. This can either be an X or a Y chromosome. When sperm are formed, half contain the X chromosome and half the Y chromosome. D aughters inherit their fathers X chromosome and sons inherit his Y chromosome.

Karyogram A karyogram shows the chromosomes of an organism in homologous pairs of decreasing length. The chromosomes of an organism are visible in cells that are in mitosis, with cells in metaphase giving the clearest view. S tains have to be used to make the chromosomes show up. S ome stains give each chromosome type a distinctive banding pattern. If dividing cells are stained and placed on a microscope slide and are then burst by pressing on the cover slip, the chromosomes become spread. O ften they overlap each other, but with careful searching a cell can usually be found with no overlapping chromosomes. A micrograph can be taken of the stained chromosomes.

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TOK To what extent is determining gender or sporting competition a scientifc question? Gender testing was introduced at the 1968 Olympic games to address concerns that women with ambiguous physiological genders would have an unair advantage. This has proven to be problematic or a number o reasons. The chromosomal standard is problematic as non-disjunction can lead to situations where an individual might technically be male, but might not defne hersel in that way. People with two X chromosomes can develop hormonally as a male and people with an X and a Y can develop hormonally as a emale.

Originally analysis involved cutting out all the chromosomes and arranging them manually but this process can now be done digitally. The chromosomes are arranged according to their size and structure. The position of the centromere and the pattern of banding allow chromosomes that are of a different type but similar size to be distinguished. As most cells are diploid, the chromosomes are usually in homologous pairs. They are arranged by size, starting with the longest pair and ending with the smallest.

The practice o gender testing was discontinued in 1996 in part because o human rights issues including the right to sel-expression and the right to identiy one's own gender. Rather than being a scientifc question, it is more airly a social question. Figure 11 Karyogram o a human emale, with fuorescent staining

Karyotypes and Down syndrome Use o karyotypes to deduce sex and diagnose Down syndrome in humans. A karyogram is an image of the chromosomes of an organism, arranged in homologous pairs of decreasing length. A karyotype is a property of an organism  it is the number and type of chromosomes that the organism has in its nuclei. Karyotypes are studied by looking at karyograms. They can be used in two ways: 1

To deduce whether an individual is male or female. If two XX chromosomes are present the individual is female whereas one X and one Y indicate a male.

2

To diagnose Down syndrome and other chromosome abnormalities. This is usually done using fetal cells taken from the uterus during pregnancy. If there are three copies of chromosome 2 1 in the karyotype instead of two, the child has Down syndrome. This is sometimes called trisomy 21 . While individuals vary, some of the component features of the syndrome are hearing loss, heart and vision disorders. Mental and growth retardation are also common.

Figure 12 Child with trisomy 21 or Down syndrome

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3.3 mEiOsis

Data-based questions: A human karyotype The karyogram shows the karyotype of a fetus. 1

S tate which chromosome type is a) longest b) shortest.

2

[2 ]

D istinguish between the structure of a) human chromosome 2 and chromosome 1 2 b) the human X and Y chromosome.

[4]

3

D educe with a reason the sex of the fetus.

[2 ]

4

E xplain whether the karyotype shows any abnormalities.

[2 ]

Figure 13

3.3 meo Udertadig  One diploid nucleus divides by meiosis to   

 

 

produce our haploid nuclei. The halving o the chromosome number allows a sexual lie cycle with usion o gametes. DNA is replicated beore meiosis so that all chromosomes consist o two sister chromatids. The early stages o meiosis involve pairing o homologous chromosomes and crossing over ollowed by condensation. Orientation o pairs o homologous chromosomes prior to separation is random. Separation o pairs o homologous chromosomes in the rst division o meiosis halves the chromosome number. Crossing over and random orientation promotes genetic variation. Fusion o gametes rom diferent parents promotes genetic variation.

Applicatio  Non-disjunction can cause Down syndrome

and other chromosome abnormalities. Studies showing age o parents inuences chances o non-disjunction.  Methods used to obtain cells or karyotype analysis e.g. chorionic villus sampling and amniocentesis and the associated risks.

skill  Drawing diagrams to show the stages o

meiosis resulting in the ormation o our haploid cells.

nature of ciece  Making careul observations: meiosis was

discovered by microscope examination o dividing germ-line cells.

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the discovery of meiosis Making careful observations: meiosis was discovered by microscope examination of dividing germ-line cells. When improved microscopes had been developed in the 1 9th century that gave detailed images o cell structures, it was discovered that some dyes specifcally stained the nucleus o the cell. These dyes revealed thread-like structures in dividing nuclei that were named chromosomes. From the 1 880s onwards a group o German biologists carried out careul and detailed observations o dividing nuclei that gradually revealed how mitosis and meiosis occur. We can appreciate the considerable achievements o these biologists i we try to repeat the observations that they made. The preparation o microscope slides showing meiosis is challenging. Suitable tissue can be obtained rom the developing anthers inside a lily bud or rom the testis o a dissected locust. The tissue must be fxed, stained and then squashed on a microscope slide. Oten no cells in meiosis are visible or the images are not clear enough to show details o the process. Even with prepared slides made by experts it is difcult to understand the images as chromosomes orm a variety o bizarre shapes during the stages o meiosis. A key observation was that in the horse threadworm (Parascaris equorum) there are two chromosomes in the nuclei o egg and sperm cells, whereas the ertilized egg contains our. This indicated that the

one diploid cell

Nuclear divisions unlike mitosis had already been observed during gamete development in both animals and plants. These divisions were identifed as the method used to halve the chromosome number and they were named meiosis. The sequence o events in meiosis was eventually worked out by careul observation o cells taken rom the ovaries o rabbits ( Oryctolagus cuniculus) between 0 and 2 8 days old. The advantage o this species is that in emales meiosis begins at birth and occurs slowly over many days.

 Figure 1

Meiosis in ouline

2n

One diploid nucleus divides by meiosis to produce four haploid nuclei.

meiosis I two haploid cells

n

n

meiosis II four haploid cells

chromosome number is doubled by ertilization. The observation led to the hypothesis that there must be a special nuclear division in every generation that halves the chromosome number.

n

n

Figure 2 Overview of meiosis

n

n

Meiosis is one o the two ways in which the nucleus o a eukaryotic cell can divide. The other method is mitosis, which was described in sub- topic 1 . 6. In meiosis the nucleus divides twice. The frst division produces two nuclei, each o which divides again to give a total o our nuclei. The two divisions are known as meiosis I and meiosis II. The nucleus that undergoes the frst division o meiosis is diploid  it has two chromosomes o each type. C hromosomes o the same type are known as homologous chromosomes. Each o the our nuclei produced by meiosis has j ust one chromosome o each type  they are haploid. Meiosis involves a halving o the chromosome number. It is thereore known as a reduction division. The cells produced by meiosis I have one chromosome o each type, so the halving o the chromosome number happens in the frst division,

160

3.3 mEiOsis not the second division. The two nuclei produced by meiosis I have the haploid number o chromosomes, but each chromosome still consists o two chromatids. These chromatids separate during meiosis II, producing our nuclei that have the haploid number o chromosomes, with each chromosome consisting o a single chromatid.

Meiosis and sexual life cycles The halving of the chromosome number allows a sexual life cycle with fusion of gametes. The lie cycles o living organisms can be sexual or asexual. In an asexual lie cycle the ospring have the same chromosomes as the parent so are genetically identical. In a sexual lie cycle there are dierences between the chromosomes o the ospring and the parents, so there is genetic diversity. In eukaryotic organisms, sexual reproduction involves the process o ertilization. Fertilization is the union o sex cells, or gametes, usually rom two dierent parents. Fertilization doubles the number o chromosomes each time it occurs. It would thereore cause a doubling o chromosome number every generation, i the number was not also halved at some stage in the lie cycle. This halving o chromosome number happens during meiosis. Meiosis can happen at any stage during a sexual lie cycle, but in animals it happens during the process o creating the gametes. B ody cells are thereore diploid and have two copies o most genes. Meiosis is a complex process and it is not at the moment clear how it developed. What is clear is that its evolution was a critical step in the origin o eukaryotes. Without meiosis there cannot be usion o gametes and the sexual lie cycle o eukaryotes could not occur.

Figure 4 Fledgling owls (bottom) produced by a sexual life cycle have diploid body cells but mosses ( top) have haploid cells

Data-baed queton: Life cycles Figure 3 shows the lie cycle o humans and mosses, with n being used to represent the haploid number o chromosomes and 2 n to represent the diploid number. Sporophytes o mosses grow on the main moss plant and consist o a stalk and a capsule in which spores are produced.

1

2

O utline fve similarities between the lie cycle o a moss and o a human.

[5 ]

D istinguish between the lie cycles o a moss and a human by giving fve dierences.

[5 ]

egg n sperm n human male 2n

sperm n

egg n zygote 2n

human female 2n

moss plant n

Key

Figure 3

mitosis meiosis fertilization

zygote 2n

spore n

sporophyte 2n

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Replicatio of DnA before meiosis 2n

2n

2n

n

n

n

DNA is replicated before meiosis so that all chromosomes consist of two sister chromatids.

interphase

homologous chromosomes

D uring the early stages o meiosis the chromosomes gradually shorten by supercoiling. As soon as they become visible it is clear that each chromosome consists o two chromatids. This is because all D NA in the nucleus is replicated during the interphase beore meiosis, so each chromosome consists o two sister chromatids. Initially the two chromatids that make up each chromosome are genetically identical. This is because D NA replication is very accurate and the number o mistakes in the copying o the D NA is extremely small.

meiosis I

n

meiosis II

n

n

We might expect the D NA to be replicated again between the frst and the second division o meiosis, but it does not happen. This explains how the chromosome number is halved during meiosis. O ne diploid nucleus, in which each chromosome consists o two chromatids, divides twice to produce our haploid nuclei in which each chromosome consists o one chromatid.

Figure 5 Outline of meiosis

Bivalets formatio ad crossig over The early stages of meiosis involve pairing of homologous chromosomes and crossing over followed by condensation. Some o the most important events o meiosis happen at the start o meiosis I while the chromosomes are still very elongated and cannot be seen with a microscope. Firstly homologous chromosomes pair up with each other. Because DNA replication has already occurred, each chromosome consists o two chromatids and so there are our DNA molecules associated in each pair o homologous chromosomes. A pair o homologous chromosomes is bivalent and the pairing process is sometimes called synapsis. Soon ater synapsis, a process called crossing over takes place. The molecular details o this need not concern us here, but the outcome is very important. A junction is created where one chromatid in each o the homologous chromosomes breaks and rejoins with the other chromatid. Crossing over occurs at random positions anywhere along the chromosomes. At least one crossover occurs in each bivalent and there can be several.

Figure 6 A pair of homologous chromosomes contains four chromatids and is sometimes called a tetrad. Five chiasmata are visible in this tetrad, showing that crossing over can occur more than once

B ecause a crossover occurs at precisely the same position on the two chromatids involved, there is a mutual exchange o genes between the chromatids. As the chromatids are homologous but not identical, some alleles o the exchanged genes are likely to be dierent. C hromatids with new combinations o alleles are thereore produced.

Radom orietatio of bivalets Orientation of pairs of homologous chromosomes prior to separation is random. While pairs o homologous chromosomes are condensing inside the nucleus o a cell in the early stages o meiosis, spindle microtubules are growing rom the poles o the cell. Ater the nuclear membrane has

162

3.3 mEiOsis

broken down, these spindle microtubules attach to the centromeres o the chromosomes. The attachment o the spindle microtubules is not the same as in mitosis. The principles are these: 

E ach chromosome is attached to one pole only, not to both.



The two homologous chromosomes in a bivalent are attached to dierent poles.



The pole to which each chromosome is attached depends on which way the pair o chromosomes is acing. This is called the orientation.



The orientation o bivalents is random, so each chromosome has an equal chance o attaching to each pole, and eventually o being pulled to it.



The orientation o one bivalent does not aect other bivalents. The consequences o the random orientation o bivalents are discussed in the section on genetic diversity later in this topic.

MITOSIS

Halving the chromosome number Separation o pairs o homologous chromosomes in the frst division o meiosis halves the chromosome number. The movement o chromosomes is not the same in the frst division o meiosis as in mitosis. Whereas in mitosis the centromere divides and the two chromatids that make up a chromosome move to opposite poles, in meiosis the centromere does not divide and whole chromosomes move to the poles.

either

or MEIOSIS

Figure 7 Comparison of attachment of chromosomes to spindle microtubules in mitosis and meiosis

Initially the two chromosomes in each bivalent are held together by chiasmata, but these slide to the end o the chromosomes and then the chromosomes can separate. This separation o homologous chromosomes is called disj unction. O ne chromosome rom each bivalent moves to one o the poles and the other chromosome to the other pole. The separation o pairs o homologous chromosomes to opposite poles o the cell halves the chromosome number o the cell. It is thereore the frst division o meiosis that is the reduction division. B ecause one chromosome o each type moves to each pole, both o the two nuclei ormed in the frst division o meiosis contain one o each type o chromosome, so they are both haploid.

Obtaining cells from a fetus Methods used to obtain cells or karyotype analysis e.g. chorionic villus sampling and amniocentesis and the associated risks. Two procedures are used or obtaining cells containing the etal chromosomes needed or producing a karyotype. Amniocentesis involves passing a nee dle through the mothe r' s ab domen wall, using ultrasound to guide the needle. The needle is used to withdraw a sample o amniotic luid containing etal cells rom the amniotic sac.

The second procedure is chorionic villus sampling. A sampling tool that enters through the vagina is used to obtain cells rom the chorion, one o the membranes rom which the placenta develops. This can be done earlier in the pregnancy than amniocentesis, but whereas the risk o miscarriage with amniocentesis is 1 % , with chorionic villus sampling it is 2 % .

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Diagrams of the stages of meiosis Drawing diagrams to show the stages of meiosis resulting in the formation of four haploid cells. In mitosis our stages are usually recognized: prophase, metaphase, anaphase and telophase. Meiosis can also be divided into these stages, but each stage happens twice: in meiosis I and then a second time in meiosis II. The main events o each stage in mitosis also happen in meiosis: 

prophase: condensation o chromosomes;



metaphase: attachment o spindle microtubules;



anaphase: movement o chromosomes to the poles;



telophase: decondensation o chromosomes.

Usually we draw biological structures rom actual specimens, oten looking at them down a microscope. Preparation o microscope slides showing meiosis is worth attempting but it is challenging. Permanent slides usually have more cells visible in meiosis than temporary mounts, but even then it is difcult to interpret the structure o bivalents rom their appearance. This is why we usually construct diagrams o meiosis rather than draw stages rom specimens on microscope slides!

The frst division o meiosis Prophase i 

Cell has 2n chromosomes (double chromatid) : n is haploid number of chromosomes.



Homologous chromosomes pair (synapsis) .



Crossing over occurs.

nuclear membrane spindle microtubules and centriole Prophase I

metaphase i 



Spindle microtubules move homologous pairs to equator of cell. Orientation of paternal and maternal chromosomes on either side of equator is random and independent of other homologous pairs.

bivalents aligned on the equator

Metaphase I

Anaphase i 

Homologous pairs are separated. One chromosome of each pair moves to each pole.

homologous chromosomes being pulled to opposite poles

Anaphase I

Telophase i 

164

Chromosomes uncoil. During interphase that follows, no replication occurs.



Reduction of chromosome number from diploid to haploid completed.



Cytokinesis occurs.

cell has divided across the equator

Telophase I

3.3 mEiOsis

The second division of meiosis Prophae ii 

Chromosomes, which still consist of two chromatids, condense and become visible.

Prophase II

metaphae ii

Metaphase II

Anaphae ii 

Centromeres separate and chromatids are moved to opposite poles.

Anaphase II

Telophae ii 

Chromatids reach opposite poles.



Nuclear envelope forms.



Cytokinesis occurs.

Telophase II

Meiosis and genetic variation Crossing over and random orientation promotes genetic variation. When two parents have a child, they know that it will inherit an unpredictable mixture of characteristics from each of them. Much of the unpredictability is due to meiosis. E very gamete produced by a parent has a new combination of alleles  meiosis is a source of endless genetic variation. Apart from the genes on the X and Y chromosomes, humans have two copies of each gene. In some cases the two copies are the same allele and there will be one copy of that allele in every gamete produced by the parent. There are likely to be thousands of genes in the parents genome

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Activity I g is the number o genes in a genome with diferent alleles, 2 g is the number o combinations o these alleles that can be generated by meiosis. I there were just 69 genes with diferent alleles (3 in each o the 23 chromosome types in humans) there would be 590,295,810,358,705, 700,000 combinations. Assuming that all humans are genetically diferent, and that there are 7,000,000 humans, calculate the percentage o all possible genomes that currently exist.

where the two alleles are dierent. Each o the two alleles has an equal chance o being passed on in a gamete. Let us suppose that there is a gene with the alleles A and a. Hal o the gametes produced by the parent will contain A and hal will contain a. Let us now suppose that there is another gene with the alleles B and b. Again hal o the gametes will contain B and hal b. However, meiosis can result in gametes with dierent combinations o these genes: AB , Ab, aB and ab. There are two processes in meiosis that generate this diversity.

50% probability

a

B

A

b

B a b A

B b

telophase I

A a

prophase I

50% probability

a

b

A

B

b a B A

metaphase I  Figure 8

Random orientation in metaphase I

1. Random orientation o bivalents In metaphase I the orientation o bivalents is random and the orientation o one bivalent does not infuence the orientation o any o the others. Random orientation o bivalents is the process that generates genetic variation among genes that are on dierent chromosome types. For every additional bivalent, the number o possible chromosome combinations in a cell produced by meiosis doubles. For a haploid number o n, the number o possible combinations is 2 n. For humans with a haploid number o 2 3 this amounts to 2 23 or over 8 million combinations.

2. Crossing over Without crossing over in prophase I, combinations o alleles on chromosomes would be orever linked together. For example, i one chromosome carried the combination C D and another carried cd, only these combinations could occur in gametes. C rossing over allows linked genes to be reshufed, to produce new combinations such as C d and cD . It increases the number o allele combinations that can be generated by meiosis so much that it is eectively innite.

Fertilization and genetic variation Fusion o gametes rom diferent parents promotes genetic variation. The usion o gametes to produce a zygote is a highly signicant event both or individuals and or species.

Figure 9

166



It is the start o the lie o a new individual.



It allows alleles rom two dierent individuals to be combined in one new individual.

3.3 mEiOsis



The combination o alleles is unlikely ever to have existed beore.



Fusion o gametes thereore promotes genetic variation in a species.



Genetic variation is essential or evolution.

no-disjuctio ad Dow sydrome Non-disjunction can cause Down syndrome and other chromosome abnormalities. Meiosis is sometimes subj ect to errors. O ne example o this is when homologous chromosomes ail to separate at anaphase. This is termed non- disj unction. This can happen with any o the pairs o homologous chromosomes. B oth o the chromosomes move to one pole and neither to the other pole. The result will be a gamete that either has an extra chromosome or is defcient in a chromosome. I the gamete is involved in human ertilization, the result will be an individual with either 45 or 47 chromosomes. An abnormal number o chromosomes will oten lead to a person possessing a syndrome, i.e. a collection o physical signs or symptoms. For example trisomy 2 1 , also known as D own syndrome, is due to a non- disj unction event that leaves the individual with three o chromosome number 2 1 instead o two. While individuals vary, some o the component eatures o the syndrome include hearing loss, heart and vision disorders. Mental and growth retardation are also common.

Most other trisomies in humans are so serious that the ospring do not survive. B abies are sometimes born with trisomy 1 8 and trisomy 1 3 . Non-disj unction can also result in the birth o babies with abnormal numbers o sex chromosomes. Klineelters syndrome is caused by having the sex chromosomes XXY. Turners syndrome is caused by having only one sex chromosome, an X. diploid parent cell with two chromosome 21 non-disjunction during meiosis

gamete with no chromosome 21

gamete with two chromosome 21 cell dies fusion of gametes

normal haploid gamete



trisomy: zygote with three chromosome 21

Figure 10 How non-disjunction can give rise to Down syndrome

trisomy 21 all chromosomal abnormalities

Studies showing age o parents infuences chances o non-disjunction The data presented in fgure 1 1 shows the relationship between maternal age and the incidence o trisomy 2 1 and o other chromosomal abnormalities. 1

O utline the relationship between maternal age and the incidence o chromosomal abnormalities in live births. [2 ]

2

a) For mothers 40 years o age, determine the probability that they will give birth to a child with trisomy 2 1 . [1 ] b) Using the data in fgure 1 1 , calculate the probability that a mother o 40 years o age will give birth to a child with a chromosomal abnormality other than trisomy 2 1 .

incidence (% of all live births)

Paretal age ad o-disjuctio 14 12 10 8 6 4 2 0 20

40 60 maternal age (years)

 Figure 11 The incidence of trisomy 21

[2 ]

and other chromosomal abnormalities as a function of maternal age

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3

4

O nly a small number of possible chromosomal abnormalities are ever found among live births, and trisomy 2 1 is much the commonest. S uggest reasons for these trends.

[3 ]

D iscuss the risks parents face when choosing to postpone having children.

[2 ]

3.4 inhertance Udertadig  Mendel discovered the principles o inheritance

  

   

 

168

with experiments in which large numbers o pea plants were crossed. Gametes are haploid so contain one allele o each gene. The two alleles o each gene separate into diferent haploid daughter nuclei during meiosis. Fusion o gametes results in diploid zygotes with two alleles o each gene that may be the same allele or diferent alleles. Dominant alleles mask the efects o recessive alleles but co-dominant alleles have joint efects. Many genetic diseases in humans are due to recessive alleles o autosomal genes. Some genetic diseases are sex-linked and some are due to dominant or co-dominant alleles. The pattern o inheritance is diferent with sex-linked genes due to their location on sex chromosomes. Many genetic diseases have been identied in humans but most are very rare. Radiation and mutagenic chemicals increase the mutation rate and can cause genetic disease and cancer.

Applicatio  Inheritance o ABO blood groups.  Red-green colour-blindness and hemophilia as

examples o sex-linked inheritance.  Inheritance o cystic brosis and Huntingtons disease.  Consequences o radiation ater nuclear bombing o Hiroshima and Nagasaki and the nuclear accidents at Chernobyl.

skill  Construction o Punnett grids or predicting the

outcomes o monohybrid genetic crosses.  Comparison o predicted and actual outcomes o genetic crosses using real data.  Analysis o pedigree charts to deduce the pattern o inheritance o genetic diseases.

nature of ciece  Making quantitative measurements with

replicates to ensure reliability: Mendels genetic crosses with pea plants generated numerical data.

3 . 4 i N h E r i TAN CE

Mendel and the principles of inheritance Mendel discovered the principles of inheritance with experiments in which large numbers of pea plants were crossed. When living organisms reproduce, they pass on characteristics to their ospring. For example, when blue whales reproduce, the young are also blue whales  they are members o the same species. More than this, variations, such as the markings on the skin o a blue whale, can be passed on. We say that the ospring inherit the parents characteristics. However, some characteristics cannot be inherited. S cars seen on the tails o some blue whales caused by killer whale attacks and cosmetic surgery in humans are examples o this. According to current theories, acquired characteristics such as these cannot be inherited. Inheritance has been discussed since the time o Hippocrates and earlier. For example, Aristotle observed that children sometimes resemble their grandparents more than their parents. Many o the early theories involved blending inheritance, in which ospring inherit characters rom both parents and so have characters intermediate between those o their parents. S ome o the observations that biologists made in the rst hal o the 1 9th century could not be explained by blending inheritance, but it was not until Mendel published his paper E xperiments in Plant Hybridization that an alternative theory was available.

 Figure 1

Hair styles are acquired characteristics and are ortunately not inherited by ofspring

Mendels experiments were done using varieties o pea plant, each o which reliably had the same characters when grown on its own. Mendel careully crossed varieties o pea together by transerring the male pollen rom one variety to the emale parts in fowers o another variety. He collected the pea seeds that were ormed as a result and grew them to nd out what their characters were. Mendel repeated each cross with many pea plants. He also did this experiment with seven dierent pairs o characters and so his results reliably demonstrated the principles o inheritance in peas, not j ust an isolated eect. In 1 866 Mendel published his research. For over thirty years his ndings were largely ignored. Various reasons have been suggested or this. One actor was that his experiments used pea plants and there was not great interest in the pattern o inheritance in that species. In 1 900 several biologists rediscovered Mendels work. They quickly did cross-breeding experiments with other plants and with animals. These conrmed that Mendels theory explained the basis o inheritance in all plants and animals.

Replicates and reliability in Mendels experiments Making quantitative measurements with replicates to ensure reliability: Mendel's genetic crosses with pea plants generated numerical data. Gregor Mendel is regarded by most biologists as the ather o genetics. His success is sometimes attributed to being the rst to use pea plants or research into inheritance. Peas have clear

characteristics such as red or white fower colour that can easily be ollowed rom one generation to the next. They can also be crossed to produce hybrids or they can be allowed to sel- pollinate.

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In act Mendel was not the rst to use pea plants. Thomas Andrew Knight, an English horticulturalist, had conducted research at D ownton C astle in Hereordshire in the late 1 8th century and published his results in the Philosophical Transactions o the Royal S ociety. Knight made some important discoveries:

pollen is collected from the anthers



male and emale parents contribute equally to the ospring;



characters such as white fower colour that apparently disappear in ospring can reappear in the next generation, showing that inheritance is discrete rather than blending;

lower petal  called the keel

one character such as red fower colour can show a stronger tendency than the alternative character.

self pollinating peas:  if the ower is left untouched, the anthers inside the keel pollinate the stigma



Although Mendel was not as pioneering in his experiments as sometimes thought, he deserves credit or another aspect o his research. Mendel was a pioneer in obtaining quantitative results and in having large numbers o replicates. He also did seven dierent cross experiments, not just one. Table 1 shows the results o his monohybrid crosses. It is now standard practice in science to include repeats in experiments to demonstrate the reliability o results. Repeats can be compared to see how close they are. Anomalous results can be identied and excluded rom analysis. S tatistical tests can be done to assess the signicance o dierences between treatments. It is also standard practice to repeat whole experiments, using a dierent organism or dierent treatments, to test a hypothesis in dierent ways. Mendel should thereore be regarded as one o the athers o genetics, but even more we should think o him as a pioneer o research methods in biology.

Paental plants

 Figure 2

Cross and sel pollination (a) Prediction based on blending inheritance tall plants 3 dwarf plants

pea plants with an intermediate height (b) Actual results tall plants 3 dwarf plants

pea plants as tall as the tall parent  Figure 3

Example o a monohybrid cross experiment. All the hybrid plants produced by crossing two varieties together had the same character as one o the parents and the character o the other parent was not seen. This is a clear alsifcation o the theory o blending inheritance

hybid plants Ofsping om sel-pollinating te ybids

ratio

Tall stem  dwar stem

All tall

787 tall : 277 dwar

2.84 : 1

Round seed  wrinkled seed

All round

5474 round : 1850 wrinkled

2.96 : 1

Yellow cotyledons  green cotyledons

All yellow

6022 yellow : 2001 green

3.01 : 1

Purple fowers  white fowers

All purple

705 purple : 224 white

3.15 : 1

Full pods  constricted pods

All ull

882 ull : 299 constricted

2.95 : 1

Green unripe pods  yellow unripe pods

All green

428 green : 152 yellow

2.82 : 1

Flowers along stem  fowers at stem tip

All along stem 651 along stem : 207 at tip

 Table 1

170

cross pollinating peas: pollen from another plant is dusted on to the stigma here

3.14 : 1

3 . 4 i N h E r i TAN CE

Gamete Gametes are haploid so contain one allele o each gene. Gametes are cells that fuse together to produce the single cell that is the start of a new life. They are sometimes called sex cells, and the single cell produced when male and female gametes fuse is a zygote. Male and female gametes are different in size and motility. The male gamete is generally smaller than the female one. It is usually able to move whereas the female gamete moves less or not at all. In humans, for example, the sperm has a much smaller volume than the egg cell and uses its tail to swim to the egg. Parents pass genes on to their offspring in gametes. Gametes contain one chromosome of each type so are haploid. The nucleus of a gamete therefore only has one allele of each gene. This is true of both male and female gametes, so male and female parents make an equal genetic contribution to their offspring, despite being very different in overall size.

Figure 4 Pollen on the anthers o a fower contains the male gamete o the plant. The male gametes contain one allele o each o the plants

Zygote Fusion o gametes results in diploid zygotes with two alleles o each gene that may be the same allele or diferent alleles. When male and female gametes fuse, their nuclei j oin together, doubling the chromosome number. The nucleus of the zygote contains two chromosomes of each type so is diploid. It contains also two alleles of each gene. If there were two alleles of a gene, A and a, the zygote could contain two copies of either allele or one of each. The three possible combinations are AA, Aa and aa. S ome genes have more than two alleles. For example, the gene for AB O blood groups in humans has three alleles: I A, I B and i. This gives six possible combinations of alleles: 

three with two of the same allele, IAIA, IB I B and ii



three with two different alleles, IAIB , I Ai and I B i.

segregation of allele The two alleles o each gene separate into diferent haploid daughter nuclei during meiosis. D uring meiosis a diploid nucleus divides twice to produce four haploid nuclei. The diploid nucleus contains two copies of each gene, but the haploid nuclei contain only one. 

If two copies of one allele of a gene were present, each of the haploid nuclei will receive one copy of this allele. For example, if the two alleles were PP, every gamete will receive one copy of P.



If two different alleles were present, each haploid nucleus will receive either one of the alleles or the other allele, not both. For example, if the two alleles were Pp, 5 0% of the haploid nuclei would receive P and 5 0% would receive p.

Figure 5 Most crop plants are pure-bred strains with two o the same allele o each gene

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TOK Did mendel alter his results for publication? In 1936, the English statistician R.A. Fisher published an analysis o Mendels data. His conclusion was that the data o most, i not all, o the experiments have been alsied so as to agree closely with Mendels expectations. Doubts still persist about Mendel's data  a recent estimate put the chance o getting seven ratios as close to 3:1 as Mendels at 1 in 33,000. 1

2

To get ratios as close to 3:1 as Mendel's would have required a miracle o chance. What are the possible explanations apart rom a miracle o chance? Many distinguished scientists, including Louis Pasteur, are known to have discarded results when they did not t a theory. Is it acceptable to do this? How can we distinguish between results that are due to an error and results that alsiy a theory? What standard do you use as a student in rejecting anomalous data?

The separation o alleles into dierent nuclei is called segregation. It breaks up existing combinations o alleles in a parent and allows new combinations to orm in the ospring.

Dominant, recessive and co-dominant alleles Dominant alleles mask the efects o recessive alleles but co-dominant alleles have joint efects. In each o Mendels seven crosses between dierent varieties o pea plant, all o the ospring showed the character o one o the parents, not the other. For example, in a cross between a tall pea plant and a dwar pea plant, all the ospring were tall. The dierence in height between the parents is due to one gene with two alleles: 

the tall parents have two copies o an allele that makes them tall, TT



the dwar parents have two copies o an allele that makes them dwar, tt



they each pass on one allele to the ospring, which thereore has one o each allele, Tt



when the two alleles are combined in one individual, it is the allele or tallness that determines the height because the allele or tallness is dominant



the other allele, that does not have an eect i the dominant allele is present, is recessive.

In each o Mendels crosses one o the alleles was dominant and the other was recessive. However, some genes have pairs o alleles where both have an eect when they are present together. They are called co-dominant alleles. A well-known example is the fower colour o Mirabilis jalapa. I a red-fowered plant is crossed with a white-fowered plant, the ospring have pink fowers. 

there is an allele or red fowers, C R



there is an allele or white fowers, C W



these alleles are co-dominant so C RC W gives pink fowers.

The usual reason or dominance o one allele is that this allele codes or a protein that is active and carries out a unction, whereas the recessive allele codes or a non- unctional protein.

Figure 6 There are co-dominant alleles of the gene for coat colour in Icelandic horses.

172

3 . 4 i N h E r i TAN CE parents:

Monohybrid crosses only involve one character, or example the height o a pea plant, so they involve only one gene. Most crosses start with two pure- breeding parents. This means that the parents have two o the same allele, not two dierent alleles. E ach parent thereore produces j ust one type o gamete, containing one copy o the allele. Their ospring are also identical, although they have two dierent alleles. The ospring obtained by crossing the parents are called F 1 hybrids or the F 1 generation.

eggs or pollen T

lle po t

tt dwarf  Figure 7

Explanation of Mendels 3:1 ratio

parents: genotype phenotype

C WC W CRCR white owers red owers

D educe the colour o coat that is due to a recessive allele, with two reasons or your answer. [3 ]

3

C hoose suitable symbols or the alleles or grey and albino coat and list the possible combinations o alleles o mice using your symbols, together with the coat colours associated with each combination o alleles. [3 ]

CW

CRCW pink owers

CW

po

l le

n

F1 hybrids genotype phenotype

C WC R pink

CRCR red C WC W white

gs CW

2

CR

R

C alculate the ratio between grey and albino ospring, showing your working. [2 ]

Tt tall

eg

1

t

tT tall

C

In the early years o the 2 0th century, many crossing experiments were done in a similar way to those o Mendel. The French geneticist Lucien C unot used the house mouse, Mus musculus, to see whether the principles that Mendel had discovered also operated in animals. He crossed normal grey- coloured mice with albino mice. The hybrid mice that were produced were all grey. These grey hybrids were crossed together and produced 1 98 grey and 72 albino ospring.

gs

TT tall

Figure 8 shows the results o a cross between red and white fowered plants o Mirabilis jalapa. It explains the F 2 ratio o one red to two pink to one white fowered plant.

Data-based questons: Coat colour in the house mouse

T

n

Tt tall stem

eg

Figure 7 shows Mendels cross between tall and dwar plants. It explains the F 2 ratio o three tall to one dwar plant.

t

F1 hybrids genotype phenotype

The F 1 hybrids have two dierent alleles o the gene, so they can each produce two types o gamete. I two F 1 hybrids are crossed together, or i an F 1 plant is allowed to sel-pollinate, there are our possible outcomes. This can be shown using a 2  2 table, called a Punnett grid ater the geneticist who rst used this type o table. The ospring o a cross between two F 1 plants are called the F 2 generation. To make a Punnett grid as clear as possible the gametes should be labeled and both the alleles and the character o the our possible outcomes should be shown on the grid. It is also useul to give an overall ratio below the Punnett grid.

tt dwarf stem

TT tall stem

CR

Construction of Punnett grids for predicting the outcomes of monohybrid genetic crosses.

genotype phenotype

T

Punnett grids

C WC W pink

 Figure 8 A cross involving co-dominance

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4 t ica

annulata

5

 Figure 9

Using a Punnett grid, explain how the observed ratio o grey and albino mice was produced.

[5 ]

The albino mice had red eyes in addition to white coats. S uggest how one gene can determine whether the mice had grey ur and black eyes or white ur and red eyes. [2 ]

Data-based questions: The two-spot ladybird Adalia bipunctata is a species o ladybird. In North America ladybirds are called ladybugs. The commonest orm o this species is known as typica. There is a rarer orm called annulata. B oth orms are shown in fgure 9.  Figure 10 F 1

 Figure 11

hybrid ofspring

1

C ompare the typica and annulata orms o Adalia bipunctata.

[2 ]

2

The dierences between the two orms are due to a single gene. I male and emale typica are mated together, all the ospring are typica. S imilarly, the ospring produced when annulata orms are mated are all annulata. Explain the conclusions that can be drawn.

[2 ]

3

When typica is mated with annulata, the F 1 hybrid ospring are not identical to either parent. Examples o these F 1 hybrid ospring are shown in fgure 1 0. D istinguish between the F 1 hybrid ospring and the typica and annulata parents. [3 ]

4

I F 1 hybrid ospring are mated with each other, the ospring include both typica and annulata orms, and also ospring with the same wing case markings as the F 1 hybrid ospring.

F2 ofspring

Activity ABO blood groups It is possible for two parents to have an equal chance of having a child with blood group A, B, AB or O. What would be the genotypes of the parents?

a) Use a genetic diagram to explain this pattern o inheritance. [6] b) Predict the expected ratio o phenotypes.

[2 ]

ABO blood groups Inheritance of ABO blood groups. The AB O blood group system in humans is an example o co- dominance. It is o great medical importance: beore blood is transused, it is vital to fnd out the blood group o a patient and ensure that it is matched. Unless this is done, there may be complications due to coagulation o red blood cells. O ne gene determines the AB O blood group o a person. The genotype IA IA gives blood group A and the genotype IBIB gives group B . Neither IA nor IB is dominant over the other allele and a person with the genotype IA IB has a dierent blood group, called AB . There is a third allele o the AB O blood group gene, usually called i. A person with the genotype ii is in blood group O . The genotypes IA i and IBi give blood groups A and B respectively, showing that i is

174

recessive to both IA and IB. The reasons or two alleles being co- dominant and the other allele being recessive are as ollows: 

All o the three alleles cause the production o a glycoprotein in the membrane o red blood cells.



IA alters the glycoprotein by addition o acetylgalactosamine. This altered glycoprotein is absent rom people who do not have the allele IA so i exposed to it they make anti-A antibodies.



IB alters the glycoprotein by addition o galactose. This altered glycoprotein is not present in people who do not have the allele IB so i exposed to it they make anti-A antibodies.

3 . 4 i N h E r i TAN CE





The genotype IAIB causes the glycoprotein to be altered by addition o acetyl-galactosamine and galactose. As a consequence neither anti-A nor anti- B antibodies are produced. This genotype thereore gives a dierent phenotype to IA IA and IBIB so the alleles IA and IB are co- dominant.

either o the IA or IB alleles is also present the glycoprotein is altered by addition o acetyl-galactosamine or galactose. IAIA and IA i thereore give the same phenotype, as do IBIB and IBi. 

The allele i is recessive because it causes production o the basic glycoprotein: i

The allele i is recessive because it does not cause the production o a glycoprotein. IA IA and IA i thereore give the same phenotype and so do IBIB and IBi. Group O

Group A

anti-A

anti-B

anti-A

Group B

anti-A  Figure 12

anti-B Group AB

anti-B

anti-A

anti-B

Blood group can easily be determined using test cards

tesing predicions in cross-breeding experimens Comparison of predicted and actual outcomes of genetic crosses using real data. It is in the nature o science to try to fnd general principles that explain natural phenomena and not j ust to describe individual examples o a phenomenon. Mendel discovered principles o inheritance that have great predictive power. We can still use them to predict the outcomes o genetic crosses. Table 2 lists possible predictions in monohybrid crosses. The actual outcomes o genetic crosses do not usually correspond exactly with the predicted outcomes. This is because there is an element o chance involved in the inheritance o genes. The tossing o a coin is a simple analogy. We expect the coin to land 5 0% o times with each o its two aces uppermost, but i we toss it 1 , 000 times we do not expect it to land precisely 5 00 times with

one ace showing and 5 00 times with the other ace showing. An important skill in biology is deciding whether the results o an experiment are close enough to the predictions or us to accept that they ft, or whether the dierences are too great and either the results or the predictions must be alse. An obvious trend is that the greater the dierence between observed and expected results, the less likely that the dierence is due to chance and the more likely that the predictions do not ft the results. To assess obj ectively whether results ft predictions, statistical tests are used. For genetic crosses the chi-squared test can be used. This test is described later in the book in sub- topic 4.1 .

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Cross

Predicted outcome

Example

Pure-breeding parents one with dominant alleles and one with recessive alleles are crossed.

All o the ofspring will have the same character as the parent with dominant alleles.

All ofspring o a cross between purebreeding tall and dwar pea plants will be tall.

Pure-breeding parents that have diferent co-dominant alleles are crossed.

All o the ofspring will have the same character and the character will be diferent rom either parent.

All ofspring o a cross between red and white owered Mirabilis jalapa plants will have pink owers.

Two parents each with one dominant and one recessive allele are crossed.

Three times as many ofspring have the character o the parent with dominant alleles as have the character o the parent with the recessive alleles.

3:1 ratio o tall to dwar pea plants rom a cross between two parents that each have one allele or tall height and one allele or dwar height.

A parent with one dominant and one recessive allele is crossed with a parent with two recessive alleles.

Equal proportions o ofspring with the character o an individual with a dominant allele and the character o an individual with recessive alleles.

1:1 ratio rom a cross between a dwar pea plant and a tall plant with one allele or tall height and one or dwar height .

Table 2

Data-based questions: Analysing genetic crosses 1

Figure 13 Antirrhinum fowers  (a) wild type, (b) peloric

C harles D arwin crossed pure breeding wild- type Antirrhinum majus plants, which have bilaterally symmetric fowers, with pure breeding plants with peloric fowers that are radially symmetric. All the F 1 ospring produced bilaterally symmetric fowers. D arwin then crossed the F 1 plants together. In the F 2 generation there were 8 8 plants with bilaterally symmetric fowers and 3 7 with peloric fowers. a) C onstruct a Punnett grid to predict the outcome o the cross between the F 1 plants. [3 ] b) D iscuss whether the actual results o the cross are close enough to support the predicted outcome.

[2 ]

c) Peloric Antirrhinum majus plants are extremely rare in wild populations o this species. Suggest reasons or this. [1 ] 2

176

There are three varieties o pheasant with eather coloration called light, ring and bu. When light pheasants were bred together, only light ospring were produced. S imilarly, when ring were crossed with ring, all the ospring were ring. When bu pheasants were crossed with bu there were 75 light ospring, 68 ring and 1 41 bu. a) C onstruct a Punnett grid to predict the outcome o breeding together bu pheasants.

[3 ]

b) D iscuss whether the actual results o the cross are close enough to support the predicted outcome.

[2 ]

3 . 4 i N h E r i TAN CE

3

Mary and Herschel Mitchell investigated the inheritance o a character called poky in the ungus Neurospora crassa. Poky strains o the ungus grow more slowly than the wild- type. The results are shown in table 3 .

male paent

Feale paent

Nube o wld type ofspng

Nube o poky ofspng

Wild type

Wild type

9,691

90

Poky

Poky

0

10,591

Wild type

Poky

0

7,905

Poky

Wild type

4,816

43

Table 3 a) D iscuss whether the data fts any o the Mendelian ratios in table 1 ( page 1 70) . [2 ] b) S uggest a reason or all the ospring being poky in a cross between wild type and poky strains when a wild type is the male parent. [2 ] c) S uggest a reason or a small number o poky ospring in a cross between wild type and poky strains when a wild type is the emale parent. [1 ]

Figure 14 Feather coloration rom a buf pheasant

Genetic diseases due to recessive alleles Many genetic diseases in humans are due to recessive alleles of autosomal genes. A genetic disease is an illness that is caused by a gene. Most genetic diseases are caused by a recessive allele o a gene. The disease thereore only develops in individuals that do not have the dominant allele o the gene, usually because they have two copies o the recessive allele. I a person has one allele or the genetic disease and one dominant allele, they will not show symptoms o the disease, but they can pass on the recessive allele to their ospring. These individuals are called carriers. Genetic diseases caused by a recessive allele usually appear unexpectedly. B oth parents o a child with the disease must be carriers, but as they do not show symptoms o the disease, they are unaware o this. The probability o these parents having a child with the disease is 2 5 per cent ( see fgure 1 5 ) . C ystic fbrosis is an example o a genetic disease caused by a recessive allele. It is described later in this sub- topic.

Other causes of genetic diseases Some genetic diseases are sex-linked and some are due to dominant or co-dominant alleles. A small proportion o genetic diseases are caused by a dominant allele. It is not possible to be a carrier o these diseases. I a person has one dominant allele then they themselves will develop the disease. I one

Aa

Aa

A

a

AA not carrier

Aa

A

a

aA

aa

carrier

do not develop the disease develops the genetic disease  Figure 15 Genetic diseases caused

by a recessive allele

177

3

G e n e ti cs bb

Bb

B

b

b

Bb develops the disease

b

bb does not develop the disease

 Figure 16 Genetic diseases caused

by a dominant allele

parent has the allele or the disease, the chance o a child inheriting it is 5 0 per cent ( see fgure 1 6) . Huntingtons disease is an example o a genetic disease caused by a dominant allele. It is described later in this sub- topic. A very small proportion o genetic diseases are caused by co- dominant alleles. An example is sickle- cell anemia. The molecular basis o this disease was described in sub- topic 3 . 1 . The normal allele or hemoglobin is Hb A and the sickle cell allele is Hb S . Figure 1 7 shows the three possible combinations o alleles and the characteristics that result. Individuals that have one Hb A and one Hb S allele do not have the same characteristics as those who have two copies o either allele, so the alleles are co- dominant. Most genetic diseases aect males and emales in the same way but some show a dierent pattern o inheritance in males and emales. This is called sex linkage. The causes o sex linkage and two examples, red- green colour- blindness and hemophilia, are described later in this sub- topic.

alleles : Hb A Hb A

alleles : Hb A Hb s

characteristics : - susceptible to malaria - not anemic

characteristics : - increased resistance to malaria - mild anemia

alleles : Hb S Hb S characteristics : - susceptible to malaria - severe anemia

normal red blood cell shape  Figure 17

sickle-cell shape

Efects o Hb A and Hb S alleles

Cystic fbrosis and Huntingtons disease Inheritance o cystic fbrosis and Huntingtons disease. C ystic fbrosis is the commonest genetic disease in parts o E urope. It is due to a recessive allele o the C FTR gene. This gene is located on chromosome 7 and the gene product is a chloride ion channel that is involved in secretion o sweat, mucus and digestive j uices. The recessive alleles o this gene result in chloride channels being produced that do not unction properly. S weat containing excessive amounts o sodium chloride is produced, but digestive j uices and mucus are secreted with insufcient sodium chloride. As a result not enough water moves by osmosis into the

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secretions, making them very viscous. S ticky mucus builds up in the lungs causing inections and the pancreatic duct is usually blocked so digestive enzymes secreted by the pancreas do not reach the small intestine. In some parts o E urope one in twenty people have an allele or cystic fbrosis. As the allele is recessive, a single copy o the allele does not have any eects. The chance o two parents 1 1 both being a carrier o the allele is __  __ , 20 20 1 which is ___ . The chance o such parents having 40 0 a child with cystic fbrosis can be ound using a Punnett grid.

3 . 4 i N h E r i TAN CE

father Cc

C

c

C

CC normal

Cc normal (carrier)

c

cC normal (carrier)

cc cystic brosis

mother Cc

B ecause of the late onset, many people diagnosed with Huntingtons disease have already had children. A genetic test can show before symptoms would develop whether a young person has the dominant allele, but most people at risk choose not to have the test. About one in 1 0, 000 people have a copy of the Huntingtons allele, so it is very unlikely for two parents both to have a copy. A person can nonetheless develop the disease if only one of their parents has the allele because it is dominant.

ratio 3 normal : 1 cystic brosis

father Hh

Huntingtons disease is due to a dominant allele of the HTT gene. This gene is located on chromosome 4 and the gene product is a protein named huntingtin. The function of huntingtin is still being researched. The dominant allele of HTT causes degenerative changes in the brain. S ymptoms usually start when a person is between 3 0 and 5 0 years old. C hanges to behaviour, thinking and emotions become increasingly severe. Life expectancy after the start of symptoms is about 2 0 years. A person with the disease eventually needs full nursing care and usually succumbs to heart failure, pneumonia or some other infectious disease.

H

h

h

Hh Huntingtons disease

hh normal

h

Hh Huntingtons disease

hh normal

mother hh

ratio 1 normal : 1 Huntingtons disease

sex-linked gene The pattern o inheritance is diferent with sex-linked genes due to their location on sex chromosomes. Plants such as peas are hermaphrodite  they can produce both male and female gametes. When Thomas Andrew Knight did crossing experiments between pea plants in the late 1 8th century, he discovered that the results were the same whichever character was in the male gamete and which in the female gamete. For example, these two crosses gave the same results: 

pollen from a plant with green stems placed onto on the stigma of a plant with purple stems;



pollen from a plant with purple stems placed onto on the stigma of a plant with green stems.

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red eye XRY

X rX R red XrY white

XrXR red

Y

Xr

R

X

Xr

white eye XrXr

XrY white

XR XR

XR Xr red

Y

red

XR Y red XR Y red

Key XR X chromosome with allele for red eye (dominant) Xr X chromosome with allele for white eye (recessive) Y Y chromosome  Figure 18 Reciprocal

crosses



normal-winged males  vestigial- winged emales;



vestigial-winged males  normal- winged emales.

These crosses gave dierent results: 

red-eyed males  white- eyed emales gave only red- eyed ospring;



white- eyed males  red- eyed emales gave red-eyed emales and white- eyed males.

r

XR

white eye X rY

O ne o the rst examples o sex linkage was discovered by Thomas Morgan in the ruit fy, Drosophila. This small insect is about 4 mm long and completes its lie cycle in two weeks, allowing crossing experiments to be done quickly with large numbers o fies. Most crosses in Drosophila do not show sex linkage. For example, these reciprocal crosses give the same results:

X

XR

red eye XRXR

Plants always give the same results when reciprocal crosses such as these are carried out, but in animals the results are sometimes dierent. An inheritance pattern where the ratios are dierent in males and emales is called sex linkage.

sex-linkage

Geneticists had observed that the inheritance o genes and o chromosomes showed clear parallels and so genes were likely to be located on chromosomes. It was also known that emale Drosophila have two copies o a chromosome called X and males only have one copy. Morgan deduced that sex linkage o eye colour could thereore be due to the eye colour gene being located on the X chromosome. Male Drosophila also have a Y chromosome, but this does not carry the eye- colour gene. Figure 1 8 explains the inheritance o eye colour in Drosophila. In crosses involving sex linkage, the alleles should always be shown as a superscript letter on a letter X to represent the X chromosome. The Y chromosome should also be shown though it does not carry an allele o the gene.

Red-green colour-blindness and hemophilia Red-green colour-blindness and hemophilia as examples of sex-linked inheritance. Many examples o sex linkage have been discovered in humans. They are almost all due to genes located on the X chromosome, as there are very ew genes on the Y chromosome. Two examples o sex-linked conditions due to genes on the X chromosomes are described here: red- green colour- blindness and hemophilia. Red- green colour- blindness is caused by a recessive allele o a gene or one o the photoreceptor proteins. These proteins are made by cone cells in the retina o the eye and detect specic wavelength ranges o visible light.

 Figure 19 A person with red-green colour-blindness cannot clearly

distinguish between the colours o the fowers and the leaves

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3 . 4 i N h E r i TAN CE

proteins involved in the clotting o blood. Lie expectancy is only about ten years i hemophilia is untreated. Treatment is by inusing Factor VIII, purifed rom the blood o donors.

XH Xh

Xh

XH Y

XH

Y

H

XH XH normal Y

Whereas red- green colour-blindness is a mild disability, hemophilia is a lie- threatening genetic disease. Although there are some rarer orms o the disease, most cases o hemophilia are due to an inability to make Factor VIII, one o the

XH

KEY XH X chromosome carrying the allele for normal blood clotting Xh X chromosome carrying the allele for hemophilia.

X

Males have only one X chromosome, which they inherit rom their mother. I that X chromosome carries the red- green colour- blindness allele then the son will be red- green colour- blind. In parts o northern E urope the percentage o males with this disability is as high as 8% . Girls are red- green colour- blind i their ather is red-green colourblind and they also inherit an X chromosome carrying the recessive gene rom their mother. We can predict that the percentage o girls with colour- blindness in the same parts o E urope to be 8%  8% = 0.64% . The actual percentage is about 0.5 % , ftting this prediction well.

XH

Blood should stop quickly owing rom a pricked fnger but in hemophiliacs bleeding continues or much longer as blood does not clot properly

Xh

 Figure 20

The gene or Factor VIII is located on the X chromosome. The allele that causes hemophilia is recessive. The requency o the hemophilia allele is about 1 in 1 0, 000. This is thereore the requency o the disease in boys. Females can be carriers o the recessive hemophilia allele but they only develop the disease i both o their X chromosomes carry the allele. The requency in 1 2 = 1 in 1 00, 000, 000. girls theoretically is ( _____ 1 0,000 ) In practice, there have been even ewer cases o girls with hemophilia due to lack o Factor VIII than this. O ne reason is that the ather would have to be hemophiliac and decide to risk passing on the condition to his children.

XH Xh carrier

XH Y normal Xh Y hemophiliac

Pedigree charts Analysis of pedigree charts to deduce the pattern of inheritance of genetic diseases. It isnt possible to investigate the inheritance o genetic diseases in humans by carrying out cross experiments. Pedigree charts can be used instead to deduce the pattern o inheritance. These are the usual conventions or constructing pedigree charts: 

males are shown as squares;



emales are shown as circles;



squares and circles are shaded or crosshatched to indicate whether an individual is aected by the disease;



parents and children are linked using a T, with the top bar o the T between the parents;



Roman numerals indicate generations;

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

Arabic numbers are used or individuals in each generation.

their children will be albino, we could only expect to see that ratio i the parents had very large numbers o children. The actual ratio o 1 in 2 is not unexpected and does not show that our deductions about the inheritance o albinism are incorrect.

Example 1 Albinism in humans generation I 1

2

Example 2 Vitamin D-resistant rickets D eductions:

generation II 1

2

3



Two unaected parents only have unaected children but two aected parents have children with vitamin D - resistant rickets, suggesting that this disease is caused by a dominant allele.



The ospring o the parents in generation I are all aected daughters and unaected sons. This suggests sex linkage although the number o ospring is too small to be sure o the inheritance pattern.



I vitamin D - resistant rickets is caused by a dominant X- linked allele, daughters o the ather in generation I would inherit his X chromosome carrying the dominant allele, so all o his daughters would have the disease. This data in the pedigree shows that this and so supports the theory.



S imilarly i vitamin D -resistant rickets is caused by a dominant X- linked allele, the mother with the disease in generation II would have one X chromosome carrying the dominant allele or the disease and one with the recessive allele. All o her ospring would have a 5 0% chance o inheriting this X chromosome and o having the disease. The data in the pedigree fts this and so supports the theory.

4

Key: normal pigmentation albino

D eductions: 



Two o the children are albino and yet the parents both have normal pigmentation. This suggests that albinism is caused by a recessive allele ( m) and normal pigmentation by a dominant allele ( M) . There are both daughters and sons with albinism suggesting that the condition is not sex- linked. B oth males and emales are albino only i they have two copies o the recessive albinism allele ( mm) .



The albino children must have inherited an allele or albinism rom both parents.



B oth parents must also have one allele or normal pigmentation as they are not albino. The parents thereore have the alleles Mm.



The chance o a child o these parents having albinism is 1 . Although on average 1 in 4 o 4

Key: vitamin D-resistant rickets not aected  Figure 21

182

Pedigree of a family with cases of vitamin D-resistant rickets

3 . 4 i N h E r i TAN CE

Data-based questons: Deducing genotypes from pedigree charts The pedigree chart in fgure 2 2 shows fve generations o a amily aected by a genetic disease. 1

2

Explain, using evidence rom the pedigree, whether the condition is due to a recessive or a dominant allele. [3 ] Explain what the probability is o the individuals in generation V having:

I 1

2

3

4

II 1

2

3

4

5

6

7

8

9

10 11

12

13

14

15

III 1

2

3

4

IV 1

2

3

4

5

6

7

8

V ?

?

?

?

1

2

3

4

unaected male unaected female aected male aected female

 Figure 22

Example of a pedigree chart

a) two copies o a recessive allele;

3

b) one recessive and one dominant allele; c) two copies o the dominant allele.

D educe, with reasons, the possible alleles o: a) 1 in generation III; b) 1 3 in generation II.

[3 ]

4

[2 ]

S uggest two examples o genetic diseases that would ft this inheritance pattern. [2 ]

Genetic diseases in humans Many genetic diseases have been identifed in humans but most are very rare. S everal genetic diseases have already been described in this sub- topic, including sickle- cell anemia, cystic fbrosis, hemophilia and Huntingtons disease. There are other well- known examples, such as phenylketonuria ( PKU) , Tay-S achs disease and Marans syndrome. Medical research has already identifed more than 4, 000 genetic diseases and more no doubt remain to be ound. Given this large number o genetic diseases, it might seem surprising that most o us do not suer rom any o them. The reason or this is that most genetic diseases are caused by very rare recessive alleles which ollow Mendelian patterns o inheritance. The chance o inheriting one allele or any specifc disease is small but to develop the disease two alleles must be inherited and the chance o this is extremely small. It is now possible to sequence the genome o an individual human relatively cheaply and quickly and large numbers o humans are being sequenced to allow comparisons. This research is revealing the number o rare recessive alleles that a typical individual is carrying that could cause a genetic disease. C urrent estimates are that the number is between 75 and 2 00 alleles among the 2 5 , 000 or so genes in the human genome. An individual can only produce a child with a genetic disease due to one o these recessive alleles i the other parent o the child has the same rare allele.

 Figure 23

Alleles from two parents come together when they have a child. There is a small chance that two recessive alleles will come together and cause a genetic disease

183

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Causes of mutation Radiation and mutagenic chemicals increase the mutation rate and can cause genetic disease and cancer.  Figure 24 Abraham

Lincolns eatures resemble Marans syndrome but a more recent theory is that he sufered rom MEN2B, another genetic disease

A gene consists o a length o D NA, with a base sequence that can be hundreds or thousands o bases long. The dierent alleles o a gene have slight variations in the base sequence. Usually only one or a very small number o bases are dierent. New alleles are ormed rom other alleles by gene mutation. A mutation is a random change to the base sequence o a gene. Two types o actor can increase the mutation rate. 

Radiation increases the mutation rate i it has enough energy to cause chemical changes in D NA. Gamma rays and alpha particles rom radioactive isotopes, short- wave ultraviolet radiation and X- rays are all mutagenic.



S ome chemical substances cause chemical changes in D NA and so are mutagenic. Examples are benzo[a] pyrene and nitrosamines ound in tobacco smoke and mustard gas used as a chemical weapon in the First World War.

Mutations are random changes  there is no mechanism or a particular mutation being carried out. A random change to an allele that has developed by evolution over perhaps millions o years is unlikely to be benefcial. Almost all mutations are thereore either neutral or harmul. Mutations o the genes that control cell division can cause a cell to divide endlessly and develop into a tumour. Mutations are thereore a cause o cancer.

 Figure 25 The risk o mutations due to

radiation rom nuclear waste is minimized by careul storage

Mutations in body cells, including those that cause cancer, are eliminated when the individual dies, but mutations in cells that develop into gametes can be passed on to ospring. This is the origin o genetic diseases. It is thereore particularly important to minimize the number o mutations in gamete- producing cells in the ovaries and testes. C urrent estimates are that one or two new mutations occur each generation in humans, adding to the risk o genetic diseases in children.

Consequences of nuclear bombing and accidents at nuclear power stations Consequences of radiation after nuclear bombing of Hiroshima and Nagasaki and the nuclear accidents at Chernobyl. The common eature o the nuclear bombing o Hiroshima and Nagasaki and the nuclear accidents at Three Mile Island and C hernobyl is that radioactive isotopes were released into the environment and as a result people were exposed to potentially dangerous levels o radiation. When the atomic bombs were detonated over Hiroshima and Nagasaki 1 5 0 , 0 0 0 2 5 0 , 00 0

184

people either died directly or within a ew months. The health o nearly 1 0 0 , 0 0 0 survivors has been ollowed since then by the Radiation E ects Research Foundation in Japan. Another 2 6 , 0 0 0 people who were not exposed to radiation have been used as a control group. B y 2 0 1 1 the survivors had developed 1 7 , 448 tumours, but only 8 5 3 o these could be

3 . 4 i N h E r i TAN CE

attributed to the eects o radiation rom the atomic bombs.

into the atmosphere in total. The eects were widespread and severe:

Apart rom cancer the other main eect o the radiation that was predicted was mutations, leading to stillbirths, malormation or death. The health o 1 0, 000 children that were etuses when the atomic bombs were detonated and 77, 000 children that were born later in Hiroshima and Nagasaki has been monitored. No evidence has been ound o mutations caused by the radiation. There are likely to have been some mutations, but the number is too small or it to be statistically signifcant even with the large numbers o children in the study.



4 km 2 o pine orest downwind o the reactor turned ginger brown and died.



Horses and cattle near the plant died rom damage to their thyroid glands.



Lynx, eagle owl, wild boar and other wildlie subsequently started to thrive in a zone around C hernobyl rom which humans were excluded.



B ioaccumulation caused high levels o radioactive caesium in fsh as ar away as Scandinavia and Germany and consumption o lamb contaminated with radioactive caesium was banned or some time as ar away as Wales.



C oncentrations o radioactive iodine in the environment rose and resulted in drinking water and milk with unacceptably high levels.



More than 6, 000 cases o thyroid cancer have been reported that can be attributed to radioactive iodine released during the accident.



According to the report C hernobyls Legacy Health, Environmental and S ocio- Economic Impacts, produced by The C hernobyl Forum, there is no clearly demonstrated increase in solid cancers or leukemia due to radiation in the most aected populations.

D espite the lack o evidence o mutations due to the atomic bombs, survivors have sometimes elt that they were stigmatized. S ome ound that potential wives or husbands were reluctant to marry them or ear that their children might have genetic diseases. The accident at C hernobyl, Ukraine, in 1 986 involved explosions and a fre in the core o a nuclear reactor. Workers at the plant quickly received atal doses o radiation. Radioactive isotopes o xenon, krypton, iodine, caesium and tellurium were released and spread over large parts o E urope. About six tonnes o uranium and other radioactive metals in uel rom the reactor was broken up into small particles by the explosions and escaped. An estimated 5 , 2 00 million GB q o radioactive material was released

Incidence per 100,000 in Belarus

12

Actvty

adults (1934) 10

Cangng ates of tyod cance

adolescents (1518)

When would you expect the cases o thyroid cancer in young adults to start to drop, based on the data in fgure 26?

Cases per 100,000

children (014) 8 6 4 2 0 1984

1986

1988

1990

 Figure 26 Incidence of thyroid

1992

1994

1996

1998

2000

2002

2004v

cancer in Belarus after the Chernobyl accident

185

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G e n e ti cs

Data-baed quetion: The aftermath of Chernobyl Mutations can cause a cell to become a tumour cell. The release of 6 . 7 tonnes of radioactive material from the nuclear power station at C hernobyl in 1 9 8 6 was therefore the cause of large numbers of deaths due to cancer. The UN C hernob yl Forum stated that  up to 4, 0 0 0 people may ultimately die as a result of the disaster, but Green Party members of the E uropean Parliament commissioned a report from a radiation scientist, which gave an estimate of 3 0 , 0 0 0 to 6 0 , 0 0 0 extra deaths. O ne way of obtaining an estimate is to use data from previous radiation exposures, such as the detonation of nuclear warheads at Hiroshima and Nagasaki in 1 9 45 . The data below is an analysis of deaths due to leukemia and cancer b etween 1 9 5 0 and 1 9 9 0 among those exposed to radiation from these warheads. It was published by the Radiation E ffects Research Foundation.

 Figure 27

Humans have been excluded from a large zone near the Chernobyl reactor. Some plants and animals have shown deformities that may be due to mutations

radiation Numbe of death Etimate of exce Pecentage of death doe ange in people expoed death ove contol attibutable to (sv) to adiation goup adiation expoue Leukemia 0.0050.2 70 10 0.20.5 27 13 48 0.51 23 17 74 56 47 >1 Cancer 0.0050.2 3391 63 2 0.20.5 646 76 12 0.51 342 79 23 308 121 39 >1 1

C alculate the percentage of excess deaths over control groups due to leukemia in people exposed to ( a) 0. 005 - 0. 02 Sv ( sieverts) of radiation ( b) >1 Sv of radiation. [4]

2

C onstruct a suitable type of graph or chart to represent the data in the right- hand column of the table, including the two percentages that you have calculated. There should be two y- axes, for the leukemia deaths and the cancer deaths. [4]

3

C ompare the effect of radiation on deaths due to leukemia and deaths due to cancer.

[3 ]

D iscuss, with reasons, what level of radiation might be acceptable in the environment.

[4]

4

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3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y

3.5 Genetc odfcaton and botecnoog Udertadig  Gel electrophoresis is used to separate proteins    

 



or ragments o DNA according to size. PCR can be used to ampliy small amounts o DNA. DNA proling involves comparison o DNA. Genetic modication is carried out by gene transer between species. Clones are groups o genetically identical organisms, derived rom a single original parent cell. Many plant species and some animal species have natural methods o cloning. Animals can be cloned at the embryo stage by breaking up the embryo into more than one group o cells. Methods have been developed or cloning adult animals using diferentiated cells.

Applicatio  Use o DNA proling in paternity and orensic

investigations.  Gene transer to bacteria with plasmids using restriction endonucleases and DNA ligase.  Assessment o the potential risks and benets associated with genetic modication o crops.  Production o cloned embryos by somatic-cell nuclear transer.

skill  Design o an experiment to assess one actor

afecting the rooting o stem-cuttings.  Analysis o examples o DNA proles.  Analysis o data on risks to monarch butteries o Bt crops.

nature of ciece  Assessing risks associated with scientic research: scientists attempt to assess the risks associated

with genetically modied crops or livestock.

Gel electrophorei Gel electrophoresis is used to separate proteins or ragments o DNA according to size. Gel electrophoresis involves separating charged molecules in an electric eld, according to their size and charge. Samples are placed in wells cast in a gel. The gel is immersed in a conducting fuid and an electric eld is applied. Molecules in the sample that are charged will move through the gel. Molecules with negative and positive charges move in opposite directions. Proteins may be positively or negatively charged so can be separated according to their charge. The gel used in gel electrophoresis consists o a mesh o laments that resists the movement o molecules in a sample. D NA molecules rom eukaryotes are too long to move through the gel, so they must be broken up into smaller ragments. All D NA molecules carry negative charges so move in the same direction during gel electrophoresis, but not

DNA samples negative electrode

2

sample well gel

1 positive electrode large fragments 2 direction of migration small fragments 1  Figure 1

Procedure for gel electrophoresis

187

3

G e n e ti cs at the same rate. S mall ragments move aster than large ones so they move urther in a given time. Gel electrophoresis can thereore be used to separate ragments o D NA according to size.

DnA amplifcatio by PCR PCR can be used to amplify small amounts of DNA.

 Figure 2

Small samples o DNA being extracted rom ossil bones o a Neanderthal or amplifcation by PCR

The polymerase chain reaction is used to make large numbers o copies o D NA. It is almost always simply called PC R. The details o this technique are described in sub- topic 2 . 7 . O nly a very small amount o D NA is needed at the start o the process  in theory j ust a single molecule. Within an hour or two, millions o copies can be made. This makes it possible to study the D NA urther without the risk o using up a limited sample. For example, D NA extracted rom ossils can be amplifed using PC R. Very small amounts o D NA rom blood, semen or hairs can also be amplifed or use in orensic investigations. PC R is not used to copy the entire set o D NA molecules in a sample such as blood or semen. White blood cells contain all chromosomes o the person rom whom the blood came, or example, and together the sperm cells in a sample o semen contain a mans entire genome. Instead PC R is used to copy specifc D NA sequences. A sequence is selected or copying by using a primer that binds to the start o the desired sequence. The primer binds by complementary base pairing. The selectivity o PC R allows particular desired sequences to be copied rom a whole genome or even greater mixture o DNA. One test or the presence o genetically modifed ingredients in oods involves the use o a primer that binds to the genetically modifed D NA. Any such DNA present is amplifed by the PC R, but i there is none present the PC R has no eect.

Data-based questions: PCR and Neanderthals

Samples o D NA were recently obtained rom ossil bones o a Neanderthal ( Homo neanderthalensis) . They were amplifed using PC R. A section o the Neanderthal mitochondrial D NA was sequenced and compared with sequences rom 994 humans and 1 6 chimpanzees. The bar chart in fgure 3 shows how many basesequence dierences were ound within the sample o humans, between the humans and the

188

Neanderthal and between the humans and the chimpanzees. frequency of number of dierences / %

The evolution o groups o living organisms can be studied by comparing the base sequences o their D NA. I a species separates into two groups, dierences in base sequence between the two species accumulate gradually over long periods o time. The number o dierences can be used as an evolutionary clock.

25 humanNeanderthal

20

15 humanhuman

humanchimp

10 5 0

0

 Figure3

5 10 15 20 25 30 35 40 45 50 55 60 65 number of dierences in base sequence

Number o dierences in base sequences between humans, chimps and Neanderthals

3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y

1

S tate the most common number o dierences in base sequence between pairs o humans. [1 ]

2

Humans and Neanderthals are both classifed in the genus Homo and chimpanzees are classifed in the genus Pan. D iscuss whether

this classifcation is supported by the data in the bar chart. [3 ] 3

Suggest a limitation to drawing any conclusion rom the humanNeanderthal comparison.

[1 ]

DnA proflig DNA profling involves comparison o DNA. D NA profling involves these stages: 

A sample o D NA is obtained, either rom a known individual or rom another source such as a ossil or a crime scene.



Sequences in the D NA that vary considerably between individuals are selected and are copied by PC R.



The copied D NA is split into ragments using restriction endonucleases.



The ragments are separated using gel electrophoresis.



This produces a pattern o bands that is always the same with D NA taken rom one individual. This is the individual' s D NA profle. The profles o dierent individuals can be compared to see which bands are the same and which are dierent.



 Figure 4 DNA profles are oten

reerred to as DNA fngerprints as they are used in a similar way to real fngerprints to distinguish one individual rom all others

Paterity ad oresic ivestigatios Use o DNA profling in paternity and orensic investigations. D NA profling is used in orensic investigations. 

B lood stains on a suspects clothing could be shown to come rom the victim.



B lood stains at the crime scene that are not rom the victim could be shown to come rom the suspect.



A single hair at the crime scene could be shown to come rom the suspect.



S emen rom a sexual crime could be shown to come rom the suspect.

In each example the DNA profle o material rom the crime scene is compared with the DNA profle o a sample o DNA taken rom the suspect or the victim. I the pattern o bands matches exactly it is highly likely that the two samples o DNA are rom the same person. This can provide very strong evidence o who committed the crime. Some countries now have databases o DNA profles, which have allowed many criminal cases to be solved.

D NA profling is also used in paternity investigations. These are done to fnd out whether a man is the ather o a child. There are various reasons or paternity investigations being requested. 

Men sometimes claim that they are not the ather o a child to avoid having to pay the mother to raise the child.



Women who have had multiple partners may wish to identiy the biological ather o a child.



A child may wish to prove that a deceased man was their ather in order to show that they are their heir.

D NA profles o the mother, the child and the man are needed. D NA profles o each o the samples are prepared and the patterns o bands are compared. I any bands in the childs profle do not occur in the profle o the mother or man, another person must be the ather.

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Aalysis o DnA profles Analysis o examples o DNA profles. Analysis o D NA profles in orensic investigations is straightorward: two D NA samples are very likely to have come rom the same person i the pattern o bands on the profle is the same. victim

specimen

1 2

suspects

3  Figure 5 Which

o the three suspects DNA fngerprints matches the specimen recovered rom the crime scene?

Analysis o DNA profles in paternity investigations is more complicated. Each o the bands in the childs DNA profle must be the same as a band in the biological mother or athers profle. Every band in the childs profle must be checked to make sure that it occurs either in the mothers profle or in the profle o the man presumed to be the ather. I one or more bands do not, another man must have been the biological ather.

Geetic modifcatio Genetic modifcation is carried out by gene transer between species. Molecular biologists have developed techniques that allow genes to be transerred between species. The transer o genes rom one species to another is known as genetic modifcation. It is possible because the genetic code is universal, so when genes are transerred between species, the amino acid sequence translated rom them is unchanged  the same polypeptide is produced. Genes have been transerred rom eukaryotes to bacteria. O ne o the early examples was the transer o the gene or making human insulin to a bacterium. This was done so that large quantities o this hormone can be produced or treating diabetics. Genetic modifcation has been used to introduce new characteristics to animal species. For example, goats have been produced that secrete milk containing spider silk protein. S pider silk is immensely strong, but spiders could not be used to produce it commercially.

 Figure 6 Genes have been

transerred rom daodil plants to rice, to make the rice produce a yellow pigment in its seeds

190

Genetic modifcation has also been used to produce many new varieties o crop plant. These are known as genetically modifed or GM crops. For example genes rom snapdragons have been transerred to tomatoes to produce ruits that are purple rather than red. The production o golden rice involved the transer o three genes, two rom daodil plants and

3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y one rom a bacterium, so that the yellow pigment - carotene is produced in the rice grains.

Actvt Scientists have an obligation to consider the ethical implications o their research. Discuss the ethics o the development o golden rice. -carotene is a precursor to vitamin A. The development o golden rice was intended as a solution to the problem o vitamin A defciency, which is a signifcant cause o blindness among children globally.

techniques for gene ransfer o baceria Gene transer to bacteria with plasmids using restriction endonucleases and DNA ligase. Genes can be transerred rom one species to another by a variety o techniques. Together these techniques are known as genetic engineering. Gene transer to bacteria usually involves plasmids, restriction enzymes and D NA ligase. 





A plasmid is a small extra circle o D NA. The smallest plasmids have about 1 , 000 base pairs ( 1 kbp) , but they can have over 1 , 000 kbp. They occur commonly in bacteria. The most abundant plasmids are those with genes that encourage their replication in the cytoplasm and transer rom one bacterium to another. There are thereore some parallels with viruses but plasmids are not pathogenic and natural selection avours plasmids that coner an advantage on a bacterium rather than a disadvantage. B acteria use plasmids to exchange genes, so naturally absorb them and incorporate them into their main circular D NA molecule. Plasmids are very useul in genetic engineering.

Bacterial cell Plasmid mRNA extracted from human pancreatic cells Plasmid obtained from bacteria mRNA

cDNA mRNA treated with reverse transcriptase to make complementary DNA (cDNA)

Restriction enzymes, also known as endonucleases, are enzymes that cut D NA molecules at specifc base sequences. They can be used to cut open plasmids and also to cut out desired genes rom larger D NA molecules. S ome restriction enzymes have the useul property o cutting the two strands o a D NA molecule at dierent points. This leaves single- stranded sections called sticky ends. The sticky ends created by any one particular restriction enzyme have complementary base sequences so can be used to link together pieces o D NA, by hydrogen bonding between the bases.

Plasmid and cDNA fused using DNA ligase Recombinant plasmid introduced into host cells Bacteria multiply in a fermenter and produce insulin Separation and purication of human insulin

D NA ligase is an enzyme that j oins D NA molecules together frmly by making sugarphosphate bonds between nucleotides. When the desired gene has been inserted into a plasmid using sticky ends there are still nicks in each sugarphosphate backbone o the D NA but D NA ligase can be used to seal these nicks.

An obvious requirement or gene transer is a copy o the gene being transerred. It is usually easier to obtain messenger RNA transcripts o genes than the genes themselves. Reverse transcriptase is an enzyme that makes D NA copies o RNA molecules called cD NA. It can be used to make the D NA needed or gene transer rom messenger RNA.

Plasmid cut with restriction enzyme

Human insulin can be used by diabetic patients

 Figure 7

shows the steps involved in one example o gene transer. It has been used to create genetically modifed E. coli bacteria that are able to manuacture human insulin, or use in treating diabetes

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Assessing the risks o genetic modifcation Assessing risks associated with scientifc research: scientists attempt to assess the risks associated with genetically modifed crops or livestock.

 Figure 8 The biohazard

symbol indicates any organism or material that poses a threat to the health of living organisms especially humans

There have been many ears expressed about the possible dangers o genetic modifcation. These ears can be traced back to the 1 970s when the frst experiments in gene transer were being conducted. Paul B erg planned an experiment in which D NA rom the monkey virus S V40 was going to be inserted into the bacterium E. coli. O ther biologists expressed serious concerns because SV40 was known to cause cancer in mice and E. coli lives naturally in the intestines o humans. There was thereore a risk o the genetically engineered bacterium causing cancer in humans. S ince then many other risks associated with genetic modifcation have been identifed. There has been ferce debate both among scientists and between scientists and non- scientists about the saety o the research and the saety o using genetically modifed organisms. This has led to bans being imposed in some countries, with potentially useul applications o GM crops or livestock let undeveloped. Almost everything that we do carries risks and it is not possible to eliminate risk entirely, either in science or in other aspects o our lives. It is natural or humans to assess the risk o an action and decide whether or not go ahead with it. This is what scientists must do  assess the risks associated with their research beore carrying it out. The risks can be assessed in two ways:

GM corn (maize) is widely grown in North America



What is the chance o an accident or other harmul consequence?



How harmul would the consequence be?

 Figure 9

I there is a high chance o harmul consequences or a signifcant chance o very harmul consequences then research should not be done.

Risks and benefts o GM crops

192

Assessment o the potential risks and benefts associated with genetic modifcation o crops.

is disagreement, because gene transer to crop plants is a relatively recent procedure, the issues involved are very complex and in science it oten takes decades or disputes to be resolved.

GM crops have many potential benefts. These have been publicized widely by the corporations that produce GM seed, but they are questioned by opponents o the technology. Even basic issues such as whether GM crops increase yields and reduce pesticide and herbicide use have been contested. It is not surprising that there

Potential benefts can be grouped into environmental benefts, health benefts and agricultural benefts. Economic benefts o GM crops are not included here, because they cannot be assessed on a scientifc basis using experimental evidence. It would be impossible in the time available or IB students to assess all claimed

3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y

benefts or all GM crops. Instead it is better to select one claim rom the list given here and assess it or one crop. Much o the evidence relating to potential benefts and also to risks is reely available. C laims about environmental benefts o GM crop s: 

Pest- resistant crop varieties can be produced by transerring a gene or making a toxin to the plants. Less insecticide then has to be sprayed on to the crop so ewer bees and other benefcial insects are harmed.



Use o GM crop varieties reduces the need or plowing and spraying crops, so less uel is needed or arm machinery.



The shel- lie o ruit and vegetables can be improved, reducing wastage and reducing the area o crops that have to be grown.

C laims about the health benefts o GM crop s: 

The nutritional value o crops can be improved, or example by increasing the vitamin content.



Varieties o crops could be produced lacking allergens or toxins that are naturally present in them.



GM crops could be engineered that produce edible vaccines so by eating the crop a person would be vaccinated against a disease.

C laims about agricultural benefts o GM crop s: 

Varieties resistant to drought, cold and salinity can be produced by gene transer, expending the range over which crops can be produced and increasing total yields.



A gene or herbicide resistance can be transerred to crop plants allowing all other plants to be killed in the growing crop by spraying with herbicide. With less weed competition crop yields are higher. Herbicides that kill all plants can be used to create weed- ree conditions or sowing non- GM crops but they cannot be used once the crop is growing.



C rop varieties can be produced that are resistant to diseases caused by viruses.

 Figure 10 Wild

plants growing next to a crop of GM maize

These diseases currently reduce crop yields signifcantly and the only current method o control is to reduce transmission by killing insect vectors o the viruses with insecticides. A wide variety o concerns about GM crops have been raised. S ome o these, such as the eect on armers incomes, cannot be assessed on scientifc grounds so are not relevant here. The remaining concerns can be grouped into health risks, environmental risks and agricultural risks. To make overall j udgments about the saety o GM crops, each risk needs to be assessed careully, using all the available experimental evidence. This needs to be done on a case by case basis as it is not possible to assess the risks and benefts o one GM crop rom experiments perormed on another one. There is no consensus among all scientists or non- scientists yet about GM crops and it is thereore important or as many o us as possible to look at the evidence or the claims and counter- claims, rather than the publicity. Any o the risks that are included here could be selected or detailed scrutiny. C laims made about health risks o GM crop s: 

Proteins produced by transcription and translation o transerred genes could be

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plants, plant-eating insects and organisms that eed on them where GM rather than non-GM crops are being grown.

toxic or cause allergic reactions in humans or livestock that eat GM crops. 



Antibiotic resistance genes used as markers during gene transer could spread to pathogenic bacteria. Transerred genes could mutate and cause unexpected problems that were not riskassessed during development o GM crops.

C laims made about agricultural risks of GM crop s: 

Some seed rom a crop is always spilt and germinates to become unwanted volunteer plants that must be controlled, but this could become very dicult i the crop contains herbicide resistance genes.



Widespread use o GM crops containing a toxin that kills insect pests will lead to the spread o resistance to the toxin in the pests that were the initial problem and also to the spread o secondary pests that are resistant to the toxin but were previously scarce.



Farmers are not permitted by patent law to save and re-sow GM seed rom crops they have grown, so strains adapted to local conditions cannot be developed.

C laims made about environmental risks of GM crop s: 

Non- target organisms could be aected by toxins that are intended to control pests in GM crop plants.



Genes transerred to crop plants to make them herbicide resistant could spread to wild plants, turning them into uncontrollable super- weeds.



Biodiversity could be reduced i a lower proportion o sunlight energy passes to weed

Analysing risks to monarch butterfies o Bt corn Analysis o data on risks to monarch butterfies o Bt crops. Insect pests o crops can be controlled by spraying with insecticides but varieties have been recently been produced by genetic engineering that produce a toxin that kills insects. A gene was transerred rom the bacterium Bacillus thuringiensis that codes or Bt toxin. The toxin is a protein. It kills members o insect orders that contain butterfies, moths, fies, beetles, bees and ants. The genetically engineered corn varieties produce Bt toxin in all parts o the plant including pollen. Bt varieties o many crops have been produced, including Zea mays. In North America this crop is called corn, while in B ritain it is known as maize, or corn on the cob. The crop is attacked by various insect pests including corn borers, which are the larvae o the moth Ostrinia nubilalis. C oncerns have been expressed about the eects o Bt corn on non-target species o insect. O ne particular species o concern is the monarch butterfy, Danaus plexippus. The larvae o the monarch butterfy eed on leaves o milkweed, Asclepias curassavica. This plant sometimes grows close enough to corn crops to become dusted with the wind- dispersed corn pollen. There is thereore a risk that monarch larvae might be poisoned by Bt toxin in pollen rom GM corn crops. This risk has been investigated experimentally. D ata rom these experiments is available or analysis.

194

To investigate the eect o pollen rom Bt corn on the larvae o monarch butterfies the ollowing procedure was used. Leaves were collected rom milkweed plants and were lightly misted with water. A spatula o pollen was gently tapped over the leaves to deposit a ne dusting. The leaves were placed in water- lled tubes. Five three-dayold monarch butterfy larvae were placed on each lea. The area o lea eaten by the larvae was monitored over our days. The mass o the larvae was measured ater our days. The survival o the larvae was monitored over our days. Three treatments were included in the experiment, with ve repeats o each treatment: 

leaves not dusted with pollen ( blue)



leaves dusted with non- GM pollen ( yellow)



leaves dusted with pollen rom Bt corn ( red)

100 75 50 25 0

2

3

4 5 6 7

2 3 Time (days)

1

2 3 Time (days)

4

1.5 1 0.5 0

The results are shown in the table, bar chart and graph on the right. 1

1 2 Cumulative leaf consumption per larva

Data-based questons: Transgenic pollen and monarch larvae

Survival of monarch larvae (%)

3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y

a) List the variables that were kept constant in the experiment.

[3 ]

b) Explain the need to keep these variables constant.

[2 ]

a) C alculate the total number o larvae used in the experiment.

[2 ]

b) Explain the need or replicates in experiments.

[2 ]

The bar chart and the graph show mean results and error bars. Explain how error bars help in the analysis and evaluation o data. [2 ] Explain the conclusions that can be drawn rom the percentage survival o larvae in the three treatments.

[2 ]

Suggest reasons or the dierences in lea consumption between the three treatments.

[3 ]

Predict the mean mass o larvae that ed on leaves dusted with non- GM pollen.

[2 ]

O utline any dierences between the procedures used in this experiment and processes that occur in nature, which might aect whether monarch larvae are actually harmed by Bt pollen.

[2 ]

4

Source: Losey JE, Rayor LS, Carter ME (May 1999) . Transgenic pollen harms monarch larvae. Nature 399 (6733) : 214. Treatment

Mean mass of surviving larvae (g)

Leaves not dusted with pollen

0.38

Leaves dusted with Not available non-GM pollen Leaves dusted with 0.16 pollen from Bt corn

Actvt Estatng te sze of a cone A total of 130,000 hectares of Russet Burbank potatoes were planted in Idaho in 2011. The mean density of planting of potato tubers was 50,000 per hectare. Estimate the size of the clone at the time of planting and at the time of harvest.

Clones Clones are groups of genetically identical organisms, derived from a single original parent cell. A zygote, produced by the usion o a male and emale gamete, is the rst cell o a new organism. B ecause zygotes are produced by sexual reproduction, they are all genetically dierent. A zygote grows and develops into an adult organism. I it reproduces sexually, its

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Activity

ospring will be genetically dierent. In some species organisms can also reproduce asexually. When they do this, they produce genetically identical organisms. The production o genetically identical organisms is called cloning and a group o genetically identical organisms is called a clone.

How many potato clones are there in this photo?

Although we do not usually think o them in this way, a pair o identical twins is the smallest clone that can exist. They are either the result o a human zygote dividing into two cells, which each develop into separate embryos, or an embryo splitting into two parts which each develop into a separate individual. Identical twins are not identical in all their characteristics and have, or example, dierent fngerprints. A better term or them is monozygotic. More rarely identical triplets, quadruplets and even quintuplets have been produced. S ometimes a clone can consist o very large numbers o organisms. For example, commercially grown potato varieties are huge clones. Large clones are ormed by cloning happening again and again, but even so all the organisms may be traced back to one original parent cell.

natural methods of cloig Many plant species and some animal species have natural methods of cloning.

 Figure 11

Identical twins are an example

of cloning

Although the word clone is now used or any group o genetically identical organisms, it was frst used in the early 2 0th century or plants produced by asexual reproduction. It comes rom the Greek word or twig. Many plants have a natural method o cloning. The methods used by plants are very varied and can involve stems, roots, leaves or bulbs. Two examples are given here: 

A single garlic bulb, when planted, uses its ood stores to grow leaves. These leaves produce enough ood by photosynthesis to grow a group o bulbs. All the bulbs in the group are genetically identical so they are a clone.



A strawberry plant grows long horizontal stems with plantlets at the end. These plantlets grow roots into the soil and photosynthesize using their leaves, so can become independent o the parent plant. A healthy strawberry plant can produce ten or more genetically identical new plants in this way during a growing season.

Natural methods o cloning are less common in animals but some species are able to do it. 

 Figure 12

One bulb of garlic clones itself to produce a group of bulbs by the end of the growing season

196

Hydra clones itsel by a process called budding ( sub- topic 1 .6, fgure 1 , page 5 1 ) .

Female aphids can give birth to ospring that have been produced entirely rom diploid egg cells that were produced by mitosis rather than meiosis. The ospring are thereore clones o their mother.

3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y

Investigating actors afecting the rooting o stem-cuttings Design o an experiment to assess one actor afecting the rooting o stem-cuttings. S tem- cuttings are short lengths o stem that are used to clone plants artifcially. I roots develop rom the stem, the cutting can become an independent new plant. 1

2

3

Many plants can be cloned rom cuttings. Ocimum basilicum roots particularly easily. Nodes are positions on the stem where leaves are attached. With most species the stem is cut below a node. Leaves are removed rom the lower hal o the stem. I there are many large leaves in the upper hal they can also be reduced.

4

The lowest third o the cutting is inserted into compost or water. C ompost should be sterile and contain plenty o both air and water.

5

A clear plastic bag with a ew holes cut in it prevents excessive water loss rom cuttings inserted in compost.

6

Rooting normally takes a ew weeks. Growth o new leaves usually indicates that the cutting has developed roots.



whether the cutting is placed in water or compost



what type o compost is used



how warm the cuttings are kept



whether a plastic bag is placed over the cuttings



whether holes are cut in the plastic bag.

You should think about these questions when you design your experiment: 1

What is your independent variable?

2

How will you measure the amount o root ormation, which is your dependent variable?

3

Which variables should you keep constant?

4

How many dierent types o plant should you use?

5

How many cuttings should you use or each treatment?

Not all gardeners have success when trying to clone plants using root cuttings. S uccessul gardeners are sometimes said to have  green fngers but a biologist would rej ect this as the reason or their success. E xperiments can give evidence about the actors that determine whether cuttings root or not. You can design and carry out an experiment to investigate one o the actors on the list below, or another actor o your own. Possible actors to investigate: 

whether the stem is cut above or below a node



how long the cutting is



whether the end o the stem is let in the air to callus over



how many leaves are let on the cutting



whether a hormone rooting powder is used

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Cloning animal embryos Animals can be cloned at the embryo stage by breaking up the embryo into more than one group o cells. At an early stage o development all cells in an animal embryo are pluripotent ( capable o developing into all types o tissue) . It is thereore theoretically possible or the embryo to divide into two or more parts and each part to develop into a separate individual with all body parts. This process is called splitting or ragmentation. C oral embryos have been observed to clone themselves by breaking up into smaller groups o cells or even single cells, presumably because this increases the chance o one embryo surviving. Formation o identical twins could be regarded as cloning by splitting, but most animal species do not appear to do this naturally. However, it is possible to break up animal embryos artifcially and in some cases the separated parts develop into multiple embryos. In livestock, an egg can be ertilized in vitro and allowed to develop into a multicellular embryo. Individual cells can be separated rom the embryo while they are still pluripotent and transplanted into surrogate mothers. Only a limited number o clones can be obtained this way, because ater a certain number o divisions the embryo cells are no longer pluripotent. Splitting o embryos is usually most successul at the eight-cell stage.

 Figure 13

Sea urchin embryo (a) 4-cell stage (b) blastula stage consisting of a hollow ball of cells

There has been little interest in this method o artifcial cloning because at the embryo stage it is not possible to assess whether a new individual produced by sexual reproduction has desirable characteristics.

Cloning adult animals using diferentiated cells Methods have been developed or cloning adult animals using diferentiated cells. It is relatively easy to clone animal embryos, but at that stage it is impossible to know whether the embryos will have desirable characteristics. O nce the embryos have grown into adults it is easy to assess their characteristics, but it is much more difcult to clone them. This is because the cells that make up the body o an adult animal are dierentiated. To produce all the tissues in a new animal body undierentiated pluripotent cells are needed. The biologist John Gurdon carried out experiments on cloning in the rog Xenopus as a postgraduate student in Oxord during the 1 950s. He removed nuclei rom body cells o Xenopus tadpoles and transplanted them into egg cells rom which the nucleus had been removed. The egg cells into which the nuclei were transplanted developed as though they were zygotes. They carried out cell division, cell growth and dierentiation to orm all the tissues o a normal Xenopus rog. In 201 2 Gurdon was awarded the Nobel Prize or Physiology or Medicine or his pioneering research.

 Figure 14 Xenopus tadpoles

198

C loning using dierentiated cells prove d to b e much more diicult in mammals. The irst cloned mammal was D olly the shee p in 1 9 9 6 . Apart rom the ob vious reproductive use s o this type o cloning, there is also interest in it or therape utic reasons. I this procedure

3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y

was done with humans, the embryo would consist of pluripotent stem cells, which could be used to regenerate tissues for the adult. B e cause the cells would be genetically ide ntical to those of the adult from whom the nucleus was obtaine d they would not cause rej ection problems.

Methods used to produce Dolly Production of cloned embryos by somatic-cell nuclear transfer. The production of D olly was a pioneering development in animal cloning. The method that was used is called somatic-cell nuclear transfer. A somatic cell is a normal body cell with a diploid nucleus. The method has these stages: 

Adult cells were taken from the udder of a Finn D orset ewe and were grown in the laboratory, using a medium containing a low concentration of nutrients. This made genes in the cells inactive so that the pattern of differentiation was lost.



Unfertilized eggs were taken from the ovaries of a S cottish B lackface ewe. The nuclei were removed from these eggs. O ne of the cultured cells from the Finn D orset was placed next to each egg cell, inside the zona pellucida around the egg, which is a protective coating of gel. A small electric pulse was used to cause the two cells to fuse together. About 1 0% of the fused cells developed like a zygote into an embryo.

 Figure 15 Dolly

with Dr Ian Wilmut, the embryologist who led the team that produced her



The embryos were then inj ected when about seven days old into the uteri of other ewes that could act as surrogate mothers. This was done in the same way as in IVF. O nly one of the 2 9 embryos implanted successfully and developed through a normal gestation. This was D olly.

egg without a nucleus fused with donor cell using a pulse of electricity cell taken from udder of donor adult and cultured in laboratory for six days

unfertilized egg taken from another sheep. Nucleus removed from the egg  Figure 16 A method

embryo resulting from fusion of udder cell and egg transfered to the uterus of a third sheep which acts as the surrogate mother

surrogate mother gives birth to lamb. Dolly is genetically identical with the sheep that donated the udder cell (the donor)

or cloning an adult sheep using diferentiated cells

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Questions 1

Human somatic cells have 46 chromosomes, while our closest primate relatives, the chimpanzee, the gorilla and the orangutan all have 48 chromosomes. One hypothesis is that the human chromosome number 2 was ormed rom the usion o two chromosomes in a primate ancestor. The image below shows human chromosome 2 compared to chromosome 1 2 and 1 3 rom the chimpanzee. a) C ompare the human chromosome 2 with the two chimpanzee chromosomes ( fgure 1 7) . [3 ]

The cheetah ( Acinonyx jubatus) is an endangered species o large cat ound in S outh and East Arica. A study o the level o variation o the cheetah gene pool was carried out. In one part o this study, blood samples were taken rom 1 9 cheetahs and analysed or the protein transerrin using gel electrophoresis. The results were compared with the electrophoresis patterns or blood samples rom 1 9 domestic cats ( Felis sylvestris) . Gel electrophoresis can be used to separate proteins using the same principles as in D NA profling. The bands on the gel which represent orms o the protein transerrin are indicated.

transferrin

H

C

b) The ends o chromosomes, called telomeres, have many repeats o the same short D NA sequence. I the usion hypothesis were true, predict what would be ound in the region o the chromosome where the usion is hypothesized to have occurred. [2 ]

3

 Figure 17 origin

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

cheetahs

The pedigree in fgure 1 8 shows the AB O groups o three generations o a amily. I

II

III

AB

B

O

B

1

2

3

4

B

A

B

O

O

1

2

3

4

5

O

A

B

O

?

1

2

3

4

5

 Figure 18

transferrin

origin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

a) D educe the genotype o each person in the amily. [4] b) D educe the possible blood groups o individual III 5 , with the percentage chance o each. [2 ] c) D educe the possible blood groups and the percentage chance o each blood group:

200

domestic cats  Figure 19

Using fgure 1 9, deduce with reasons: a) the number o domestic cats and the number o cheetahs that were heterozygous or the transerrin gene; [2 ]

( i) o children o individual III 1 and his partner who is also in blood group O [2 ]

b) the number o alleles o the transerrin gene in the gene pool o domestic cats; [2 ]

( ii) o children o III 2 and her partner who is in blood group AB . [2 ]

c) the number o alleles o the transerrin gene in the gene pool o cheetahs. [1 ]

4 E co lo gy Intrdutin E cosystems require a continuous supply o energy to uel lie processes and to replace energy lost as heat. C ontinued availability o carbon and other chemical elements in ecosystems depends on cycles. The uture

survival o living organisms including humans depends on sustainable ecological communities. C oncentrations o gases in the atmosphere have signifcant eects on climates experienced at the Earths surace.

4.1 Species, communities and ecosystems Understandin  Species are groups o organisms that can  

  



   

potentially interbreed to produce ertile ofspring. Members o a species may be reproductively isolated in separate populations. Species have either an autotrophic or heterotrophic method o nutrition (a ew species have both methods) . Consumers are heterotrophs that eed on living organisms by ingestion. Detritivores are heterotrophs that obtain organic nutrients rom detritus by internal digestion. Saprotrophs are heterotrophs that obtain organic nutrients rom dead organic matter by external digestion. A community is ormed by populations o diferent species living together and interacting with each other. A community orms an ecosystem by its interactions with the abiotic environment. Autotrophs and heterotrophs obtain inorganic nutrients rom the abiotic environment. The supply o inorganic nutrients is maintained by nutrient cycling. Ecosystems have the potential to be sustainable over long periods o time.

Skis  Classiying species as autotrophs, consumers,

detritivores or saprotrophs rom a knowledge o their mode o nutrition.  Testing or association between two species using the chi-squared test with data obtained by quadrat sampling.  Recognizing and interpreting statistical signicance.  Setting up sealed mesocosms to try to establish sustainability. (Practical 5)

Nature f siene  Looking or patterns, trends and discrepancies:

plants and algae are mostly autotrophic but some are not.

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Species Species are groups o organisms that can potentially interbreed to produce ertile ofspring. B irds o paradise inhabit Papua New Guinea and other Australasian islands. In the breeding season the males do elaborate and distinctive courtship dances, repeatedly carrying out a series o movements to display their exotic plumage. O ne reason or this is to show to a emale that they are ft and would be a suitable partner. Another reason is to show that they are the same type o bird o paradise as the emale.

 Figure 1

A bird of paradise in Papua New Guinea

There are orty- one dierent types o bird o paradise. E ach o these usually only reproduces with others o its type and hybrids between the dierent types are rarely produced. For this reason each o the orty- one types o bird o paradise remains distinct, with characters that are dierent to those o other types. B iologists call types o organism such as these sp ecies. Although ew species have as elaborate courtship rituals as birds o paradise, most species have some method o trying to ensure that they reproduce with other members o their species. When two members o the same species mate and produce ospring they are interbreeding. O ccasionally members o dierent species breed together. This is called cross- breeding. It happens occasionally with birds o paradise. However, the ospring produced by cross- breeding between species are almost always inertile, which prevents the genes o two species becoming mixed. The reproductive separation between species is the reason or each species being a recognizable type o organism with characters that distinguish it rom even the most closely related other species. In summary, a species is a group o organisms that interbreed to produce ertile ospring.

Populations Members o a species may be reproductively isolated in separate populations. A population is a group o organisms o the same species who live in the same area at the same time. I two populations live in dierent areas they are unlikely to interbreed with each other. This does not mean that they are dierent species. I they potentially could interbreed, they are still members o the same species. I two populations o a species never interbreed then they may gradually develop dierences in their characters. Even i there are recognizable dierences, they are considered to be the same species until they cannot interbreed and produce ertile ospring. In practice it can be very difcult to decide whether two populations have reached this point and biologists sometimes disagree about whether populations are the same or dierent species.

202

4 . 1 S P e c i e S , c o m m u n i t i e S a n d e c o S yS t e m S

arph  hrrph r Species have either an autotrophic or heterotrophic method o nutrition (a ew species have both methods) . All organisms need a supply o organic nutrients, such as glucose and amino acids. They are needed or growth and reproduction. Methods o obtaining these carbon compounds can be divided into two types: 

some organisms make their own carbon compounds rom carbon dioxide and other simple substances  they are autotrophic, which means sel-eeding;



some organisms obtain their carbon compounds rom other organisms  they are heterotrophic, which means eeding on others.

S ome unicellular organisms use both methods o nutrition. Euglena gracilis or example has chloroplasts and carries out photosynthesis when there is sufcient light, but can also eed on detritus or smaller organisms by endocytosis. O rganisms that are not exclusively autotrophic or heterotrophic are mixotrophic.

 Figure 3

Arabidopsis thaliana the autotroph that molecular biologists use as a model plant

 Figure 4 Humming birds

are heterotrophic; the plants from which they obtain nectar are autotrophic

 Figure 5 Euglena  an

unusual organism as it can feed both autotrophically and heterotrophically

trs  pl  lgl r Looking or patterns, trends and discrepancies: plants and algae are mostly autotrophic but some are not. Almost all plants and algae are autotrophic  they make their own complex organic compounds using carbon dioxide and other simple substances. A supply o energy is needed to do this, which plants and algae obtain by absorbing light. Their method o autotrophic nutrition is thereore photosynthesis and they carry it out in chloroplasts.

av Glpgs rss The tortoises that live on the Galpagos islands are the largest in the world. They have sometimes been grouped together into one species, Chelinoidis nigra, but more recently have been split into separate species. Discuss whether each o these observations indicates that populations on the various islands are separate species: 

The Galpagos tortoises are poor swimmers and cannot travel rom one island to another so they do not naturally interbreed.



Tortoises rom diferent islands have recognizable diferences in their characters, including shell size and shape.



Tortoises rom diferent islands have been mated in zoos and hybrid ofspring have been produced but they have lower ertility and higher mortality than the ofspring o tortoises rom the same island.

 Figure 2

Galpagos tortoise

This trend or plants and algae to make their own carbon compounds by photosynthesis in chloroplasts is ollowed by the majority o species. However there are small numbers o both plants and algae that do not ft the trend, because although they are recognizably plants or algae, they

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do not contain chloroplasts and they do not carry out photosynthesis. These species grow on other plants, obtain carbon compounds rom them and cause them harm. They are thereore parasitic. To decide whether parasitic plants alsiy the theory that plants and algae are groups o autotrophic species or whether they are j ust minor and insignifcant discrepancies we need to consider how many species there are and how they evolved. 

The number o parasitic plants and algae is relatively small  only about 1 % o all plant and algal species.



It is almost certain that the original ancestral species o plant and alga were autotrophic and that the parasitic species evolved rom them. C hloroplasts can quite easily be lost rom cells, but cannot easily be developed. Also, parasitic species are diverse and occur in many dierent amilies. This pattern suggests that parasitic plants have evolved repeatedly rom photosynthetic species.

B ecause o this evidence, ecologists regard plants and algae as groups o autotrophs, with a small number o exceptional species that are parasitic.

data-base questions: Unexpected diets Although we usually expect plants to be autotrophs and animals to be consumers, living organisms are very varied and do not always conorm to our expectations. Figures 6 to 9 show our organisms with diets that are unexpected. 1

Which o the organisms is autotrophic?

[4]

2

Which o the organisms is heterotrophic?

[4]

3

O  the organisms that are heterotrophic, deduce which is a consumer, which a detritivore and which a saprotroph. [4]

 Figure 7

Ghost orchid: grows underground in woodland, eeding of dead organic matter, occasionally growing a stem with owers above ground

204

 Figure 8

Euglena: unicell that lives in ponds, using its chloroplasts or photosynthesis, but also ingesting dead organic matter by endocytosis

 Figure 6 Venus y

trap: grows in swamps, with green leaves that carry out photosynthesis and also catch and digest insects, to provide a supply o nitrogen

 Figure 9

Dodder: grows parasitically on gorse bushes, using small root-like structures to obtain sugars, amino acids and other substances it requires, rom the gorse

4 . 1 S P e c i e S , c o m m u n i t i e S a n d e c o S yS t e m S

csrs Consumers are heterotrophs that feed on living organisms by ingestion. Heterotrophs are divided into groups by ecologists according to the source o organic molecules that they use and the method o taking them in. O ne group o heterotrophs is called consumers. C onsumers eed o other organisms. These other organisms are either still alive or have only been dead or a relatively short time. A mosquito sucking blood rom a larger animal is a consumer that eeds on an organism that is still alive. A lion eeding o a gazelle that it has killed is a consumer.

 Figure 10

Red kite (Milvus milvus) is a consumer that feeds on live prey but also on dead animal remains (carrion)

C onsumers ingest their ood. This means that they take in undigested material rom other organisms. They digest it and absorb the products o digestion. Unicellular consumers such as Paramecium take the ood in by endocytosis and digest it inside vacuoles. Multicellular consumers such as lions take ood into their digestive system by swallowing it. C onsumers are sometimes divided up into trophic groups according to what other organisms they consume. Primary consumers eed on autotrophs; secondary consumers eed on primary consumers and so on. In practice, most consumers do not ft neatly into any one o these groups because their diet includes material rom a variety o trophic groups.

 Figure 11

Yellow-necked mouse (Apodemus favicollis) is a consumer that feeds mostly on living plant matter, especially seeds, but also on living invertebrates

drvrs

Sprrphs

Detritivores are heterotrophs that obtain organic nutrients from detritus by internal digestion.

Saprotrophs are heterotrophs that obtain organic nutrients from dead organic matter by external digestion.

O rganisms discard large quantities o organic matter, or example:

Saprotrophs secrete digestive enzymes into the dead organic matter and digest it externally. They then absorb the products o digestion. Many types o bacteria and ungi are saprotrophic. They are also known as decomposers because they break down carbon compounds in dead organic matter and release elements such as nitrogen into the ecosystem so that they can be used again by other organisms.



dead leaves and other parts o plants



eathers, hairs and other dead parts o animal bodies



eces rom animals.

This dead organic matter rarely accumulates in ecosystems and instead is used as a source o nutrition by two groups o heterotroph  detritivores and saprotrophs. D etritivores ingest dead organic matter and then digest it internally and absorb the products o digestion. Large multicellular detritivores such as earthworms ingest the dead matter into their gut. Unicellular organisms ingest it into ood vacuoles. The larvae o dung beetles eed by ingestion o eces rolled into a ball by their parent.

 Figure 12

Saprotrophic fungi growing over the surfaces of dead leaves and decomposing them by secreting digestive enzymes

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TOK

Identifying modes of nutrition

to wh exen do he lssifion sysems (lbels nd egories) we use se limis o wh we pereive?

Classiying species as autotrophs, consumers, detritivores or saprotrophs rom a knowledge o their mode o nutrition.

There are innite ways to divide up our observations. Organisms can be organized in a number o ways by scientists: by morphology (physical similarity to other organisms) , phylogeny (evolutionary history) and niche (ecological role) . In everyday language, we classiy organisms such as domesticated or wild; dangerous or harmless; edible or toxic.

By answering a series o simple questions about an organisms mode o nutrition it is usually possible to deduce what trophic group it is in. These questions are presented here as a dichotomous key, which consists o a series o pairs o choices. The key works or unicellular and multicellular organisms but does not work or parasites such as tapeworms or ungi that cause diseases in plants. All multicellular autotrophs are photosynthetic and have chloroplasts containing chlorophyll. Feeds on living or recently killed organisms = CONSUMERS

Feeds on dead organic matter = DETRITIVORES

Either ingests organic matter by endocytosis (no cell walls) or by taking it into its gut.

aiviy

START HERE

cleruing

Cell walls present. No ingestion of organic matter. No gut.

 Figure 14

Secretes enzymes into its environment to digest dead organic matter = SAPROTROPHS

Enzymes not secreted. Only requires simple ions and compounds such as CO 2 = AUTOTROPHS

In a classic essay written in 1972, the physicist Philip Anderson stated this: The ability to reduce everything to simple fundamental laws does not imply the ability to start from those laws and reconstruct the universe. At each level of complexity entirely new properties appear. Clearcutting is the most common and economically protable orm o logging. It involves clearing every tree in an area so that no canopy remains. With reerence to the concept o emergent properties, suggest why the ecological community oten ails to recover ater clearcutting.

206

communiies A community is ormed by populations o diferent species living together and interacting with each other. An important part o ecology is research into relationships between organisms. These relationships are complex and varied. In some cases the interaction between two species is o benet to one species and harms the other, or example the relationship between a parasite and its host. In other cases both species benet, as when a hummingbird eeds on nectar rom a fower and helps the plant by pollinating it. All species are dependent on relationships with other species or their long- term survival. For this reason a population o one species can never live in isolation. Groups o populations live together. A group

4 . 1 S P e c i e S , c o m m u n i t i e S a n d e c o S yS t e m S o populations living together in an area and interacting with each other is known in ecology as a community. Typical communities consist o hundreds or even thousands o species living together in an area.

 Figure 13

A coral reef is a complex community with many interactions between the populations. Most corals have photosynthetic unicellular algae called zooxanthellae living inside their cells

Field work  associations between species Testing for association between two species using the chi-squared test with data obtained by quadrat sampling. Quadrats are square sample areas, usually marked out using a quadrat rame. Quadrat sampling involves repeatedly placing a quadrat rame at random positions in a habitat and recording the numbers o organisms present each time.



The quadrat is placed precisely at the distances determined by the two random numbers.

I this procedure is ollowed correctly, with a large enough number o replicates, reliable estimates o

The usual procedure or randomly positioning quadrats is this: 

A base line is marked out along the edge o the habitat using a measuring tape. It must extend all the way along the edge o the habitat.



Random numbers are obtained using either a table or a random number generator on a calculator.



A frst random number is used to determine a distance along the measuring tape. All distances along the tape must be equally likely.



A second random number is used to determine a distance out across the habitat at right angles to the tape. All distances across the habitat must be equally likely.

 Figure 15 Quadrat sampling of seaweed

populations on a

rocky shore

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population sizes are obtained. The method is only suitable or plants and other organisms that are not motile. Quadrat sampling is not suitable or populations o most animals, or obvious reasons. I the presence or absence o more than one species is recorded in every quadrat during sampling o a habitat, it is possible to test or an association between species. Populations are oten unevenly distributed because some parts o the habitat are more suitable or a species than others. I two species occur in the same parts o a habitat, they will tend to be ound in the same quadrats. This is known as a positive association. There can also be negative associations, or the distribution o two species can be independent. There are two possible hypotheses:

2

C alculate the expe cted requencies, assuming indepe ndent distribution, or each o the our species combinations. E ach e xpe cted requency is calculated rom value s on the contingency table using this equation: row total  column total expected =  ___ grand total requency

3

C alculate the number o degrees o reedom using this equation. degrees o reedom = ( m  1 ) ( n  1 ) where m and n are the numbe r o rows and number o columns in the contingency table.

4

Find the critical region or chi- squared rom a table o chi- squared values, using the degrees o reedom that you have calculated and a signifcance level ( p) o 0.05 ( 5 % ) . The critical region is any value o chi-squared larger than the value in the table.

5

C alculate chi-squared using this equation:

H 0 : two species are distributed independently ( the null hypothesis) . H 1 : two species are associated ( either positively so they tend to occur together or negatively so they tend to occur apart) . We can test these hypotheses using a statistical procedure  the chi- squared test.

( fo - fe) 2 X2 =  _ fe

The chi- squared test is only valid i all the expected requencies are 5 or larger and the sample was taken at random rom the population.

where fo is the observed requency fe is the expected requency and

Method for chi-squared test 1

 is the sum o.

Draw up a contingency table o observed requencies, which are the numbers o quadrats containing or not containing the two species.

Species A present

Species A absent

6

C ompare the calculated value o chi- squared with the critical region. 

I the calculated value is in the critical region, there is evidence at the 5 % level or an association between the two species. We can rej ect the hypothesis H 0 .



I the calculated value is not in the critical region, because it is equal or below the value obtained rom the table o chisquared values, H 0 is not rej ected. There is no evidence at the 5 % level or an association between the two species.

Row totals

Species B present Species B absent Column totals C alculate the row and column totals. Adding the row totals or the column totals should give the same grand total in the lower right cell.

208

4 . 1 S P e c i e S , c o m m u n i t i e S a n d e c o S yS t e m S

d-bs qss: Chi-squared testing Figure 1 6 shows an area on the summit o C aer C aradoc, a hill in S hropshire, E ngland. The area is grazed by sheep in summer and hill walkers cross it on grassy paths. There are raised hummocks with heather (Calluna vulgaris) growing in them. A visual survey o this site suggested that Rhytidiadelphus squarrosus, a species o moss growing in this area, was associated with these heather hummocks. The presence or absence o the heather and the moss was recorded in a sample o 1 00 quadrats, positioned randomly.

Results Sps

Frq

Heather only

9

Moss only

7

Both species

57

Neither species

27

3

Calculate the number o degrees o reedom. [2 ]

4

Find the critical region or chi- squared at a signifcance level o 5 % . [2 ]

5

C alculate chi- squared.

6

S tate the two alternative hypotheses, H 0 and H 1 , and evaluate them using the calculated value or chi- squared. [4]

7

Suggest ecological reasons or an association between the heather and the moss. [4]

8

Explain the methods that should have been used to position quadrats randomly in the area o study. [3 ]

[4]

Questions 1

C onstruct a contingency table o observed values. [4]

2

C alculate the expected values, assuming no association between the species. [4]

 Figure 16 Caer Caradoc, Shropshire

Statistical signifcance Recognizing and interpreting statistical signifcance. B iologists oten use the phrase statistically signifcant when discussing results o an experiment. This reers to the outcome o a statistical hypothesis test. There are two alternative types o hypothesis: 



H 0 is the null hypothesis and is the belie that there is no relationship, or example that two means are equal or that there is no association or correlation between two variables. H 1 is the alternative hypothesis and is the belie that there is a relationship, or example that two means are dierent or that there is an association between two variables.

The usual procedure is to test the null hypothesis, with the expectation o showing

that it is alse. A statistic is calculated using the results o the research and is compared with a range o possible values called the critical region. I the calculated statistic exceeds the critical region, the null hypothesis is considered to be alse and is thereore rej ected, though we cannot say that this has been proved with certainty. When a biologist states that results were statistically signifcant it means that i the null hypothesis ( H 0 ) was true, the probability o getting results as extreme as the observed results would be very small. A decision has to be made about how small this probability needs to be. This is known as the signifcance level. It is the cut- o point or the probability o rej ecting the null

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hypothesis when in act it was true. A level o 5 % is oten chosen, so the probability is less than one in twenty. That is the minimum acceptable signicance level in published research. 

I there is a dierence between the mean results or the two treatments in an experiment, a statistical test will show whether the dierence is signicant at the 5 % level. I it is, there is a less than 5 % probability o such a large dierence between the sample means arising by chance, even when the population means are equal. We say that there is statistically signicant evidence that the population means dier.



In the example o testing or an association between two species, described on previous pages, the chi-squared test shows whether there is a less than 5 % probability o the dierence between the observed and the expected results being as large as it is without the species being either positively or negatively associated.

When results o biological research are displayed on a bar chart, letters are oten used to indicate statistical signicance. Two dierent letters, usually a and b, indicate mean results with a statistically signicant dierence. Two o the same letter such as a and a indicates that any dierence is not statistically signicant.

Ecosystems A community forms an ecosystem by its interactions with the abiotic environment. A community is composed o all organisms living in an area. These organisms could not live in isolation  they depend on their nonliving surroundings o air, water, soil or rock. Ecologists reer to these surroundings as the abiotic environment. In some cases the abiotic environment exerts a powerul infuence over the organisms. For example the wave action on a rocky shore creates a very specialized habitat and only organisms adapted to it can survive. On clis, the rock type determines whether there are ledges on which birds can nest. There are also many cases where living organisms infuence the abiotic environment. Sand dunes are an example o this. They develop along coasts where sand is blown up the shore and specialized plants grow in the loose wind-blown sand. The roots o these plants stabilize the sand and their leaves break the wind and encourage more sand to be deposited. So, not only are there complex interactions within communities, there are also many interactions between organisms and the abiotic environment. The community o organisms in an area and their non-living environment can thereore be considered to be a single highly complex interacting system, known as an ecosystem. Ecologists study both the components o ecosystems and the interactions between them.

inorganc nutrents Autotrophs and heterotrophs obtain inorganic nutrients from the abiotic environment. Living organisms need a supply o chemical elements:  Figure 17

Grasses in an area of developing sand dunes

210



C arbon, hydrogen and oxygen are needed to make carbohydrates, lipids and other carbon compounds on which lie is based.

4 . 1 S P e c i e S , c o m m u n i t i e S a n d e c o S yS t e m S



Nitrogen and phosphorus are also needed to make many o these compounds.



Approximately fteen other elements are needed by living organisms. S ome o them are used in minute traces only, but they are nonetheless essential.

Autotrophs obtain all o the elements that they need as inorganic nutrients rom the abiotic environment, including carbon and nitrogen. Heterotrophs on the other hand obtain these two elements and several others as part o the carbon compounds in their ood. They do however obtain other elements as inorganic nutrients rom the abiotic environment, including sodium, potassium and calcium.

nr ls The supply of inorganic nutrients is maintained by nutrient cycling. There are limited supplies on Earth o chemical elements. Although living organisms have been using the supplies or three billion years, they have not run out. This is because chemical elements can be endlessly recycled. O rganisms absorb the elements that they require as inorganic nutrients rom the abiotic environment, use them and then return them to the environment with the atoms unchanged. Recycling o chemical elements is rarely as simple as shown in this diagram and oten an element is passed rom organism to organism beore it is released back into the abiotic environment. The details vary rom element to element. The carbon cycle is dierent rom the nitrogen cycle or example. E cologists reer to these schemes collectively as nutrient cycles. The word nutrient is oten ambiguous in biology but in this context it simply means an element that an organism needs. The carbon cycle is described as an example o a nutrient cycle in subtopic 4.2 and the nitrogen cycle in O ption C .

Reserves of an element in the abiotic environment

Element forming part of a living organism

Ssbl f sss Ecosystems have the potential to be sustainable over long periods of time. The concept o sustainability has risen to prominence recently because it is clear that some current human uses o resources are unsustainable. S omething is sustainable i it can continue indefnitely. Human use o ossil uels is an example o an unsustainable activity. Supplies o ossil uels are fnite, are not currently being renewed and cannot thereore carry on indefnitely. Natural ecosystems can teach us how to live in a sustainable way, so that our children and grandchildren can live as we do. There are three requirements or sustainability in ecosystems: 

nutrient availability



detoxifcation o waste products



energy availability.

 Figure 18 Living organisms have been recycling

for billions of years

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41

E c o lo g y Nutrients can be recycled indefnitely and i this is done there should not be a lack o the chemical elements on which lie is based. The waste products o one species are usually exploited as a resource by another species. For example, ammonium ions released by decomposers are absorbed and used or an energy source by Nitrosomonas bacteria in the soil. Ammonium is potentially toxic but because o the action o these bacteria it does not accumulate.

 Figure 19

Sunlight supplies energy to a forest ecosystem and nutrients are recycled

ativity

E nergy cannot be recycled, so sustainability depends on continued energy supply to ecosystems. Most energy is supplied to ecosystems as light rom the sun. The importance o this supply can be illustrated by the consequences o the eruption o Mount Tambora in 1 81 5 . D ust in the atmosphere reduced the intensity o sunlight or some months aterwards, causing crop ailures globally and deaths due to starvation. This was only a temporary phenomenon, however, and energy supplies to ecosystems in the orm o sunlight will continue or billions o years.

cve eosystems Organisms have been ound living in total darkness in caves, including eyeless fsh. Discuss whether ecosystems in dark caves are sustainable. Figure 20 shows a small ecosystem with photosynthesizing plants near artifcial lighting in a cave that is open to visitors in Cheddar Gorge. Discuss whether this is more or less sustainable than ecosystems in dark caves.

Mesocosms Setting up sealed mesocosms to try to establish sustainability. (Practical 5) Mesocosms are small experimental areas that are set up as ecological experiments. Fenced- o enclosures in grassland or orest could be used as terrestrial mesocosms; tanks set up in the laboratory can be used as aquatic mesocosms. E cological experiments can be done in replicate mesocosms, to fnd out the eects o varying one or more conditions. For example, tanks could be set up with and without fsh, to investigate the eects o fsh on aquatic ecosystems. Another possible use o mesocosms is to test what types o ecosystems are sustainable. This involves sealing up a community o organisms together with air and soil or water inside a container. You should consider these questions beore setting up either aquatic or terrestrial mesocosms:

 Figure 20

212



Large glass j ars are ideal but transparent plastic containers could also be used. S hould the sides o the container be transparent or opaque?



Which o these groups o organisms must be included to make up a sustainable community: autotrophs, consumers, saprotrophs and detritivores?



How can we ensure that the oxygen supply is sufcient or all the organisms in the mesocosm as once it is sealed, no more oxygen will be able to enter.



How can we prevent any organisms suering as a result o being placed in the mesocosm?

4. 2 e n erG y Flo w

4.2 eg f Understanding  Most ecosystems rely on a supply o energy       

rom sunlight. Light energy is converted to chemical energy in carbon compounds by photosynthesis. Chemical energy in carbon compounds fows through ood chains by means o eeding. Energy released by respiration is used in living organisms and converted to heat. Living organisms cannot convert heat to other Nature of science orms o energy. Experimental design: accurate quantitative Heat is lost rom ecosystems. measurements in osmosis experiments Energy losses between trophic levels restrict are essential. the length o ood chains and the biomass o higher trophic levels.

Skills  Quantitative representations o energy fow

using pyramids o energy.

Nature of science  Use theories to explain natural phenomena:

the concept o energy fow explains the limited length o ood chains.

Sunlight and ecosystems Most ecosystems rely on a supply o energy rom sunlight. For most biological communities, the initial source of energy is sunlight. Living organisms can harvest this energy by photosynthesis. Three groups of autotroph carry out photosynthesis: plants, eukaryotic algae including seaweeds that grow on rocky shores, and cyanobacteria. These organisms are often referred to by ecologists as producers. Heterotrophs do not use light energy directly, but they are indirectly dependent on it. There are several groups of heterotroph in ecosystems: consumers, saprotrophs and detritivores. All of them use carbon compounds in their food as a source of energy. In most ecosystems all or almost all energy in the carbon compounds will originally have been harvested by photosynthesis in producers. The amount of energy supplied to ecosystems in sunlight varies around the world. The percentage of this energy that is harvested by producers and therefore available to other organisms also varies. In the S ahara D esert, for example, the intensity of sunlight is very high but little of it becomes available to organisms because there are very few producers. In the redwood forests of C alifornia the intensity of sunlight is less than in the S ahara but much more energy becomes available to organisms because producers are abundant.

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ativity cynobteri in ves Cyanobacteria are photosynthetic bacteria that are oten very abundant in marine and reshwater ecosystems. Figure 1 shows an area o green cyanobacteria on an area o wall in a cave that is illuminated by artifcial light. The surrounding areas are normally dark. I the artifcial light was not present, what other energy sources could be used by bacteria in caves?

dt-bse questions: Insolation Insolation is a measure o solar radiation The two maps in fgure 2 show annual mean insolation at the top o the Earths atmosphere (upper map) and at the Earths surace (lower map) .

Questions 1

State the relationship between distance rom the equator and insolation at the top o the Earths atmosphere. [1 ]

2

S tate the mean annual insolation in Watts per square metre or the most northerly part o Australia

3

4

a) at the top o the atmosphere

[1 ]

b) at the Earths surace.

[1 ]

S uggest reasons or dierences in insolation at the Earths surace between places that are at the same distance rom the equator.

[2 ]

Tropical rainorests are ound in equatorial regions o all continents. They have very high rates o photosynthesis. Evaluate the hypothesis that this is due to very high insolation. Include named parts o the world in your answer.

[5 ]

 Figure 1

0

40

 Figure 2

214

80

120

160

200

240

280

320

360

400 w/m 2

4. 2 e n erG y Flo w

Energy conversion Light energy is converted to chemical energy in carbon compounds by photosynthesis.

activit Bush d st fs

Producers absorb sunlight using chlorophyll and other photosynthetic pigments. This converts the light energy to chemical energy, which is used to make carbohydrates, lipids and all the other carbon compounds in producers. Producers can release energy rom their carbon compounds by cell respiration and then use it or cell activities. Energy released in this way is eventually lost to the environment as waste heat. However, only some o the carbon compounds in producers are used in this way and the largest part remains in the cells and tissues o producers. The energy in these carbon compounds is available to heterotrophs.

Energy in food chains Chemical energy in carbon compounds fows through ood chains by means o eeding. A ood chain is a sequence o organisms, each o which eeds on the previous one. There are usually between two and ve organisms in a ood chain. It is rare or there to be more organisms in the chain. As they do not obtain ood rom other organisms, producers are always the rst organisms in a ood chain. The subsequent organisms are consumers. Primary consumers eed on producers; secondary consumers eed on primary consumers; tertiary consumers eed on secondary consumers, and so on. No consumers eed on the last organism in a ood chain. Consumers obtain energy rom the carbon compounds in the organisms on which they eed. The arrows in a ood chain thereore indicate the direction o energy fow.

 Figure 3

Figure 3 shows a bush re in Australia. What energy conversion is happening in a bush re? Bush and orest res occur naturally in some ecosystems. Suggest two reasons or this hypothesis: There are ewer heterotrophs in ecosystems where res are common compared to ecosystems where res are not common.

Figure 4 is an example o a ood chain rom the orests around Iguazu alls in northern Argentina.



Figure 4

Respiration and energy release Energy released by respiration is used in living organisms and converted to heat. Living organisms need energy or cell activities such as these: 

Synthesizing large molecules like D NA, RNA and proteins.



Pumping molecules or ions across membranes by active transport.



Moving things around inside the cell, such as chromosomes or vesicles, or in muscle cells the protein bres that cause muscle contraction.

ATP supplies energy or these activities. Every cell produces its own ATP supply.

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E c o lo g y All cells can produce ATP by cell respiration. In this process carbon compounds such as carbohydrates and lipids are oxidized. These oxidation reactions are exothermic and the energy released is used in endothermic reactions to make ATP. So cell respiration transers chemical energy rom glucose and other carbon compounds to ATP. The reason or doing this is that the chemical energy in carbon compounds such as glucose is not immediately usable by the cell, but the chemical energy in ATP can be used directly or many dierent activities. The second law o thermodynamics states that energy transormations are never 1 00% efcient. Not all o the energy rom the oxidation o carbon compounds in cell respiration is transerred to ATP. The remainder is converted to heat. S ome heat is also produced when ATP is used in cell activities. Muscles warm up when they contract or example. Energy rom ATP may reside or a time in large molecules when they have been synthesized, such as D NA and proteins, but when these molecules are eventually digested the energy is released as heat.

data-base questions

a) D escribe the relationship between external temperature and respiration rate in yellowbilled magpies. [3 ] b) Explain the change in respiration rate as temperature drops rom + 1 0 C to 1 0 C . c) S uggest a reason or the change in respiration rate as temperature increased rom 3 0 C to 40 C .

respiration rate (mW g1 )

20

Figure 5 shows the results o an experiment in which yellow- billed magpies (Pica nuttalli) were put in a cage in which the temperature could be controlled. The birds rate o respiration was measured at seven dierent temperatures, rom 1 0 C to + 40 C . B etween 1 0 C and 3 0 C the magpies maintained constant body temperature, but above 3 0 C body temperature increased.

15

10

5

0

-10

0

10 20 30 temperature (C)

40

50

 Figure 5 Cell

[3 ]

[2 ]

respiration rates at diferent temperatures in yellow-billed magpies

d) S uggest two reasons or the variation in respiration rate between the birds at each temperature.

[2 ]

Heat energy in ecosystems Living organisms cannot convert heat to other forms of energy. Living organisms can perorm various energy conversions: 

Light energy to chemical energy in photosynthesis.



C hemical energy to kinetic energy in muscle contraction.



C hemical energy to electrical energy in nerve cells.



C hemical energy to heat energy in heat-generating adipose tissue.

They cannot convert heat energy into any other orm o energy.

216

4. 2 e n erG y Flo w

Heat losses from ecosystems Heat is lost rom ecosystems. Heat resulting rom cell respiration makes living organisms warmer. This heat can be useul in making cold-blooded animals more active. B irds and mammals increase their rate o heat generation i necessary to maintain their constant body temperatures. According to the laws o thermodynamics in physics, heat passes rom hotter to cooler bodies, so heat produced in living organisms is all eventually lost to the abiotic environment. The heat may remain in the ecosystem or a while, but ultimately is lost, or example when heat is radiated into the atmosphere. Ecologists assume that all energy released by respiration or use in cell activities will ultimately be lost rom an ecosystem.

acivi thikig bu g chgs What energy conversions are required to shoot a basketball? What is the nal orm o the energy?

expiig h gh f fd chis Use theories to explain natural phenomena: the concept o energy fow explains the limited length o ood chains. I we consider the diet o a top carnivore that is at the end o a ood chain, we can work out how many stages there are in the ood chain leading up to it. For example, i an osprey eeds on sh such as salmon that ed on shrimps, which ed on phytoplankton, there are our stages in the ood chain. There are rarely more than our or ve stages in a ood chain. We might expect ood chains to be limitless, with one species being eaten by another ad innitum. This does not happen. In ecology, as in all branches o science, we try to explain natural phenomena such as the restricted length o ood chains using scientic theories. In this case it is the concept o energy fow along ood chains and the energy losses that occur between trophic levels that can provide an explanation.

Energy losses and ecosystems Energy losses between trophic levels restrict the length o ood chains and the biomass o higher trophic levels.

 Figure 6 An

inrared camera image o an Arican grey parrot (Psittacus erithacus) shows how much heat is being released to the environment by dierent parts o its body

Biomass is the total mass o a group o organisms. It consists o the cells and tissues o those organisms, including the carbohydrates and other carbon compounds that they contain. Because carbon compounds have chemical energy, biomass has energy. Ecologists can measure how much energy is added per year by groups o organisms to their biomass. The results are calculated per square metre o the ecosystem so that dierent trophic levels can be compared. When this is done, the same trend is always ound: the energy added to biomass by each successive trophic level is less. In secondary consumers, or example, the amount o energy is always less per year per square metre o ecosystem than in primary consumers. The reason or this trend is loss o energy between trophic levels. 

Most o the energy in ood that is digested and absorbed by organisms in a trophic level is released by them in respiration or

 Figure 7

The osprey (Pandion halietus) is a fsh-eating top carnivore

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E c o lo g y use in cell activities. It is thereore lost as heat. The only energy available to organisms in the next trophic level is chemical energy in carbohydrates and other carbon compounds that have not been used up in cell respiration.

activity Slmon nd soy Most salmon eaten by humans is produced in sh arms. The salmon have traditionally been ed on sh meal, mostly based on anchovies harvested o the coast o South America. These have become scarce and expensive. Feeds based on plant products such as soy beans are increasingly being used. In terms o energy ow, which o these human diets is most and least efcient? 1 Salmon ed on sh meal 2 Salmon ed on soy beans 3 Soy beans.



The organisms in a trophic level are not usually entirely consumed by organisms in the next trophic level. For example, locusts sometimes consume all the plants in an area but more usually only parts o some plants are eaten. Predators may not eat material rom the bodies o their prey such as bones or hair. E nergy in uneaten material passes to saprotrophs or detritivores rather than passing to organisms in the next trophic level.



Not all parts o ood ingested by the organisms in a trophic level are digested and absorbed. Some material is indigestible and is egested in eces. Energy in eces does not pass on along the ood chain and instead passes to saprotrophs or detritivores.

B ecause o these losses, only a small proportion o the energy in the biomass o organisms in one trophic level will ever become part o the biomass o organisms in the next trophic level. The fgure o 1 0%  is oten quoted, but the level o energy loss between trophic levels is variable. As the losses occur at each stage in a ood chain, there is less and less energy available to each successive trophic level. Ater only a ew stages in a ood chain the amount o energy remaining would not be enough to support another trophic level. For this reason the number o trophic levels in ood chains is restricted. B iomass, measured in grams, also diminishes along ood chains, due to loss o carbon dioxide and water rom respiration and loss rom the ood chain o uneaten or undigested parts o organisms. The biomass o higher trophic levels is thereore usually smaller than that o lower levels. There is generally a higher biomass o producers, the lowest trophic level o all, than o any other trophic level.

decomposers (16,000 kJ m 2 yr1 )

secondary consumer (200 kJ m 2 yr1 ) primary consumer (2,500 kJ m 2 yr1 )

plankton (150,000 kJ m 2 yr1 )  Figure 8 An

energy pyramid for an aquatic ecosystem (not to scale)

secondary consumer (3,000 MJ m 2 yr1 ) primary consumer (7,000 MJ m 2 yr1 ) producers (50,000 MJ m 2 yr1 )  Figure 9

218

Pyramid of energy for grassland

Pyramids of energy Quantitative representations o energy ow using pyramids o energy. The amount o energy converted to new biomass by each trophic level in an ecological community can be represented with a pyramid o energy. This is a type o bar chart with a horizontal bar or each trophic level. The amounts o energy should be per unit area per year. Oten the units are kilojoules per metre squared per year (kJ m -2 yr-1 ) . The pyramid should be stepped, not triangular, starting with the producers in the lowest bar. The bars should be labelled producer, frst consumer, second consumer and so on. I a suitable scale is chosen, the length o each bar can be proportional to the amount o energy that it shows. Figure 8 shows an example o a pyramid o energy or an aquatic ecosystem. To be more accurate, the bars should be drawn with relative widths that match the relative energy content at each trophic level. Figure 9 shows a pyramid o energy or grassland, with the bars correctly to scale.

4. 2 e n erG y Flo w

dt-bs qustis: a simple food web A sinkhole is a surace eature which orms when an underground cavern collapses. Montezuma Well in the Sonoran desert in Arizona is a sinkhole flled with water. It is an aquatic ecosystem that lacks fsh, due in part to the extremely high concentrations o dissolved C O 2 . The dominant top predator is Belostoma bakeri, a giant water insect that can grow to 70 mm in length. Figure 1 0 shows a ood web or Montezuma Well. 1 2 3

4 5 6 7

C ompare the roles o Belostoma bakeri and Ranatra montezuma within the ood web.

[2 ]

D educe, with a reason, which organism occupies more than one trophic level.

[2 ]

D educe using P values: a) what would be the most common ood chain in this web

[2 ]

b) what is the preerred prey o B. bakeri?

[1 ]

C onstruct a pyramid o energy or the frst and second trophic levels.

[3 ]

C alculate the percentage o energy lost between the frst and second trophic levels.

[2 ]

D iscuss the difculties o classiying organisms into trophic levels.

[2 ]

Outline the additional inormation that would be required to complete the pyramid o energy or the third and ourth trophic level.

[1 ]

Ranatra montezuma 235,000 kJ ha 1 yr1 P = 1.0 gm 2 yr1

Belostoma bakeri 588,000 kJ ha 1 yr1 P = 2.8 gm 2 yr1

Telebasis salva 1,587,900 kJ ha 1 yr1 P = 7.9 gm 2 yr1

Hyalella montezuma 30,960,000 kJ ha 1 yr1 P = 215 gm 2 yr1

phytoplankton - Metaphyton 234,342,702 kJ ha 1 yr1 P = 602 g C m 2 yr1

piphyton 427,078,320 kJ ha 1 yr1 P = 1,096 g C m 2 yr1

 Figure 10

A food web for Montezuma Well. P values represent the biomass stored in the population of that organism each year. Energy values represent the energy equivalent of that biomass. Arrows indicate trophic linkages and arrow thickness indicates the relative amount of energy transferred between trophic levels

219

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E c o lo g y

4.3 carbon yling Understanding

Appliations

 Autotrophs convert carbon dioxide into   



 



 

carbohydrates and other carbon compounds. In aquatic habitats carbon dioxide is present as a dissolved gas and hydrogen carbonate ions. Carbon dioxide diuses rom the atmosphere or water into autotrophs. Carbon dioxide is produced by respiration and diuses out o organisms into water or the atmosphere. Methane is produced rom organic matter in anaerobic conditions by methanogenic archaeans and some diuses into the atmosphere. Methane is oxidized to carbon dioxide and water in the atmosphere. Peat orms when organic matter is not ully decomposed because o anaerobic conditions in waterlogged soils. Partially decomposed organic matter rom past geological eras was converted into oil and gas in porous rocks or into coal. Carbon dioxide is produced by the combustion o biomass and ossilized organic matter. Animals such as ree-building corals and molluscs have hard parts that are composed o calcium carbonate and can become ossilized in limestone.

 Estimation o carbon fuxes due to processes in

the carbon cycle.  Analysis o data rom atmosphere monitoring stations showing annual fuctuations.

Skills  Construct a diagram o the carbon cycle.

Nature o siene  Making accurate, quantitative measurements:

it is important to obtain reliable data on the concentration o carbon dioxide and methane in the atmosphere.

carbon fxation Autotrophs convert carbon dioxide into carbohydrates and other carbon compounds. Autotrophs absorb carbon dioxide from the atmosphere and convert it into carbohydrates, lipids and all the other carbon compounds that they require. This has the effect of reducing the carbon dioxide concentration of the atmosphere. The mean C O 2 concentration of the atmosphere is currently approximately 0.03 9% or 3 90 micromoles per mole ( mol/mol) but it is lower above parts of the Earths surface where photosynthesis rates have been high.

220

4 . 3 c ar B o n c ycli n G

dt-bse quests: Carbon dioxide concentration The two maps in fgure 1 were produced by NAS A. They show the carbon dioxide concentration o the atmosphere eight kilometres above the surace o the E arth, in May and O ctober 2 01 1 . 1

S tate whether O ctober is in the spring or all( autumn) in the southern hemisphere. [1 ]

2

a) D istinguish between carbon dioxide concentrations in May and O ctober in the northern hemisphere.

[1 ]

b) Suggest reasons or the dierence.

[2 ]

a) Distinguish between the carbon dioxide concentrations in May between the northern and the southern hemisphere.

[1 ]

b) S uggest reasons or the dierence.

[2 ]

3

4

a) D educe the part o the Earth that had the lowest mean carbon dioxide concentration between May and O ctober 2 01 1 . [1 ] b) S uggest reasons or the carbon dioxide concentration being lowest in this area. [2 ]

Figure 1

carbon dioxide in solution In aquatic habitats carbon dioxide is present as a dissolved gas and hydrogen carbonate ions. C arbon dioxide is soluble in water. It can either remain in water as a dissolved gas or it can combine with water to orm carbonic acid ( H 2 C O 3 ) . C arbonic acid can dissociate to orm hydrogen and hydrogen carbonate ions ( H + and HC O -3 ) . This explains how carbon dioxide can reduce the pH o water. B oth dissolved carbon dioxide and hydrogen carbonate ions are absorbed by aquatic plants and other autotrophs that live in water. They use them to make carbohydrates and other carbon compounds.

Absorption of arbon dioxide Carbon dioxide difuses rom the atmosphere or water into autotrophs. Autotrophs use carbon dioxide in the production o carbon compounds by photosynthesis or other processes. This reduces the concentration o carbon dioxide inside autotrophs and sets up a concentration gradient between cells in autotrophs and the air or water around. C arbon dioxide thereore diuses rom the atmosphere or water into autotrophs. In land plants with leaves this diusion usually happens through stomata in the underside o the leaves. In aquatic plants the entire surace o the leaves and stems is usually permeable to carbon dioxide, so diusion can be through any part o these parts o the plant.

atvt pH hges  k ps Ecologists have monitored pH in rock pools on sea shores that contain animals and also photosynthesizing algae. The pH o the water rises and alls in a 24-hour cycle, due to changes in carbon dioxide concentration in the water. The lowest values o about pH 7 have been ound during the night, and the highest values o about pH 10 have been ound when there was bright sunlight during the day. What are the reasons or these maxima and minima? The pH in natural pools or articial aquatic mesocosms could be monitored using data loggers.

221

41

E c o lo g y

Release of carbon dioxide from cell respiration Carbon dioxide is produced by respiration and difuses out o organisms into water or the atmosphere. C arbon dioxide is a waste product o aerobic cell respiration. It is produced in all cells that carry out aerobic cell respiration. These can be grouped according to trophic level o the organism: 

non-photosynthetic cells in producers or example root cells in plants



animal cells



saprotrophs such as ungi that decompose dead organic matter.

C arbon dioxide produced by respiration diuses out o cells and passes into the atmosphere or water that surrounds these organisms.

data-base questions: Data-logging pH in an aquarium

1

E xplain the changes in light intensity during the experiment. [2 ]

2

D etermine how many days the data logging covers.

[2 ]

a) D educe the trend in pH in the light.

[1 ]

b) Explain this trend.

[2 ]

3

pH sensor (pH) 7.50

100

light intensity pH

90

7.45

80 70

7.40

60 50

7.35

40 30

7.30

20

light intensity /arbitrary units

Figure 2 shows the pH and light intensity in an aquarium containing a varied community o organisms including pondweeds, newts and other animals. The data was obtained by data logging using a pH electrode and a light meter. The aquarium was illuminated articially to give a 24-hour cycle o light and dark using a lamp controlled by a timer.

10 7.25 0.14:02:31 0.23:13:11 3.08:23:50 4.17:34:30 06 February 2013 14:02:31 absolute time (d.hh:mm:ss)

0 6.02:45:09

Figure 2 4

a) D educe the trend in pH in darkness.

[1 ]

b) Explain this trend.

[2 ]

Methanogenesis Methane is produced rom organic matter in anaerobic conditions by methanogenic archaeans and some difuses into the atmosphere. In 1 776 Alessandro Volta collected bubbles o gas emerging rom mud in a reed bed on the margins o Lake Maggiore in Italy, and ound that it was infammable. He had discovered methane, though Volta did not give it this name. Methane is produced widely in anaerobic environments, as it is a waste product o a type o anaerobic respiration. Three dierent groups o anaerobic prokaryotes are involved. 1

222

B acteria that convert organic matter into a mixture o organic acids, alcohol, hydrogen and carbon dioxide.

4 . 3 c ar B o n c ycli n G

2

B acteria that use the organic acids and alcohol to produce acetate, carbon dioxide and hydrogen.

3

Archaeans that produce methane rom carbon dioxide, hydrogen and acetate. They do this by two chemical reactions: C O 2 + 4H 2  C H 4 + 2 H 2 O C H3C O O H  C H4 + C O 2

The archaeans in this third group are thereore methanogenic. They carry out methanogenesis in many anaerobic environments: 

Mud along the shores and in the bed o lakes.



Swamps, mires, mangrove orests and other wetlands where the soil or peat deposits are waterlogged.



Guts o termites and o ruminant mammals such as cattle and sheep.



Landfll sites where organic matter is in wastes that have been buried.

S ome o the methane produced by archaeans in these anaerobic environments diuses into the atmosphere. C urrently the concentration in the atmosphere is between 1 .7 and 1 .85 micromoles per mole. Methane produced rom organic waste in anaerobic digesters is not allowed to escape and instead is burned as a uel.

Figure 3 Waterlogged woodlanda typical habitat for methanogenic prokaryotes

oxidatin f methane Methane is oxidized to carbon dioxide and water in the atmosphere. Molecules o methane released into the atmosphere persist there on average or only 1 2 years, because it is naturally oxidized in the stratosphere. Monatomic oxygen ( O ) and highly reactive hydroxyl radicals ( O H  ) are involved in methane oxidation. This explains why atmospheric concentrations are not high, despite large amounts o production o methane by both natural processes and human activities.

Peat frmatin Peat forms when organic matter is not fully decomposed because of anaerobic conditions in waterlogged soils. In many soils all o rganic matte r such as de ad le ave s rom plants is e ve ntually dige ste d by saprotrophic b acte ria and ungi. S apro trop hs o btain the oxygen that they ne ed or re spiration rom air spaces in the so il. In some e nvironments water is unable to drain o ut o  so ils so they be co me wate rlogged and anae rob ic. S ap rotrophs cannot thrive in these co nditions so de ad organic matter is not ully deco mposed. Acidic conditions te nd to de ve lo p, urthe r inhib iting sapro trop hs and also me thanogens that might b re ak down the o rganic matte r.

Figure 4 Peat deposits form a blanket on a boggy hill top at Bwlch Groes in North Wales

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E c o lo g y

data-base questions: Release of carbon from tundra soils Soils in tundra ecosystems typically contain large amounts o carbon in the orm o peat. This accumulates because o low rates o decomposition o dead plant organic matter by saprotrophs. To investigate this, ecologists collected samples o soil rom areas o tussock vegetation near Toolik Lake in Alaska. Some o the areas had been ertilized with nitrogen and phosphorus every year or the previous eight years (TF) and some had not (TC ) . The soils were incubated or 1 00-day periods at

either 7 or 1 5C. Some samples were kept moist (M) and others were saturated with water (W) . The initial carbon content o the soils was measured and the amount o carbon dioxide given o during the experiment was monitored. The bar chart in fgure 5 shows the results. 1

S tate the eect o increasing the temperature o the soils on the rate o release o carbon.

b) Explain the reasons or this eect.

40 TC percentage of initial C

a)

30

2

TF

a)

7M

7W 15M treatment group

15W

[2 ]

3

O utline the eects o ertilizers on rates o release o carbon rom the soils. [2 ]

4

D iscuss whether dierences in temperature, amount o water in the soil or amount o ertilizer have the greatest impact on the release o carbon. [2 ]

10

0

[2 ]

C ompare the rates o release o carbon in moist soils with those in soils saturated with water. [2 ]

b) S uggest reasons or the dierences.

20

[2 ]

Figure 5 Large quantities o partially decomposed organic matter have accumulated in some ecosystems and become compressed to orm a dark brown acidic material called peat. About 3 % o the Earths land surace is covered by peat and as the depth is ten metres or more in some places, the total quantities o this material are immense.

Fossilized organic matter Partially decomposed organic matter from past geological eras was converted into oil and gas in porous rocks or into coal. C arbon and some compounds o carbon are chemically very stable and can remain unchanged in rocks or hundreds o millions o years. There are large deposits o carbon rom past geological eras. These deposits are the result o incomplete decomposition o organic matter and its burial in sediments that became rock. 

Figure 6 Coal at a power station

224

C oal is ormed when deposits o peat are buried under other sediments. The peat is compressed and heated, gradually turning into coal. Large coal deposits were ormed during the Pennsylvanian subperiod o the C arbonierous. There was a cycle o sea level rises and alls; coastal swamps ormed as the level ell and were destroyed and buried when the level rose and the sea spread inland. Each cycle has let a seam o coal.

4 . 3 c ar B o n c ycli n G



Oil and natural gas are ormed in the mud at the bottom o seas and lakes. C onditions are usually anaerobic and so decomposition is oten incomplete. As more mud or other sediments are deposited the partially decomposed matter is compressed and heated. C hemical changes occur, which produce complex mixtures o liquid carbon compounds or gases. We call these mixtures crude oil and natural gas. Methane orms the largest part o natural gas. Deposits are ound where there are porous rocks that can hold them such as shales and also impervious rocks above and below the porous rocks that prevent the deposits escape.

combustion Carbon dioxide is produced by the combustion of biomass and fossilized organic matter. I organic matter is heated to its ignition temperature in the presence o oxygen it will set light and burn. The oxidation reactions that occur are called combustion. The products o complete combustion are carbon dioxide and water.

Figure 7 Carbon dioxide is released by combustion of the leaves of sugar cane

In some parts o the world it is natural or there to be periodic fres in orests or grassland. C arbon dioxide is released rom the combustion o the biomass in the orest or grassland. In these areas the trees and other organisms are oten well adapted to fres and communities regenerate rapidly aterwards. In other areas fres due to natural causes are very unusual, but humans sometimes cause them to occur. Fire is used to clear areas o tropical rainorest or planting oil palms or or cattle ranching. C rops o sugar cane are traditionally burned shortly beore they are harvested. The dry leaves burn o, leaving the harvestable stems. C oal, oil and natural gas are dierent orms o ossilized organic matter. They are all burned as uels. The carbon atoms in the carbon dioxide released may have been removed rom the atmosphere by photosynthesizing plants hundreds o millions o years ago.

limestone

Figure 8 Kodonophylluma Silurian coral, in limestone from Wenlock Edge. The calcium carbonate skeletons of the coral are clearly visible embedded in more calcium carbonate that precipitated 420 million years ago in shallow tropical seas

Animals such as reef-building corals and molluscs have hard parts that are composed of calcium carbonate and can become fossilized in limestone. S ome animals have hard body parts composed o calcium carbonate ( C aC O 3 ) : 

mollusc shells contain calcium carbonate;



hard corals that build rees produce their exoskeletons by secreting calcium carbonate.

When these animals die, their sot parts are usually decomposed quickly. In acid conditions the calcium carbonate dissolves away but in neutral or alkaline conditions it is stable and deposits o it rom hard animal parts can orm on the sea bed. In shallow tropical seas calcium

Figure 9 Chalk cliffs on the south coast of England. Chalk is a form of limestone that consists almost entirely of 90-million-yearold shells of tiny unicellular animals called foraminifera

225

41

E c o lo g y carbonate is also deposited by precipitation in the water. The result is limestone rock, where the deposited hard parts o animals are oten visible as ossils. Approximately 1 0% o all sedimentary rock on Earth is limestone. About 1 2 % o the mass o the calcium carbonate is carbon, so huge amounts o carbon are locked up in limestone rock on Earth.

carbon yle diagrams Construct a diagram of the carbon cycle. Ecologists studying the carbon cycle and the recycling o other elements use the terms pool and fux. A pool is a reserve o the element. It can be organic or inorganic. For example the carbon dioxide in the atmosphere is an inorganic pool o carbon. The biomass o producers in an ecosystem is an organic pool.



A fux is the transer o the element rom one pool to another. An example o carbon fux is the absorption o carbon dioxide rom the atmosphere and its conversion by photosynthesis to plant biomass.



D iagrams can be used to represent the carbon cycle. Text boxes can be used or pools and labeled arrows or fuxes. Figure 1 0 shows an illustrated diagram which can be converted to a diagram o text boxes and arrows. Figure 1 0 only shows the carbon cycle or terrestrial ecosystems. A separate diagram could be constructed or marine or aquatic ecosystems, or a combined diagram or all ecosystems. In marine and aquatic ecosystems, the inorganic reserve o carbon is dissolved carbon dioxide and hydrogen carbonate, which is absorbed by producers and by various means is released back into the water.

CO 2 in atmosphere

fu e l s

cell respiration in saprotrophs and detritivores

ce in

pr

ll r

od

es

uc

pi r

er

at

tos

ynt

carbon in organic compounds in producers

co m b

feeding egestion

incomplete decomposition and fossilization of organic matter coal

Figure 10 Carbon cycle

226

oil

and

gas

is

ion

death

carbon in dead organic matter

hes

s

u stio

n of f ossil

cell respiration in consumers

pho

4 . 3 c ar B o n c ycli n G

carbon fuxes Estimation o carbon fuxes due to processes in the carbon cycle. The carbon cycle diagram in gure 1 0 shows processes that transer carbon rom one pool to another but it does not show the quantities o these fuxes. It is not possible to measure global carbon fuxes precisely but as these quantities are o great interest, scientists have produced estimates or them. Estimates are based on many measurements in individual natural ecosystems or in mesocosms.

Fux/ggtes e- 1 120 119.6 92.8 90.0 1.6

Pess Photosynthesis Cell respiration Ocean uptake Ocean loss Deorestation and land use changes Burial in marine sediments Combustion o ossil uels

Global carbon fuxes are extremely large so estimates are in gigatonnes (petagrams) . One gigatonne is 1 ,01 5 grams. Table 1 shows estimates based on Ocean Biogeochemical Dynamics, Sarmiento and Gruber, 2006, Princeton University Press.

0.2 6.4

Table 1

dt-bse quests: Oak woodland and carbon dioxide concentrations C arbon fuxes have been measured since 1 998 in deciduous woodland at Alice Holt Research Forest in E ngland. The trees are mainly oaks, Quercus robur and Quercus petraea, with some ash, Fraxinus excelsior. They were planted in 1 93 5 and are now nearly 2 0 metres tall. C arbon dioxide concentrations are measured 2 0 times a second. From these measurements the net ecosystem production can be deduced. This is the net fux o carbon dioxide between the orest and the atmosphere. Positive values indicate an increase in the carbon pool o the orest and negative values indicate a decrease due to net loss o carbon dioxide. The graph shows the daily average net ecosystem production or several years and also the cumulative net ecosystem production.

1

C alculate whether the carbon pool in the biomass o the orest increases or decreases on more days in the year. [1 ]

2

D educe the months in which the carbon pool o biomass in the orest was highest and lowest. [2 ]

3

Explain the reasons or increases in the carbon pool o biomass in the orest during part o the year and decreases in other parts.

4

State the annual carbon fux to or rom the orest. [2 ]

5

Suggest a reason based on the data or encouraging the planting o more oak orests.

[1 ]

25 20

15

15 10

10

5

5 0 0

50

100

150

200

250

300

530 5

cumulative NEP (t CO 2 ha 1 )

daily average NEP (kg CO 2 ha 1 h 1 )

20

0

[4]

5

10

10 day of year

15

227

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E c o lo g y

Environmental monitoring Making accurate, quantitative measurements: it is important to obtain reliable data on the concentration o carbon dioxide and methane in the atmosphere. C arbon dioxide and methane concentrations in the atmosphere have very important eects. C arbon dioxide concentrations aect photosynthesis rates and the pH o seawater. B oth gases infuence global temperatures and as a result the extent o ice sheets at the poles. Indirectly they thereore aect sea levels and the position o coast lines. Through their eects on the amount o heat energy in the oceans and the atmosphere they aect ocean currents, the distribution o rainall and also the requency and severity o extreme weather events such as hurricanes. C onsider these hypotheses and predictions: 

The carbon dioxide concentration o the atmosphere is currently higher than at any time in the past twenty million years.



Human activities have increased the carbon dioxide and methane concentrations in the Earths atmosphere.



Human activity will cause atmospheric

carbon dioxide concentrations to rise rom 3 97 micromoles per mole in 2 01 4 to a level above 600 by the end o the century. Reliable data are an essential prerequisite or evaluating hypotheses and predictions such as these. Reliable measurements o atmospheric carbon dioxide and methane concentration are needed over as long a period as possible beore we can evaluate the past and possible uture consequences o human activity. D ata on concentrations o gases in the atmosphere is collected by the Global Atmosphere Watch programme o the World Meteorological O rganization, an agency o the United Nations. Research stations in various parts o the world now monitor the atmosphere, but Mauna Loa O bservatory on Hawaii has records rom the longest period. C arbon dioxide concentrations have been measured rom 1 95 9 onwards and methane rom 1 984. These and other reliable records are o immense value to scientists.

Trends in atmospheric carbon dioxide Analysis o data rom atmosphere monitoring stations showing annual fuctuations. D ata rom atmosphere monitoring stations is reely available allowing any person to analyse it. There are both long- term trends and annual fuctuations in the data. The Mauna Loa O bservatory in Hawaii produces vast amounts o data and data rom this and other monitoring stations are available or analysis.

Figure 11 Hawaii from space. Mauna Loa is near the centre of the largest island

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4 . 4 c l i m at e c H a n G e

4.4 c hg Understandin  Carbon dioxide and water vapour are the most  

 







signicant greenhouse gases. Other gases including methane and nitrogen oxides have less impact. The impact o a gas depends on its ability to absorb long-wave radiation as well as on its concentration in the atmosphere. The warmed Earth emits longer-wave radiation (heat) . Longer-wave radiation is reabsorbed by greenhouse gases which retains the heat in the atmosphere. Global temperatures and climate patterns are infuenced by concentrations o greenhouse gases. There is a correlation between rising atmospheric concentrations o carbon dioxide since the start o the industrial revolution two hundred years ago and average global temperatures. Recent increases in atmospheric carbon dioxide are largely due to increases in the combustion o ossilized organic matter.

Applications  Correlations between global temperatures and

carbon dioxide concentrations on Earth.  Evaluating claims that human activities are not causing climate change.  Threats to coral rees rom increasing concentrations o dissolved carbon dioxide.

Nature of science  Assessing claims: assessment o the claims

that human activities are not causing climate change.

greenhouse ases Carbon dioxide and water vapour are the most signicant greenhouse gases. The Earth is kept much warmer than it otherwise would be by gases in the atmosphere that retain heat. The effect of these gases has been likened to that of the glass that retains heat in a greenhouse and they are therefore known as greenhouse gases, though the mechanism of heat retention is not the same. The greenhouse gases that have the largest warming effect on the Earth are carbon dioxide and water vapour. 

C arbon dioxide is released into the atmosphere by cell respiration in living organisms and also by combustion of biomass and fossil

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E c o lo g y uels. It is removed rom the atmosphere by photosynthesis and by dissolving in the oceans. 

Water vapour is ormed by evaporation rom the oceans and also transpiration in plants. It is removed rom the atmosphere by rainall and snow.

Water continues to retain heat ater it condenses to orm droplets o liquid water in clouds. The water absorbs heat energy and radiates it back to the Earths surace and also refects the heat energy back. This explains why the temperature drops so much more quickly at night in areas with clear skies than in areas with cloud cover.

other greenhuse gases Other gases including methane and nitrogen oxides have less impact. Figure 1 Satellite image of Hurricane Andrew in the Gulf of Mexico. Hurricanes are increasing in frequency and intensity as a result of increases in heat retention by greenhouse gases

Although carbon dioxide and water vapour are the most signicant greenhouse gases there are others that have a smaller but nonetheless signicant eect. 

Methane is the third most signicant greenhouse gas. It is emitted rom marshes and other waterlogged habitats and rom landll sites where organic wastes have been dumped. It is released during extraction o ossil uels and rom melting ice in polar regions.



Nitrous oxide is another signicant greenhouse gas. It is released naturally by bacteria in some habitats and also by agriculture and vehicle exhausts.

The two most abundant gases in the Earths atmosphere, oxygen and nitrogen, are not greenhouse gases as they do not absorb longer- wave radiation. All o the greenhouse gases together thereore make up less than 1 % o the atmosphere.

Assessing the impact f greenhuse gases The impact of a gas depends on its ability to absorb long-wave radiation as well as on its concentration in the atmosphere. Two actors together determine the warming impact o a greenhouse gas: 

how readily the gas absorbs long- wave radiation; and



the concentration o the gas in the atmosphere.

For example, methane causes much more warming per molecule than carbon dioxide, but as it is at a much lower concentration in the atmosphere its impact on global warming is less. The concentration o a gas depends on the rate at which it is released into the atmosphere and how long on average it remains there. The rate at which water vapour enters the atmosphere is immensely rapid, but it remains there only nine days on average, whereas methane remains in the atmosphere or twelve years and carbon dioxide or even longer.

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4 . 4 c l i m at e c H a n G e

lon-waveenth emissions from Earth

TOK

The warmed Earth emits longer-wave radiation.

Qusos xs bou h ry o sf phoo. wh osqus gh hs hv or h pub prpo d udrsdg o s?

The warmed surface of the Earth absorbs short- wave energy from the sun and then re- emits it, but at much longer wavelengths. Most of the re- emitted radiation is infrared, with a peak wavelength of 1 0, 000 nm. The peak wavelength of solar radiation is 400 nm.

spectral intensity

Figure 2 shows the range of wavelengths of solar radiation that pass through the atmosphere to reach the Earths surface and warm it ( red) and the range of much longer wavelengths emitted by the Earth that pass out through the atmosphere ( blue) . The smooth red and blue curves show the range of wavelengths expected to be emitted by bodies of the temperature of the Earth and the sun.

UV 0.2

Visible

Much o what science investigates involves entities and concepts beyond everyday experience o the world, such as the nature and behaviour o electromagnetic radiation or the build-up o invisible gases in the atmosphere. This makes it difcult or scientists to convince the general public that such phenomenon actually exist  particularly when the consequences o accepting their existance might run counter to value systems or entrenched belies.

Infrared 1

10

70

wavelength (m)

Figure 2

greenhouse ases Longer-wave radiation is reabsorbed by greenhouse gases which retains the heat in the atmosphere. 2 5 3 0% of the short-wavelength radiation from the sun that is passing through the atmosphere is absorbed before it reaches the Earths surface. Most of the solar radiation absorbed is ultraviolet light, which is absorbed by ozone. 7075 % of solar radiation therefore reaches the Earths surface and much of this is converted to heat.

A far higher percentage of the longer- wavelength radiation re-emitted by the surface of the Earth is absorbed before it has passed out to space. B etween 70% and 85 % is captured by greenhouse gases in the atmosphere. This energy is re- emitted, some towards the E arth. The effect is global warming. Without it the mean temperature at the Earths surface would be about 1 8C .

Key short-wave radiation from the sun long-wave radiation from earth

Figure 3 The greenhouse efect

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E c o lo g y Greenhouse gases in the Earths atmosphere only absorb energy in specifc wavebands. Figure 4 below shows total percentage absorption o radiation by the atmosphere. The graph also shows the bands o wavelengths absorbed by

individual gases. The wavelengths re-emitted by the E arth are between 5 and 70nm. Water vapour, carbon dioxide, methane and nitrous oxide all absorb some o these wavelengths, so each o them is a greenhouse gas.

percent

100 75 Total absorption and scattering

50 25 0 0.2

1

10

70

major components

Water vapour Carbon dioxide Oxygen and ozone Methane Nitrous oxide 0.2

1

10

70

wavelength (m)

Figure 4

global temperatures and carbon dioxide concentrations Correlations between global temperatures and carbon dioxide concentrations on Earth. I the concentration o any o the greenhouse gases in the atmosphere changes, we can expect the size o its contribution to the greenhouse eect to change and global temperatures to rise or all. We can test this hypothesis using the carbon dioxide concentration o the atmosphere, because it has changed considerably. To deduce carbon dioxide concentrations and temperatures in the past, columns o ice have been drilled in the Antarctic. The ice has built up over thousands o years, so ice rom deeper down is older than ice near the surace. B ubbles o air trapped in the ice can be extracted and analysed to fnd the carbon dioxide concentration. Global temperatures can be deduced rom ratios o hydrogen isotopes in the water molecules. Figure 5 shows results or an 800, 000 year period beore the present. They were obtained rom an ice core drilled in D ome C on the Antarctic plateau by the European Proj ect or Ice C oring in

232

Antarctica. D uring this part o the current Ice Age there has been a repeating pattern o rapid periods o warming ollowed by much longer periods o gradual cooling. There is a very striking correlation between carbon dioxide concentration and global temperatures  the periods o higher carbon dioxide concentration repeatedly coincide with periods when the Earth was warmer. The same trend has been ound in other ice cores. D ata o this type are consistent with the hypothesis that rises in carbon dioxide concentration increase the greenhouse eect. It is important always to remember that correlation does not prove causation, but in this case we know rom other research that carbon dioxide is a greenhouse gas. At least some o the temperature variation over the past 800,000 years must thereore have been due to rises and alls in atmospheric carbon dioxide concentrations.

4 . 4 c l i m at e c H a n G e

CO 2 /ppmv

300 250

D/%  (temperature proxy)

200 -380

warm 9C

-410 -440

cold 800,000

600,000

400,000 age (years before present)

200,000

0

Figure 5 Data from the European Project for Ice Coring in the Antarctic Dome C ice core

d-bs qusos: CO 2 concentrations and global temperatures 0.6 temperature anomaly (C)

Figure 6 shows atmospheric carbon dioxide concentrations. The red line shows direct measurements at Mauna Loa O bservatory. The points show carbon dioxide concentrations measured rom trapped air in polar ice cores.

parts per million by volume

380 360

Annual average Five year average

0.2 0

-0.2

Direct measurments Ice core measurments

-0.4

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Figure 7

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Figure 6 Figure 7 shows a record o global average temperatures compiled by the NAS A Goddard Institute or S pace S tudies. The green points are annual averages and the red curve is a rolling ve-year average. The values are given as the deviation rom the mean temperature between 1 961 and 1 990. 1

0.4

D iscuss whether the measurements o carbon dioxide concentration rom ice cores are consistent with direct measurements at Mauna Loa.

[2 ]

4

C ompare the trends in carbon dioxide concentration and global temperatures between 1 880 and 2 008.

[2 ]

Estimate the change in global average temperature between a) 1 900 and 2 000

[1 ]

b) 1 905 and 2 005

[1 ]

a) S uggest reasons or global average temperatures alling or a ew years during a period with an overall trend o rising temperatures.

[2 ]

b) D iscuss whether these alls indicate that carbon dioxide concentration does not infuence global temperatures.

[2 ]

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greenhouse ases and climate patterns Global temperatures and climate patterns are infuenced by concentrations o greenhouse gases. The surace o the Earth is warmer than it would be with no greenhouse gases in the atmosphere. Mean temperatures are estimated to be 3 2 C higher. I the concentration o any o the greenhouse gases rises, more heat will be retained and we should expect an increase in global average temperatures. This does not mean that global average temperatures are directly proportional to greenhouse gas concentrations. O ther actors have an infuence, including Milankovitch cycles in the E arths orbit and variation in sunspot activity. Even so, increases in greenhouse gas concentrations will tend to cause higher global average temperatures and also more requent and intense heat waves. Global temperatures infuence other aspects o climate. Higher temperatures increase the

evaporation o water rom the oceans and thereore periods o rain are likely to be more requent and protracted. The amount o rain delivered during thunderstorms and other intense bursts is likely to increase very signicantly. In addition, higher ocean temperatures cause tropical storms and hurricanes to be more requent and more powerul, with aster wind speeds. The consequences o any rise in global average temperature are unlikely to be evenly spread. Not all areas would become warmer. The west coast o Ireland and Scotland might become colder i the North Atlantic C urrent brought less warm water rom the Gul Stream to north-west Europe. The distribution o rainall would also be likely to change, with some areas becoming more prone to droughts and other areas to intense periods o rainall and fooding. Predictions about changes to weather patterns are very uncertain, but it is clear that just a ew degrees o warming would cause very proound changes to the Earths climate patterns.

data-base questions: Phenology Phenologists are biologists who study the timing o seasonal activities in animals and plants, such as the opening o tree leaves and the laying o eggs by birds. Data such as these can provide evidence o climate changes, including global warming.

2

Identiy the year in which: a) the leaves opened earliest

[1 ]

b) mean temperatures in March and April were at their lowest.

[1 ]

Use the data in the graph to deduce the ollowing: a) the relationship between temperatures in March and April and the date o opening o leaves on horse chestnut trees. [1 ] b) whether there is evidence o global warming towards the end o the 2 0th century. -15

4 3 2 1 0 -1 -2 -3 -4

-10 -5 0 5 10 1970

1980

1990 year

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1

15 2000

dierence in date of leaf opening / days

dierence in mean temperature / C

The date in the spring when new leaves open on horse chestnut trees ( Aesculus hippocastaneum) has been recorded in Germany every year since 1 95 1 . Figure 8 shows the dierence between each years date o lea opening and the mean date o lea opening between 1 970 and 2 000. Negative values indicate that the date o lea opening was earlier than the mean. The graph also shows the dierence between each years mean temperature during March and April and the overall mean temperature or these two months. The data or

temperature was obtained rom the records o 3 5 German climate stations.

[2 ]

Figure 8 The relationship between temperature and horse chestnut leaf opening in Germany since 1951

Key: temperature leaf opening

4 . 4 c l i m at e c H a n G e

Industrialization and climate change There is a correlation between rising atmospheric concentrations o carbon dioxide since the start o the industrial revolution two hundred years ago and average global temperatures. The graph o atmospheric carbon dioxide concentrations over the past 800, 000 years shown in gure 5 indicates that there have been large fuctuations. D uring glaciations the concentration dropped to as low as 1 80 parts per million by volume. D uring warm interglacial periods they rose as high as 3 00 ppm. The rise during recent times to concentrations nearing 400 ppm is thereore unprecedented in this period. Atmospheric carbon dioxide concentrations were between 2 60 and 2 80 ppm until the late 1 8th century. This is when concentrations probably started to rise above the natural levels, but as the rise was initially very slight, it is impossible to say exactly when an unnatural rise in concentrations began. Much o the rise has happened since 1 95 0.

Figure 9 During the industrial revolution renewable sources of power including wind were replaced with power generated by burning fossil fuels

In the late 1 8th century the industrial revolution was starting in some countries but the main impact o industrialization globally was in the second hal o the 2 0th century. More countries became industrialized, and combustion o coal, oil and natural gas increased ever more rapidly, with consequent increases in atmospheric carbon dioxide concentration. There is strong evidence or a correlation between atmospheric carbon dioxide concentration and global temperatures, but as already explained, other actors have an eect so temperatures are not directly proportional to carbon dioxide concentration. Nevertheless, since the start o the industrial revolution the correlation between rising atmospheric carbon dioxide concentration and average global temperatures is very marked.

Burning fossil fuels Recent increases in atmospheric carbon dioxide are largely due to increases in the combustion o ossilized organic matter. As the industrial revolution spread rom the late 1 8th century onwards, increasing quantities o coal were being mined and burned, causing carbon dioxide emissions. E nergy rom combustion o the coal provided a source o heat and power. D uring the 1 9 th century the combustion o oil and natural gas became increasingly widespread in addition to coal. Increases in the burning o ossil uels were most rapid rom the 1 95 0s onwards and this coincides with the period o steepest rises in atmospheric carbon dioxide. It seems hard to doubt the conclusion that the burning o ossil uels has been a maj or contributory actor in the rise o atmospheric carbon dioxide concentrations to higher levels than experienced on Earth or more than 800, 000 years.

TOK wh osus  upb v of rsk? In situations where the public is at risk, scientists are called upon to advise governments on the setting o policies or restrictions to oset the risk. Because scientic claims are based largely on inductive observation, absolute certainty is difcult to establish. The precautionary principle argues that action to protect the public must precede certainty o risk when the potential consequences or humanity are catastrophic. Principle 15 o the 1992 Rio Declaration on the Environment and Development stated the principle in this way: Where there are threats o serious or irreversible damage, lack o ull scientic certainty shall not be used as a reason or postponing cost-efective measures to prevent environmental degradation.

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data-base questions: Comparing CO 2 emissions The bar chart in gure 1 0 shows the cumulative CO 2 emissions rom ossil uels o the European Union and ve individual countries between 1 950 and 2000. It also shows the total CO 2 emissions including orest clearance and other land use changes. 1

D iscuss reasons or higher cumulative C O 2 emissions rom combustion o ossil uels in the United States than in B razil. [3 ]

2

Although cumulative emissions between 1 95 0 and 2 000 were higher in the United S tates than any other country, there were our countries in which emissions per capita

were higher in the year 2 000: Qatar, United Arab Emirates, Kuwait and B ahrain. Suggest reasons or the dierence. [3 ] 3

Although cumulative C O 2 emissions rom combustion o ossil uels in Indonesia and B razil between 1 95 0 and 2000 were relatively low, total C O 2 emissions were signicantly higher. S uggest reasons or this. [3 ]

4

Australia ranked seventh in the world or emissions o C O 2 in 2 000, but ourth when all greenhouse gases are included. S uggest a reason or the dierence. [1 ]

30%

Figure 10 CO 2 from fossil fuels CO 2 from fossil fuels & land-use change

percent of world total

25% 20% 15% 10% 5% 0%

U.S.

EU-25

Russia

China

Indonesia

Brazil

Assessing claims and counter-claims Assessing claims: assessment of the claims that human activities are not causing climate change. C limate change has been more hotly debated than almost any other area o science. A search o the internet will quickly reveal diametrically opposed views, expressed very vocierously. The author Michael C richton portrayed climate change scientists as eco- terrorists who were prepared to use mass murder to promote their work in his novel S tate o Fear. What reasons could there be or such erce opposition to climate change science and or what reason do climate change scientists deend their ndings so vigorously? These questions are worth discussing. There are many actors that could be having an infuence: 

236

Scientists are trained to be cautious about their claims and to base their ideas on evidence. They are expected to admit when there are uncertainties and this can give the impression that evidence is weaker than it actually is.



Global climate patterns are very complex and it is dicult to make predictions about the consequences o urther increases in greenhouse gas concentrations. There can be tipping points in climate patterns where sudden massive changes occur. This makes prediction even more dicult.



The consequences o changes in global climate patterns could be very severe or humans and or other species so many eel that there is a need or immediate action even i uncertainties remain in climate change science. C ompanies make huge prots rom coal, oil and natural gas and it is in their interests or ossil uel combustion to continue to grow. It would not be surprising i they paid or reports to be written that minimized the risks o climate change.

4 . 4 c l i m at e c H a n G e

oppsitin t the climate change science Evaluating claims that human activities are not causing climate change. Many claims that human activities are not causing climate change have been made in newspapers, on television and on the internet. One example o this is: Global warming stopped in 1 998, yet carbon dioxide concentrations have continued to rise, so human carbon dioxide emissions cannot be causing global warming. This claim ignores the act that temperatures on Earth are infuenced by many actors, not j ust greenhouse gas concentrations. Volcanic activity and cycles in ocean currents can cause signicant variations rom year to year. B ecause o such actors, 1 998 was an unusually warm year and also because o them some recent years have been cooler than they otherwise would have been.

Global warming is continuing but not with equal increases each year. Humans are emitting carbon dioxide by burning ossil uels and there is strong evidence that carbon dioxide causes warming, so the claim is not supported by the evidence. C laims that human activities are not causing climate change will continue and these claims need to be evaluated. As always in science, we should base our evaluations on reliable evidence. There is now considerable evidence about emissions o greenhouse gases by humans, about the eects o these gases and about changing climate patterns. Not all sources on the internet are trustworthy and we need to be careul to distinguish between websites with objective assessments based on reliable evidence and others that show bias.

d-bs qusos: Uncertainty in temperature rise projections Figure 1 1 shows computer-generated orecasts or average global temperatures, based on eight dierent scenarios or the changes in the emissions o greenhouse gases. The light green band includes the ull range o orecasts rom research centres around the world, and the dark green band shows the range o most o the orecasts. Figure 1 2 shows orecasts or arctic temperatures, based on two o the emissions scenarios. 1

2

3

4

5

6

Identiy the code or the least optimistic emissions scenario.

6 5 4 3 2

AIB AIT AIFI A2 B1 B2 IS92a

1 0 0 0 0 0 0 0 0 0 0 0 0 0 199 200 201 202 203 204 205 206 207 208 209 210

[1 ]

S tate the minimum and maximum orecasts or average global temperature change. [2 ] C alculate the dierence between the A2 and B 2 orecasts o global average temperature rise.

[2 ]

C ompare the orecasts or arctic temperatures with those or global average temperatures.

[2 ]

S uggest uncertainties, apart rom greenhouse gas emissions, which aect orecasts or average global temperatures over the next 1 00 years.

[2 ]

Discuss how much more condent we can be in orecasts based on data rom a number o dierent research centres, rather than one. [3 ]

Figure 11 Forecast global average temperatures 7

Discuss whether the uncertainty in temperature orecasts justies action or inaction. [4]

8

D iscuss whether it is possible to balance environmental risks with socio- economic and livelihood risks or whether priorities need to be established. [4]

7 6 A2 B2 5 4 3 2 1 0 2000 2020

2040

2060

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Figure 12 Forecast arctic temperature

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coral reefs and arbon dioxide Threats to coral rees rom increasing concentrations o dissolved carbon dioxide. In addition to its contribution to global warming, emissions o carbon dioxide are having eects on the oceans. Over 500 billion tonnes o carbon dioxide released by humans since the start o the industrial revolution have dissolved in the oceans. The pH o surace layers o the Earths oceans is estimated to have been 8.1 79 in the late 1 8th century when there had been little industrialization. Measurements in the mid-1 990s showed that it had allen to 8.1 04 and current levels are approximately 8.069. This seemingly small change represents a 30% acidication. Ocean acidication will become more severe i the carbon dioxide concentration o the atmosphere continues to rise. Marine animals such as ree- building corals that deposit calcium carbonate in their skeletons need to absorb carbonate ions rom seawater. The concentration o carbonate ions in seawater is low, because they are not very soluble. D issolved carbon dioxide makes the carbonate concentration even lower as a result o some interrelated chemical reactions. C arbon dioxide reacts with water to orm carbonic acid, which dissociates into hydrogen and hydrogen carbonate ions. Hydrogen ions react with dissolved carbonate ions, reducing their concentration.

make their skeletons. Also, i seawater ceases to be a saturated solution o carbonate ions, existing calcium carbonate tends to dissolve, so existing skeletons o ree-building corals are threatened. In 2 01 2 oceanographers rom more than 2 0 countries met in S eattle and agreed to set up a global scheme or monitoring ocean acidication. There is already evidence or concerns about corals and coral rees. Volcanic vents near the island o Ischia in the Gul o Naples have been releasing carbon dioxide into the water or thousands o years, reducing the pH o the seawater. In the area o acidied water there are no corals, sea urchins or other animals that make their skeletons rom calcium carbonate. In their place other organisms fourish such as sea grasses and invasive algae. This could be the uture o coral rees around the world i carbon dioxide continues to be emitted rom burning ossil uels.

C O 2 + H 2 O  H 2 C O 3  H + + HC O -3 H + + C O 23  HC O 3

I carbonate ion concentrations drop it is more dicult or ree- building corals to absorb them to

activity Draw a graph o oceanic pH rom the 18th century onwards, using the gures given in the text above, and extrapolate the curve to obtain an estimate o when the pH might drop below 7.

238

Figure 13 Skeleton of calcium carbonate from a reef-building coral

TOK wht re the potentil impcts of funding bis? The costs o scientic research is oten met by grant agencies. Scientists submit research proposals to agencies, the application is reviewed and i successul, the research can proceed. Questions arise when the grant agency has a stake in the study's outcome. Further, grant applications might ask scientists to project outcomes or suggest applications o the research beore it has even begun. The sponsor may und several diferent research groups, suppressing results that run counter to their interests and publishing those that support their industry. For example, a 2006 review o studies examining the health efects o cell phone use revealed that studies unded by the telecommunications industry were statistically least likely to report a signicant efect. Pharmaceutical research, nutrition research and climate change research are all areas where claims o unding bias have been prominent in the media.

QueStion S

Questions

a) C alculate the energy lost by plant respiration.

[2 ]

b) C onstruct a pyramid o energy or this grassland.

[3 ]

Drought Index

The total solar energy received by a grassland is 5  l0 5 kJ m - 2 yr - 1 . The net production o the grassland is 5  1 0 2 kJ m - 2 yr - 1 and its gross production is 6  1 0 2 kJ m - 2 yr - 1 . The total energy passed on to primary consumers is 60 kJ m - 2 yr - 1 . O nly 1 0 per cent o this energy is passed on to the secondary consumers.

Area of tree mortality/km 2

1

4 Warm/dry 3 long-term average 2 1 0 1 2 3 Cool/moist

2000 1500 1000 500 0 1930 1940 1950 1960 1970 1980 1990 2000

Figure 15 Tree mortality and drought index 2

a)

Figure 1 4 shows the energy fow through a temperate orest. The energy fow is shown per square metre per year ( kJ m - 2 yr - 1 ) . lost 5,223,120

b) ( i) C ompare the beetle outbreaks in the 1 970s and 1 990s. [2 ] respiration 24,024 green plants

172 14,448 decomposers

c) consumers storage (e.g. wood) 5,036

Figure 14 a) The chart shows that 99.1 7 per cent o the sunlight energy in the temperate orest is lost. Predict with a reason whether a greater or lesser percentage o sunlight energy would be lost in desert. [2 ] b) O nly a small part o the net production o plants in the temperate orest passes to herbivores. Explain the reasons or this. [2 ]

3

Warmer temperatures avour some species o pest, or example the spruce beetle. Since the rst maj or outbreak in 1 992 , it has killed approximately 400, 000 hectares o trees in Alaska and the C anadian Yukon. The beetle normally needs two years to complete its lie cycle, but it has recently been able to do it in one year. The graphs in gure 1 5 show the drought index, a combination o temperatures and precipitation, and the area o spruce trees destroyed annually.

4

CO 2 concentration/ppm

sunlight energy 5,266,800

Identiy the two periods when the drought index remained high or three or more years. [2 ]

( ii) S uggest reasons or the dierences between the outbreaks.

[2 ]

Predict rates o destruction o spruce trees in the uture, with reasons or your answer.

[4]

Figure 1 6 shows monthly average carbon dioxide concentrations or B aring Head, New Zealand and Alert, C anada. 390 385 380 375 370 365 360 355 350 345 340 335 330

Key Alert station, Canada Baring Head, New Zealand

76 78 80 82 84 86 88 90 92 94 96 98 00 02 04 year

Figure 16 a)

S uggest why scientists have chosen such areas as Mauna Loa, B aring Head and Alert as the locations or monitoring stations. [1 ]

b) C ompare the trends illustrated in both graphs. c)

[2 ]

Explain why the graphs show dierent patterns. [3 ]

239

41

e c o lo G y 5

Figure 1 7 shows the concentration o CO 2 in the atmosphere, measured in parts per million (ppm) . In a orest, concentrations o CO 2 change over the course o the day and change with height. The top o the orest is reerred to as the canopy.

tundra

above ground

taiga

root

above ground

height/m

soil 320 330 320 310 30

Top forest canopy

320

grasslands

340 350

340350

0 0

360 6

12

soil

18 24 time of day / hours

soil

equatorial forest

above ground

( i) S tate the highest concentration o C O 2 reached in the canopy. [1 ]

soil

( ii) D etermine the range o concentration ound in the canopy. [2 ] b) ( i) State the time o day ( or night) when the highest levels o C O 2 are detected.

root

root

savannah

Figure 17

above ground soil

root

root

Figure 18 The distribution of nitrogen in the three organic matters compartments for each of six major biomes [1 ]

( ii) The highest levels o C O 2 are detected j ust above the ground. D educe two reasons why this is the case. [2 ]

240

above ground

330

10

6

deciduous forest

above ground

305

c)

soil

310 ppm

20

a)

root

Give an example o an hour when C O 2 concentrations are reasonably uniorm over the ull range o heights. [1 ]

Within an ecosystem, nitrogen can be stored in one o three organic matter compartments: above ground, in roots and in the soil. Figure 1 8 shows the distribution o nitrogen in the three organic matter compartments or each o six maj or biomes.

a)

Deduce what the above ground compartment consists o in an ecosystem. [1 ]

b) S tate which biome has the largest above ground compartment. [1 ] c)

Explain why it is difcult to grow crops in an area where equatorial orest has been cleared o its vegetation. [2 ]

d) S tate the name o the process carried out by decomposers and detritus eeders that releases C O 2 into the atmosphere. [1 ] e) f)

Suggest why most o the nitrogen in a tundra ecosystem is in the soil.

[1 ]

Explain why warming due to climate change might cause a release o C O 2 rom tundra soil. [2 ]

5C E LELvOB Lu t I O n an d B I O d I vE r s I t Y I O LO GY Iocio

There is overwhelming evidence or the theory that the diversity o lie has evolved, and continues to evolve by natural selection. The ancestry o groups o species can be deduced by

comparing their base or amino acid sequences. S pecies are named and classifed using an internationally agreed system.

5.1 Evidence for evolution ueig  Evolution occurs when heritable characteristics  



 

o a species change. The ossil record provides evidence or evolution. Selective breeding o domesticated animals shows that artifcial selection can cause evolution. Evolution o homologous structures by adaptive radiation explains similarities in structure when there are dierences in unction. Populations o a species can gradually diverge into separate species by evolution. Continuous variation across the geographical range o related populations matches the concept o gradual divergence.

applicio  Comparison o the pentadactyl limb o

mammals, birds, amphibians and reptiles with dierent methods o locomotion.  Development o melanistic insects in polluted areas.

ne of ciece  Looking or patterns, trends and discrepancies:

there are common eatures in the bone structure o vertebrate limbs despite their varied use.

241

5

E vo l u t i o n an d b i o d i vE r s i t y

Evolution in summary Evolution occurs when heritable characteristics of a species change. There is strong evidence or characteristics o species changing over time. B iologists call this process evolution. It lies at the heart o a scientifc understanding o the natural world. An important distinction should be drawn between acquired characteristics that develop during the lietime o an individual and heritable characteristics that are passed rom parent to ospring. E volution only concerns heritable characteristics.  Figure 1

Fossils o dinosaurs show there were animals on Earth in the past that had diferent characteristics rom those alive today

The mechanism o evolution is now well understood  it is natural selection. D espite the robustness o evidence or evolution by natural selection, there is still widespread disbelie among some religious groups. There are stronger obj ections to the concept that species can evolve than to the logic o the mechanism that inevitably causes evolution. It is thereore important to look at the evidence or evolution.

Evidence from fossils The fossil record provides evidence for evolution. In the frst hal o the 1 9 th century, the sequence in which layers or strata o rock were deposited was worked out and the geological eras were named. It became obvious that the ossils ound in the various layers were dierent  there was a sequence o ossils. In the 2 0th century, reliable methods o radioisotope dating revealed the ages o the rock strata and o the ossils in them. There has been a huge amount o research into ossils, which is the branch o science called palaeontology. It has given us strong evidence that evolution has occurred.

 Figure 2

Many trilobite species evolved over hundreds o millions o years but the group is now totally extinct

242



The sequence in which ossils appear matches the sequence in which they would be expected to evolve, with bacteria and simple algae appearing frst, ungi and worms later and land vertebrates later still. Among the vertebrates, bony fsh appeared about 42 0 million years ago ( mya) , amphibians 3 40 mya, reptiles 3 2 0 mya, birds 2 5 0 mya and placental mammals 1 1 0 mya.



The sequence also fts in with the ecology o the groups, with plant ossils appearing beore animal, plants on land beore animals on land, and plants suitable or insect pollination beore insect pollinators.



Many sequences o ossils are known, which link together existing organisms with their likely ancestors. For example, horses, asses and zebras, members o the genus Equus, are most closely related to rhinoceroses and tapirs. An extensive sequence o ossils, extending back over 60 million years, links them to Hyracotherium, an animal very similar to a rhinoceros.

5 .1 E vi D E n cE fo r E vo lu ti o n

Daa-based qess: Missing links An obj ection to ossil evidence or evolution has been gaps in the record, called missing links, or example a link between reptiles and birds. (a)

(b) (d)

(g)

(c)

The discovery o ossils that ll in these gaps is particularly exciting or biologists. 1

2

(i)

(h) 100 mm

Drawings o ossils recently ound in Western China. They show Dilong paradoxus, a 130-million-year-old tyrannosauroid dinosaur with protoeathers. ad: bones o skull; e: teeth; g: tail vertebrae with protoeathers; hj: limb bones

[2 ]

D educe three similarities between Dilong paradoxus and reptiles that live on Earth today.

[3 ]

3

Suggest a unction or the protoeathers o Dilong paradoxus. [1 ]

4

Suggest two eatures which Dilong paradoxus would have had to evolve to become capable o fight. [2 ]

5

Explain why it is not possible to be certain whether the protoeathers o Dilong paradoxus are homologous with the eathers o birds. [2 ]

(j) (e) (f)

C alculate the length o Dilong paradoxus, rom its head to the tip o its tail.

 Figure 3

Evidence from selective breeding Selective breeding o domesticated animals shows that artifcial selection can cause evolution. Humans have deliberately bred and used particular animal species or thousands o years. I modern breeds o livestock are compared with the wild species that they most resemble, the dierences are oten huge. Consider the dierences between modern egg-laying hens and the jungleowl o Southern Asia, or between Belgian Blue cattle and the aurochs o Western Asia. There are also many dierent breeds o sheep, cattle and other domesticated livestock, with much variation between breeds. It is clear that domesticated breeds have not always existed in their current orm. The only credible explanation is that the change has been achieved simply by repeatedly selecting or and breeding the individuals most suited to human uses. This process is called articial selection. The eectiveness o articial selection is shown by the considerable changes that have occurred in domesticated animals over periods o time that are very short, in comparison to geological time. It shows that selection can cause evolution, but it does not prove that evolution o species has actually occurred naturally, or that the mechanism or evolution is natural selection.

 Figure 4 Over the last 15,000

years many breeds o dog have been developed by artifcial selection rom domesticated wolves

243

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Homology and evolution Looking or patterns, trends and disrepanies: there are ommon eatures in the one struture o verterate lims despite their varied use. Vertebrate limbs are used in many dierent ways, such as walking, running, j umping, fying, swimming, grasping and digging. These varied uses require j oints that articulate in dierent ways, dierent velocities o movement and also dierent amounts o orce. It would be reasonable to expect them to have very dierent bone structure, but there are in act common eatures o bone structure that are ound in all vertebrate limbs. Patterns like this require explanation. The only reasonable explanation so ar proposed in this case is evolution rom a common ancestor. As a consequence, the common bone structure o vertebrate limbs has become a classic piece o evidence or evolution.

Data-based questions: Domestication of corn A wild grass called teosinte that grows in C entral America was probably the ancestor o cultivated corn, Zea mays. When teosinte is grown as a crop, it gives yields o about 1 5 0 kg per hectare. This compares with a world average yield o corn o 4, 1 00 kg per hectare at the start o the 2 1 st century. Table 1 gives the lengths o some cobs. C orn was domesticated at least 7, 000 years ago. 1

C alculate the percentage dierence in length between teosinte and S ilver Queen. [2 ]

2

C alculate the percentage dierence in yield between teosinte and world average yields o corn.

[2 ]

3

Suggest actors apart rom cob length, selected or by armers. [3 ]

4

Explain why improvement slows down over generations o selection.

corn variety and origin Teosinte  wild relative o orn Early primitive orn rom Colomia Peruvian anient orn rom 500 bc Imriado  primitive orn rom Colomia Silver Queen  modern sweetorn

[3 ]

length of ob (mm) 14 45 65 90 170

 Table 1

 Figure 5 Corn

cobs

Evidence from homologous structures Evolution o homologous strutures y adaptive radiation explains similarities in struture when there are diferenes in untion. D arwin pointed out in The Origin of Species that some similarities in structure between organisms are supercial, or example between a dugong and a whale, or between a whale and a sh. S imilarities like those between the tail ns o whales and shes are known as analogous structures. When we study them closely we nd that these structures are very dierent. An evolutionary interpretation is that they have had

244

5 .1 E vi D E n cE fo r E vo lu ti o n

dierent origins and have become similar because they perorm the same or a similar unction. This is called convergent evolution. Homologous structures are the converse o this. They are structures that may look supercially dierent and perorm a dierent unction, but which have what D arwin called a unity o type. He gave the example o the orelimbs o a human, mole, horse, porpoise and bat and asked what could be more curious than to nd that they include the same bones, in the same relative positions, despite on the surace appearing completely dierent. The evolutionary explanation is that they have had the same origin, rom an ancestor that had a pentadactyl or vedigit limb, and that they have become dierent because they perorm dierent unctions. This is called adaptive radiation. There are many examples o homologous structures. They do not prove that organisms have evolved or had common ancestry and do not reveal anything about the mechanism o evolution, but they are dicult to explain without evolution. Particularly interesting are the structures that D arwin called rudimentary organs  reduced structures that serve no unction. They are now called vestigial organs and examples o them are the beginnings o teeth ound in embryo baleen whales, despite adults being toothless, the small pelvis and thigh bone ound in the body wall o whales and some snakes, and o course the appendix in humans. These structures are easily explained by evolution as structures that no longer have a unction and so are being gradually lost.

Pentadactyl limbs Comparison o the pentadactyl limb o mammals, birds, amphibians and reptiles with dierent methods o locomotion. The pentadactyl limb consists o these structures:

Be se single bone in the proximal part

femb humerus

Hdmb emur

two bones in the distal part

radius and ulna

group o wrist/ ankle bones

carpals

series o bones in each o fve digits

metacarpals and metatarsals phalanges and phalanges

classes that have limbs: amphibians, reptiles, birds and mammals. E ach o them has pentadactyl limbs: 

crocodiles walk or crawl on land and use their webbed hind limbs or swimming



penguins use their hind limbs or walking and their orelimbs as fippers or swimming



echidnas use all our limbs or walking and also use their orelimbs or digging



rogs use all our limbs or walking and their hindlimbs or j umping.

tibia and fbula tarsals

The pattern o bones or a modication o it is present in all amphibians, reptiles, birds and mammals, whatever the unction o their limbs. The photos in gure 6 show the skeletons o one example o each o the our vertebrates

D ierences can be seen in the relative lengths and thicknesses o the bones. Some metacarpals and phalanges have been lost during the evolution o the penguins orelimb.

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Activity Pentadactyl limbs in mammals

mole

horse

 Figure 6

porpoise

speciation Populations o a species can gradually diverge into separate species by evolution. bat human  Figure 7

Pentadactyl limbs (not to scale)

Choose a colour code or the types o bone in a pentadactyl limb and colour the diagrams in fgure 7 to show the type o each bone. How is each limb used? What eatures o the bones in each limb make them well adapted to the use?

246

If two populations of a species become separated so that they do not interbreed and natural selection then acts differently on the two populations, they will evolve in different ways. The characteristics of the two populations will gradually diverge. After a time they will be recognizably different. If the populations subsequently merge and have the chance of interbreeding, but do not actually interbreed, it would be clear that they have evolved into separate species. This process is called speciation. S peciation often occurs after a population of a species extends its range by migrating to an island. This explains the large numbers of endemic species on islands. An endemic species is one that is found only in a certain geographical area. The lava lizards of the Galpagos Islands are an example of this. O ne species is present on all the main islands of the archipelago. O n six smaller islands there is a closely related but different species, formed by migration to the island and by subsequent divergence.

5 .1 E vi D E n cE fo r E vo lu ti o n

Evidence from patterns of variation

Pinta

Continuous variation across the geographical range o related populations matches the concept o gradual divergence.

Genovesa Marchena Santiago

I populations gradually diverge over time to become separate species, then at any one moment we would expect to be able to nd examples o all stages o divergence. This is indeed what we nd in nature, as C harles D arwin describes in C hapter II o The Origin of Species. He wrote:

Santa Cruz

Fernandina

Santa Fe Isabel a Espaola

Santa Maria

Many years ago, when comparing, and seeing others compare, the birds from the separate islands of the Galpagos Archipelago, both one with another, and with those from the American mainland, I was much struck how entirely vague and arbitrary is the distinction between species and varieties.

San Cristbal

key T. albemarlensis T. duncanensis

T. delanonis

T. habelii

T. pacicus

T. bivittatus

T. grayii

 Figure 8

Distribution of lava lizards in the Galpagos Islands

D arwin gave examples o populations that are recognizably dierent, but not to the extent that they are clearly separate species. O ne o his examples is the red grouse o B ritain and the willow ptarmigan o Norway. They have sometimes been classied as separate species and sometimes as varieties o the species Lagopus lagopus. This is a common problem or biologists who name and classiy living organisms. B ecause species can gradually diverge over long periods o time and there is no sudden switch rom being two populations o one species to being two separate species, the decision to lump populations together or split them into separate species remains rather arbitrary. The continuous range in variation between populations does not match either the belie that species were created as distinct types o organism and thereore should be constant across their geographic range or that species are unchanging. Instead it provides evidence or the evolution o species and the origin o new species by evolution.

Industrial melanism Development o melanistic insects in polluted areas. D ark varieties o typically light- coloured insects are called melanistic. The most amous example o an insect with a melanistic variety is Biston betularia, the peppered moth. It has been widely used as an example o natural selection, as the melanistic variety became commoner in polluted industrial areas where it is better camoufaged than the pale peppered variety. A simple explanation o industrial melanism is this: 

Adult Biston betularia moths fy at night to try to nd a mate and reproduce.



D uring the day they roost on the branches o trees.



B irds and other animals that hunt in daylight predate moths i they nd them.

TOK t wha exe a mpe mdes be sed  es hees? The useulness o a theory is the degree to which it explains phenomenon and the degree to which it allows predictions to be made. One way to test the theory o evolution by natural selection is through the use o computer models. The Blind Watchmaker computer model is used to demonstrate how complexity can evolve rom simple orms through artifcial selection. The Weasel computer model is used to demonstrate how artifcial selection can increase the pace o evolution over random events. What eatures would a computer model have to include or it to simulate evolution by natural selection realistically?

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

In unpolluted areas tree branches are covered in pale- coloured lichens and peppered moths are well camoufaged against them.



Sulphur dioxide pollution kills lichens. S oot rom coal burning blackens tree branches.



Melanic moths are well camoufaged against dark tree branches in polluted areas.



In polluted areas the melanic variety o Biston betularia replaced the peppered variety over a relatively short time, but not in nonpolluted areas.

 Figure 9

Museum specimen of the peppered form of Biston betularia mounted on tree bark with lichens from an unpolluted area

 Figure 10

The ladybug Adalia bipunctata has a melanic form which has become common in polluted areas. A melanic male is mating with a normal female here

B iologists have used industrial melanism as a classic example o evolution by natural selection. Perhaps because o this, research ndings have been repeatedly attacked. The design o some early experiments into camoufage and predation o the moths has been criticized and this has been used to cast doubt over whether natural selection ever actually occurs. Michael Majerus gives a careul evaluation o evidence about the development o melanism in Biston betularia and other species o moth in his book in the New Naturalist series (Moths, Michael Majerus, HarperCollins 2002) . His nding is that the evidence or industrial pollution causing melanism in Biston betularia and other species o moth is strong, though actors other than camoufage can also infuence survival rates o pale and melanic varieties.

Data-based questions: Predation rates in Biston betularia One o the criticisms o the original experiments into predation o Biston betularia was that the moths were placed in exposed positions on tree trunks and that this is not normally where they roost. The moths were able to move to more suitable positions but even so the criticisms have persisted on some websites. Experiments done in the 1 980s tested the eect o the position in which the moths were placed. Peppered and melanic

248

orms ( ty o each) o Biston betularia were placed in exposed positions on tree trunks and 5 0 millimetres below a joint between a maj or branch and the tree trunk. This procedure was carried out at two oak woods, one in an unpolluted area o the New Forest in southern England and another in a polluted area near Stoke-on-Trent in the Midlands. The box plots in gure 1 1 show the percentage o moths eaten and moths surviving.

5 . 2 n At u r A l s E l E c t i o n

1

a)

D educe, with a reason from the data, whether the moths were more likely to be eaten if they were placed on the exposed trunk or below the j unction of a main branch and the trunk. [2 ]

b) Suggest a reason for the difference. 2

a)

C ompare and contrast the survival rates of peppered and melanic moths in the New Forest.

b) Explain the difference in survival rate between the two varieties in the New Forest.

[1 ]

[3 ]

peppered New Forest/melanic/BJ New Forest/melanic/ET

4

D istinguish between the S toke- on- Trent and New Forest woodlands in relative survival rates of peppered and melanic moths. [2 ] Pollution due to industry has decreased greatly near S toke- on- Trent since the 1 980s. Predict the consequences of this change for Biston betularia. [4]

38

40 62

74

26

New Forest/peppered/ET

68

32

Stoke/melanic/BJ

72

28

Stoke/melanic/ET Stoke/peppered/BJ

[3 ]

60

New Forest/peppered/BJ

Stoke/peppered/ET melanic

3

Stoke on Trent and New Forest

key not eaten ET = exposed trunk

0%

60 50 42

40 50 58

20% 40% 60% 80% 100%

eaten BJ = branch junction

 Figure 11

Source: Howlett and Majerus (1987) The Understanding of industrial melanism in the peppered moth (Biston betularia) Biol. J.Linn.Soc. 30, 3144

5.2 naa ee uderstdig  Natural selection can only occur i there is    

 

variation amongst members o the same species. Mutation, meiosis and sexual reproduction cause variation between individuals in a species. Adaptations are characteristics that make an individual suited to its environment and way o lie. Species tend to produce more ospring than the environment can support. Individuals that are better adapted tend to survive and produce more ospring while the less well adapted tend to die or produce ewer ospring. Individuals that reproduce pass on characteristics to their ospring. Natural selection increases the requency o characteristics that make individuals better adapted and decreases the requency o other characteristics leading to changes within the species.

applictios  Changes in beaks o fnches on Daphne Major.  Evolution o antibiotic resistance in bacteria.

ntre of sciece  Use theories to explain natural phenomena:

the theory o evolution by natural selection can explain the development o antibiotic resistance in bacteria.

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vrition Natural selection can only occur if there is variation amongst members of the same species.

 Figure 1 Populations o bluebells (Hyacinthoides

non-scripta) mostly have blue fowers but white-fowered plants sometimes occur

C harles D arwin developed his understanding of the mechanism that causes evolution over many years, after returning to England from his voyage around the world on HMS B eagle. He probably developed the theory of natural selection in the late 1 83 0s, but then worked to accumulate evidence for it. D arwin published his great work, The Origin of Species, in 1 85 9. In this book of nearly 5 00 pages, he explains his theory and presents the evidence for it that he had found over the previous 2 0 to 3 0 years. O ne of the observations on which D arwin based the theory of evolution by natural selection is variation. Typical populations vary in many respects. Variation in human populations is obvious  height, skin colour, blood group and many other features. With other species the variation may not be so immediately obvious but careful observation shows that it is there. Natural selection depends on variation within populations  if all individuals in a population were identical, there would be no way of some individuals being favoured more than others.

source of rition Mutation, meiosis and sexual reproduction cause variation between individuals in a species. The causes of variation in populations are now well understood:

 Figure 2

Dandelions (Taraxacum ofcinale) appear to be reproducing sexually when they disperse their seed but the embryos in the seeds have been produced asexually so are genetically identical

1

Mutation is the original source of variation. New alleles are produced by gene mutation, which enlarges the gene pool of a population.

2

Meiosis produces new combinations of alleles by breaking up the existing combination in a diploid cell. Every cell produced by meiosis in an individual is likely to carry a different combination of alleles, because of crossing over and the independent orientation of bivalents.

3

S exual reproduction involves the fusion of male and female gametes. The gametes usually come from different parents, so the offspring has a combination of alleles from two individuals. This allows mutations that occurred in different individuals to be brought together.

In species that do not carry out sexual reproduction the only source of variation is mutation. It is generally assumed that such species will not generate enough variation to be able to evolve quickly enough for survival during times of environmental change.

adpttion Adaptations are characteristics that make an individual suited to its environment and way of life. O ne of the recurring themes in biology is the close relationship between structure and function. For example, the structure of a birds beak is correlated with its diet and method of feeding. The thick coat of a musk

250

5 . 2 n At u r A l s E l E c t i o n ox is obviously correlated with the low temperatures in its northerly habitats. The water storage tissue in the stem o a cactus is related to inrequent rainall in desert habitats. In biology characteristics such as these that make an individual suited to its environment or way o lie are called adaptations. The term adaptation implies that characteristics develop over time and thus that species evolve. It is important not to imply purpose in this process. According to evolutionary theory adaptations develop by natural selection, not with the direct purpose o making an individual suited to its environment. They do not develop during the lietime o one individual. C haracteristics that do develop during a lietime are known as acquired characteristics and a widely accepted theory is that acquired characteristics cannot be inherited.

Avy Adapa f bd beak The our photographs o birds show the beaks o a heron, macaw, hawk and woodpecker. To what diet and method o eeding is each adapted?

Overproduction o ofspring Species tend to produce more ofspring than the environment can support. Living organisms vary in the number o ospring they produce. An example o a species with a relatively slow breeding rate is the southern ground hornbill, Bucorvus leadbeateri. It raises one fedgling every three years on average and needs the cooperation o at least two other adults to do this. However they can live or as long as 7 0 years so in their lietime a pair could theoretically raise twenty ospring. Most species have a aster breeding rate. For example, the coconut palm, Cocos nucifera usually produces between 2 0 and 60 coconuts per year. Apart rom bacteria, the astest breeding rate o all may be in the ungus Calvatia gigantea. It produces a huge ruiting body called a giant puball in which there can be as many as 7 trillion spores ( 7, 000, 000, 000, 000) .

 Figure 3



D espite the huge variation in breeding rate, there is an overall trend in living organisms or more ospring to be produced than the environment can support. D arwin pointed out that this will tend to lead to a struggle or existence within a population. There will be competition or resources and not every individual will obtain enough to allow them to survive and reproduce.

Figure 4 The breeding rate of pairs of southern ground hornbills, Bucorvus leadbeateri, is as low as 0.3 young per year

251

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Activity simulation of natural election 





Make ten or more artifcial fsh using modelling clay, or some other malleable material. Drop each o them into a measuring cylinder o water and time how long each takes to reach the bottom. Discard the hal o the models that were slowest. Pair up the astest models and make intermediate shapes, to represent their ospring. Random new shapes can also be introduced to simulate mutation. Test the new generation and repeat the elimination o the slowest and the breeding o the astest. Does one shape gradually emerge? Describe its eatures.

diferential survival an reprouction Individuals that are better adapted tend to survive and produce more ospring while the less well adapted tend to die or produce ewer ospring. C hance plays a part in deciding which individuals survive and reproduce and which do not, but the characteristics o an individual also have an infuence. In the struggle or existence the less well- adapted individuals tend to die or ail to reproduce and the best adapted tend to survive and produce many ospring. This is natural selection. An example that is oten quoted is that o the girae. It can graze on grass and herbs but is more adapted to browse on tree leaves. In the wet season its ood is abundant but in the dry season there can be periods o ood shortage when the only remaining tree leaves are on high branches. Giraes with longer necks are better adapted to reaching these leaves and surviving periods o ood shortage than those with shorter necks.

Inheritance Individuals that reproduce pass on characteristics to their ospring. Much o the variation between individuals can be passed on to ospring  it is heritable. Maasai children inherit the dark skin colour o their parents or example and children o light- skinned north European parents inherit a light skin colour. Variation in behaviour can be heritable. The direction o migration to overwintering sites in the blackcap Sylvia atricapilla is an example. D ue to dierences in their genes, some birds o this species migrate southwestwards rom Germany to Spain or the winter and others northwestwards to B ritain. Not all eatures are passed on to ospring. Those acquired during the lietime o an individual are not usually inherited. An elephant with a broken tusk does not have calves with broken tusks or example. I a person develops darker skin colour through exposure to sunlight, the darker skin is not inherited. Acquired characteristics are thereore not signicant in the evolution o a species.

Progressive change Natural selection increases the requency o characteristics that make individuals better adapted and decreases the requency o other characteristics leading to changes within the species. B ecause better- adapted individuals survive, they can reproduce and pass on characteristics to their ospring. Individuals that are less well adapted have lower survival rates and less reproductive success. This leads to an increase in the proportion o individuals in a population with

252

5 . 2 n At u r A l s E l E c t i o n characteristics that make them well adapted. O ver the generations, the characteristics o the population gradually change  this is evolution by natural selection. Maj or evolutionary changes are likely to occur over long time periods and many generations, so we should not expect to be able to observe them during our lietime, but there are many examples o smaller but signicant changes that have been observed. The evolution o dark wing colours in moths has been observed in industrial areas with polluted air. Two examples o evolution are described in the next sections o this book: changes to beaks o nches on the Galapagos Islands and the development o antibiotic resistance in bacteria.

Avy The impulse to reproduce and pass on characteristics can be very strong. It can cause adult males to carry out infanticide. How could this behaviour pattern have evolved in lions and other species? Female cheetahs mate with two or more males so their litters have multiple paternity. How does this protect the young against infanticide?

Daa-baed qe: Evolution in rice plants The bar charts in gure 6 show the results o an investigation o evolution in rice plants. F 1 hybrid plants were bred by crossing together two rice varieties. These hybrids were then grown at ve dierent sites in Japan. Each year the date o fowering was recorded and seed was collected rom the plants, or re-sowing at that site in the ollowing year. F3

F4

F5

F

 Figure 5 A female cheetahs cubs inherit

Sapporo 43 N

characteristics from her and from one of the several males with whom she mated

Fujisaka 40 N Konasu 36 N single original population planted out at

Hiratsuka 35 N Chikugo 33 N Miyazaki 31 N 56 70 84 98 112 126

68 82 96 110 124 138

54 68 82 96 110124138

51 65 79 93 107121 135

days to owering  Figure 6

1

Why was the investigation done using hybrids rather than a single pure- bred variety?

[2 ]

2

D escribe the changes, shown in the chart, between the F 3 and F 6 generations o rice plants grown at Miyazaki. [2 ]

3

a)

S tate the relationship between fowering time and latitude in the F 6 generation. [1 ]

b) S uggest a reason or this relationship. 4

a)

[1 ]

Predict the results i the investigation had been carried on until the F 1 0 generation. [1 ]

b) Predict the results o collecting seeds rom F 1 0 plants grown at S apporo and rom F 1 0 plants grown at Miyazaki and sowing them together at Hiratsuka. [3 ]

253

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Galpagos fnches Changes in beaks o fnches on Daphne Major. Pinta (5) Rabida (8)

Marchena (4)

Genovesa (4)

Santiago (10) Daphne Major (2/3) Fernandina (9) Isabela (10)

Santa Cruz (9) Santa Fe (5) Santa Maria (8)

San Cristbal (7)

(a) G. fortis (large beak)

Espaola (3)

 Figure 7

The Galpagos archipelago with the number o species o fnch ound on each island

Darwin visited the Galpagos Islands in 1 835 and collected specimens o small birds, which were subsequently identifed as fnches. There are 1 4 species in all. Darwin observed that the sizes and shapes o the beaks o the fnches varied, as did their diet. From the overall similarities between the birds and their distribution over the Galapagos islands (see fgure 7) , Darwin hypothesized that one might really ancy that rom an original paucity o birds in this archipelago, one species had been taken and modifed or dierent ends. There has since been intense research into what have become known as D arwins fnches. In particular, Peter and Rosemary Grant have shown that beak characters and diet are closely related and when one changes, the other does also. A particular ocus o Peter and Rosemary Grants research has been a population o the medium ground fnch, Geospiza fortis, on a small island called D aphne Maj or. O n this island, the small ground fnch, Geospiza fuliginosa, is almost absent. B oth species eed on small seeds, though G. fortis can also eat larger seeds. In the absence o competition rom G. fuliginosa or small seeds, G. fortis is smaller in body size and beak size on D aphne Maj or than on other islands. In 1 977, a drought on D aphne Major caused a shortage o small seeds, so G. fortis ed instead on larger, harder seeds, which the larger-beaked individuals are able to crack open. Most o the population died in that year, with highest mortality

254

(b) G. fortis (small beak)

(c) G. magnirostris  Figure 8 Variation

in beak shape in Galpagos fnches. (a) G. fortis (large beak) . (b) G. fortis (small beak) . (c) G. magnirostris

among individuals with shorter beaks. In 1 982 83 there was a severe El Nio event, causing eight months o heavy rain and as a result an increased supply o small, sot seeds and ewer large, hard seeds. G. fortis bred rapidly, in response to the increase in ood availability. With a return to dry weather conditions and greatly reduced supplies o small seeds, breeding stopped until 1 987. In that year, only 3 7 per cent o those alive in 1 983 bred and they were not a random sample o the 1 983 population. In 1 987, G. fortis had longer and narrower beaks than the 1 983 averages, correlating with the reduction in supply o small seeds. Variation in the shape and size o the beaks ( see fgure 8) is mostly due to genes, though the

5 . 2 n At u r A l s E l E c t i o n

environment has some eect. The proportion o the variation due to genes is called heritability. Using the heritability o beak length and width and data about the birds that had survived to breed, the changes in mean beak length and width between 1 983 and 1 987 were predicted. The observed results are very close to the predictions. Average beak length was predicted to increase by 1 0 m and actually increased by 6 m. Average beak width was predicted to decrease by 1 3 0 m and actually decreased by 1 2 0 m.

O ne o the obj ections to the theory o evolution by natural selection is that signifcant changes caused by natural selection have not been observed actually occurring. It is unreasonable to expect huge changes to have occurred in a species, even i it had been ollowed since D arwins theory was published in 1 85 9, but in the case o G. fortis, signifcant changes have occurred that are clearly linked to natural selection.

Daa-baed qe: Galpagos fnches When Peter and Rosemary Grant began to study fnches on the island o D aphne Maj or in 1 973 , there were breeding populations o two species, Geospiza fortis and Geospiza scandens. Geospiza magnirostris established a breeding population on the island in 1 982 , initially with j ust two emales and three males. Figure 9 shows the numbers o G. magnirostris and G. fortis on D aphne Maj or between 1 997 and 2 006. 1500 numbers

G. fortis G. magnirostris

1000 500

the changes in the population o G. magnirostris. 2

1998

2000

2002 year

2004

Changes in numbers of G. fortis and G. magnirostris between 1996 and 2006 a)

D escribe the changes in the population o G. magnirostris between 1 997 and 2 006. [2 ]

b) C ompare the changes in population o G. fortis between 1 997 and 2 006 with

spee Yea sma Medm lage

1977 75 10 17

Geospiza fortis 1985 1989 80 77 0.0 5.1 19 16

2004 80 11 8.2

a)

O utline the diet o each o the species o fnch on D aphne Maj or. [3 ]

b) There was a very severe drought on D aphne Maj or in 2 003 and 2 004. D educe how the diet o the fnches changed during the drought, using the data in the table.

2006

 Figure 9

1

D aphne Maj or has an area o 0.3 4 km . 1 km 2 is 1 00 hectares and 1 hectare is 1 00  1 00 m. C alculate the maximum and minimum population densities o G. ortis during 1 9972 006. [4]

Table 2 shows the percentages o three types o seed in the diets o the three fnch species on D aphne Maj or. Small seeds are produced by 2 2 plant species, medium seeds by the cactus Opuntia echios, and large seeds, which are very hard, by Tribulus cistoides. 3

0 1996

[3 ] 2

4

[3 ]

Figure 1 0 shows an index o beak size o adult G. fortis rom 1 973 to 2 006, with the size in 1 973 assigned the value zero and the sizes in other years shown in comparison to this.

Geospiza magnirostris 1985 1989 2004 18 5.9 4.5 0.0 12 26 82 82 69

Geospiza scandens 1977 1985 1989 2004 85 77 23 17 15 22 70 83 0.0 0.0 0.0 0.0

 Table 2

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5

E vo l u t i o n an d b i o d i vE r s i t y

c) In the frst severe drought, the mean beak size o G. fortis increased, but in the second drought, it decreased. Using the data in this question, explain how natural selection could cause these changes in beak size in the two droughts. [3 ]

1

beak size index

0.5 0

-0.5

5

The intensity o natural selection on D aphne Maj or was calculated during the two droughts. The calculated values are called selection dierentials. They range rom 1 .08 or beak length during the second drought, to +0. 88 or beak length in the frst drought, with similar selection dierentials or beak width and depth and overall beak size. These are very large selection dierentials, compared to values calculated in other investigations o evolution. Suggest reasons or natural selection on the beak size o G. fortis being unusually intense on the island o D aphne Maj or. [2 ]

6

D iscuss the advantages o investigations o evolution over long periods and the reasons or ew long-term investigations being done. [3 ]

-1 -1.5 1975

1980

1985

1990 year

1995

2000

2005

 Figure 10

Relative beak size in G. fortis between 1973 and 2006 The graph shows two periods o very rapid change in mean beak size, both o which correspond with droughts on D aphne Maj or. a) S tate two periods o most rapid change in mean beak size o G. fortis. [2 ] b) S uggest two reasons or mean beak size changing most rapidly when there is a drought. [2 ]

natural selectio ad atibiotic resistace Use theories to explain natural phenomena: the theory of evolution by natural selection can explain the development of antibiotic resistance in bacteria. Antibiotics were one o the great triumphs o medicine in the 2 0th century. When they were frst introduced, it was expected that they would oer a permanent method o controlling bacterial diseases, but there have been increasing problems o antibiotic resistance in pathogenic bacteria.

development o antibiotic resistance is thereore an example o evolution. It can be explained in terms o the theory o natural selection. A scientifc understanding o how antibiotic resistance develops is very useul as it gives an understanding o what should be done to reduce the problem.

The ollowing trends have become established:





Ater an antibiotic is introduced and used on patients, bacteria showing resistance appear within a ew years. Resistance to the antibiotic spreads to more and more species o pathogenic bacteria. In each species the proportion o inections that are caused by a resistant strain increases.

14 12 % resistant



16

10 8 6 4 2

256

 Figure 11

2003

Percentage resistance to ciprofoxacin between 1990 and 2004

2004

2001

2002

1999

2000

1997

1998

1996

1994

1995

1992

1993

1991

1990

0

So, during the time over which antibiotics have been used to treat bacterial diseases there have been cumulative changes in the antibiotic resistance properties o populations o bacteria. The

5 . 2 n At u r A l s E l E c t i o n

antibiotic resistnce Evolution of antibiotic resistance in bacteria. Antibiotic resistance is due to genes in bacteria and so it can be inherited. The mechanism that causes antibiotic resistance to become more prevalent or to diminish is summarized in gure 1 2 . The evolution o multiple antibiotic resistance has occurred in j ust a ew decades. This rapid evolution is due to the ollowing causes: 







population with no antibiotic-resistant bacteria antibiotic resistance gene received from a bacterium in another population

population with some antibiotic-resistant bacteria

There has been very widespread use o antibiotics, both or treating diseases and in animal eeds used on arms.

antibiotic is used therefore there is strong natural selection for resistance

B acteria can reproduce very rapidly, with a generation time o less than an hour.

population with more antibiotic-resistant bacteria

Populations o bacteria are oten huge, increasing the chance o a gene or antibiotic resistance being ormed by mutation. B acteria can pass genes on to other bacteria in several ways, including using plasmids, which allow one species o bacteria to gain antibiotic resistance genes rom another species.

antibiotic resistance gene formed by mutation in one bacterium

antibiotic is not used therefore there is natural selection (weak) against resistance population with slightly fewer antibiotic-resistant bacteria  Figure 12

Evolution o antibiotic resistance

Daa-baed qe: Chlortetracycline resistance in soil bacteria

1

a)

S tate the relationship between percentage antibiotic resistance and distance rom the animal pen. [1 ]

b) E xplain the dierence in antibiotic resistance between populations o bacteria near and ar rom the pen. [4]

3.0 2.5 desistance (%)

B acteria were collected rom soil at dierent distances rom a site on a pig arm in Minnesota where manure had been allowed to overfow rom an animal pen and accumulate. The eed given to the pigs on this arm contained subtherapeutic low doses o the antibiotic chlortetracycline, in order to promote aster growth rates. The bacteria were tested to nd out what percentage o them was resistant to this antibiotic. The results are shown in the bar chart. The yellow bars show the percentage o chlortetracycline resistant bacteria that grew on nutrient-rich medium and the orange bars show the percentage on a nutrient- poor medium that encouraged dierent types o bacteria to grow.

2.0 1.5 1.0 0.5 0.0 5m 20 m 100 m distance from animal pen

Source: " The efects o subtherapeutic antibiotic use in arm animals on the prolieration and persistence o antibiotic resistance among soil bacteria", Sudeshna Ghosh and Timothy M LaPara, The International Society for Microbial Ecology Journal (2007) 1, 191203

2

Predict whether the percentage antibiotic resistance would have been lower at 200 metres rom the pen than at 1 00 metres. [3]

3

D iscuss the use o subtherapeutic doses o antibiotics in animal eeds. [2 ]

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5.3 classifation o biodiversity udertdig  The binomial system o names or species is

   







universal among biologists and has been agreed and developed at a series o congresses. When species are discovered they are given scientifc names using the binomial system. Taxonomists classiy species using a hierarchy o taxa. All organisms are classifed into three domains. The principal taxa or classiying eukaryotes are kingdom, phylum, class, order, amily, genus and species. In a natural classifcation the genus and accompanying higher taxa consist o all the species that have evolved rom one common ancestral species. Taxonomists sometimes reclassiy groups o species when new evidence shows that a previous taxon contains species that have evolved rom dierent ancestral species. Natural classifcations help in identifcation o species and allow the prediction o characteristics shared by species within a group.

applictio  Classifcation o one plant and one animal

species rom domain to species level.  External recognition eatures o bryophytes, flicinophytes, conierophytes and angiospermophytes.  Recognition eatures o poriera, cnidaria, platyhelminthes, annelida, mollusca and arthropoda, chordata.  Recognition o eatures o birds, mammals, amphibians, reptiles and fsh.

skill  Construction o dichotomous keys or use in

identiying specimens.

ntre o ciece  Cooperation and collaboration between groups

o scientists: scientists use the binomial system to identiy a species rather than the many dierent local names.

Itertiol coopertio d clifctio Cooperation and collaboration between groups o scientists: scientists use the binomial system to identiy a species rather than the many dierent local names. Recognizable groups of organisms are known to biologists as species. The same species can have many different local names, even within one language. For example, in E ngland the species of plant known to scientists as Arum maculatum has been called lords- and- ladies, cuckoopint, j ack in the pulpit, devils and angels, cows and bulls, willy lily and snakes meat. In French there is also a variety of local names:

258

la chandelle, le pied- de- veau, le manteau de la S ainte- Vierge, la pilette or la vachotte. In S panish there are even more names for this one species of which these are j ust a few: comida de culebra, alcatrax, barba de arn, dragontia menor, hoj as de fuego, vela del diablo and yerba del quemado. The name primaveras is used for Arum maculatum in S panish but for a different plant in other languages.

5 . 3 c l A s s i f i c At i o n o f B i o D i v E r s i t Y

Local names may be a valuable part o the culture o an area, but science is an international venture so scientifc names are needed that are understood throughout the world. The binomial system that has developed is a good example o cooperation and collaboration between scientists. The credit or devising our modern system o naming species is given to the Swedish biologist C arl Linnaeus who introduced a system o twopart names in the 1 8th century. This stroke o genius was the basis or the binomial system that is still in use today. In act Linnaeus was mirroring a style o nomenclature that had been used in many languages beore. The style recognizes that there are groups o similar species, so the name or each species in a group consists o a specifc name attached to the group name, as in the Ancient Greek    and    (used by Threophrastus) , Latin anagallis mas and anagallis femina (used by Pliny) , German weiss

Seeblumen and geel Seeblumen (used by Fuchs) , English wild mynte and water mynte (used by Turner) and Malayan jambu bol and jambu chilli (applied by Malays to dierent species o Eugenia) .

 Figure 1

Arum maculatum

development of the binomial system The binomial system of names for species is universal among biologists and has been agreed and developed at a series of congresses. To ensure that all biologists use the same system o names or living organisms, congresses attended by delegates rom around the world are held at regular intervals. There are separate congresses or animals and or plants and ungi. International B otanical C ongresses ( IB C ) were held every year during the late 1 9th century. The IB C held in Genoa in 1 892 proposed that 1 75 3 be taken as the starting point or both genera and species o plants and ungi as this was the year when Linnaeus published Species Plantarum, the book that gave consistent binomials or all species o the plant kingdom then known. The IB C o Vienna in 1 905 accepted by 1 5 0 votes to 1 9 the rule that La nomenclature botanique commence avec Linn, Species Plantarum ( ann. 1 75 3 ) pour les groupes de plantes vasculaires. The 1 9th IB C will be in S henzhen, C hina, in 2 01 7. The frst International Zoological C ongress was held in Paris in 1 889. It was recognized that internationally accepted rules or naming and classiying animal species were needed and these were agreed at this and subsequent congresses. 1 75 8 was chosen as the starting date or valid names o animal species as this was when Linnaeus published Systema Natura in which he gave binomials or all species known then. The current International C ode or Zoological Nomenclature is the 4th edition and there will no doubt be more editions in the uture as scientists refne the methods that they use or naming species.

 Figure 2 Linnaea borealis. Binomials

are often chosen to honour a biologist, or to describe a feature of the organism. Linnaea borealis is named in honour of Carl Linnaeus, the Swedish biologist who introduced the binomial system of nomenclature and named many plants and animals using it

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the binomial sysem When species are discovered they are given scientifc names using the binomial system. The system that biologists use is called binomial nomenclature, because the international name o a species consists o two words. An example is Linnaea borealis ( fgure 2 ) . The frst name is the genus name. A genus is a group o species that share certain characteristics. The second name is the species or specifc name. There are various rules about binomial nomenclature:

ALLIGATORIDAE mississippiensis Alligator sinensis crocodilus Caiman

latirostris yacare

Melanosuchus

niger palpebrosus

Paleosuchus  Figure 3

trigonatus

Classifcation o the alligator amily



The genus name begins with an upper- case ( capital) letter and the species name with a lower-case ( small) letter.



In typed or printed text, a binomial is shown in italics.



Ater a binomial has been used once in a piece o text, it can be abbreviated to the initial letter o the genus name with the ull species name, or example: L. borealis.



The earliest published name or a species, rom 1 75 3 onwards or plants or 1 75 8 or animals, is the correct one.

the hierarchy of axa Taxonomists classiy species using a hierarchy o taxa. The word taxon is Greek and means a group o something. The plural is taxa. In biology, species are arranged or classifed into taxa. Every species is classifed into a genus. Genera are grouped into amilies. An example o the genera and species in a amily is shown in fgure 3 . Families are grouped into orders, orders into classes and so on up to the level o kingdom or domain. The taxa orm a hierarchy, as each taxon includes taxa rom the level below. Going up the hierarchy, the taxa include larger and larger numbers o species, which share ewer and ewer eatures.

the hree domains All organisms are classifed into three domains. Traditional classifcation systems have recognized two maj or categories o organisms based on cell types: eukaryotes and prokaryotes. This classifcation is now regarded as inappropriate because the prokaryotes have been ound to be very diverse. In particular, when the base sequence o ribosomal RNA was determined, it became apparent that there are two distinct groups o prokaryotes. They were given the names Eubacteria and Archaea. Most classifcation systems thereore now recognize three major categories o organism, Eubacteria, Archaea and Eukaryota. These categories are called domains, so all organisms are classifed into three domains. Table 1 shows some o the eatures that can be used to distinguish between them. Members o the domains are usually reerred to as bacteria, archaeans and eukaryotes. B acteria and eukaryotes are relatively amiliar to most biologists but archaeans are oten less well known.

260

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feaue Baea Histones associated Absent with DNA Presence o introns Rare or absent Structure o cell walls Made o chemical called peptidoglycan Cell membrane Glycerol-ester lipids; dierences unbranched side chains; d-orm o glycerol

Dma Ahaea Proteins similar to histones bound to DNA Present in some genes Not made o peptidoglycan

Eukaya Present

Frequent Not made o peptidoglycan; not always present Glycerol-ether lipids; Glycerol-ester lipids; unbranched side chains; l-orm unbranched side chains; o glycerol d-orm o glycerol

 Table 1

Archaeans are ound in a broad range o habitats such as the ocean surace, deep ocean sediments and even oil deposits ar below the surace o the Earth. They are also ound in some airly extreme habitats such as water with very high salt concentrations or temperatures close to boiling. The methanogens are obligate anaerobes and give o methane as a waste product o their metabolism. Methanogens live in the intestines o cattle and the guts o termites and are responsible or the production o marsh gas in marshes. Viruses are not classifed in any o the three domains. Although they have genes coding or proteins using the same genetic code as living organisms they have too ew o the characteristics o lie to be regarded as living organisms. Bacteria

Archaea

Eukaryota

Green lamentous Slime bacteria molds Animals Spirochetes Gram Methanobacterium Halophiles Fungi Proteobacteria positives Methanococcus Plants Cyanobacteria Ciliates Flagellates

 Figure 4 Tree diagram

showing relationships between living organisms based on base sequences o ribosomal RNA

Ay ideyg a kgdm This is a defnition o the characteristics o organisms in one o the kingdoms. Can you deduce which kingdom it is? Multicellular; cells typically held together by intercellular junctions; extracellular matrix with brous proteins, typically collagens, between two dissimilar epithelia; sexual with production of an egg cell that is fertilized by a smaller, often monociliated, sperm cell; phagotrophic and osmotrophic; without cell wall.

Eukaryote classifcation The principal taxa or classiying eukaryotes are kingdom, phylum, class, order, amily, genus and species. E ukaryotes are classifed into kingdoms. Each kingdom is divided up into phyla, which are divided into classes, then orders, amilies and genera. The hierarchy o taxa or classiying eukaryotes is thus kingdom, phylum, class, order, amily, genus and species. Most biologists recognize our kingdoms o eukaryote: plants, animals, ungi and protoctista. The last o these is the most controversial as protoctists are very diverse and should be divided up into more kingdoms. At present there is no consensus on how this should be done.

 Figure 5 Brown

seaweeds have been classifed in the kingdom Protoctista

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Examples o classifcatio Classifcation o one plant and one animal species rom domain to species level. Animals and plants are kingdoms o the domain Eukaryota. Table 2 shows the classication o one plant and one animal species rom kingdom down to species.

taxon Kingdom Phylum Class Order Family Genus Species

Grey wolf Animalia Chordata Mammalia Carnivora Canidae Canis lupus

Dae palm Plantae Angiospermophyta Monocotyledoneae Palmales Arecaceae Phoenix dactylifera

 Table 2

Daa-based quesions: Classiying cartilaginous fsh All the sh shown in gure 6 are in the class C hondrichthyes. They are the most requently ound sh in this class in north- west Europe.

1

S tate the kingdom to which all o the species in gure 6 belong. [1 ]

2

a)

Four o the sh in gure 6 are classied in the same genus. D educe which these sh are. [1 ]

b) D educe with a reason whether these our sh are in: ( i) the same or dierent species

[2 ]

( ii) the same or dierent amilies.

[2 ]

c) State two characteristics o these our sh that are not possessed by the other our sh. [2 ] 3  Figure 6 Cartilaginous fsh in

seas in north-west Europe

The other our sh are classied into two orders. D educe, with a reason, how the our sh are split into two orders. [2 ]

natural classifcatio In a natural classifcation, the genus and accompanying higher taxa consist o all the species that have evolved rom one common ancestral species. Scientic consensus is to classiy species in a way that most closely ollows the way in which species evolved. Following this convention, all members o a genus or higher taxon should have a common ancestor. This is called a natural classication. Because o the common ancestry we can expect the members o a natural group to share many characteristics.

262

An example o an unnatural or articial classication would be one in which birds, bats and insects are grouped together, because they all fy. Flight evolved separately in these groups and as they do not share a common ancestor they dier in many ways. It would not be appropriate to classiy them together other than to place them

5 . 3 c l A s s i  i c At i o n o  B i o D i v E r s i t Y all in the animal kingdom and both birds and bats in the phylum C hordata. Plants and ungi were at one time classifed together, presumably because they have cell walls and do not move, but this is an artifcial classifcation as their cell walls evolved separately and molecular research shows that they are no more similar to each other than to animals. It is not always clear which groups o species do share a common ancestor, so natural classifcation can be problematic. C onvergent evolution can make

distantly related organisms appear superfcially similar and adaptive radiation can make closely related organisms appear dierent. In the past, natural classifcation was attempted by looking at as many visible characteristics as possible, but new molecular methods have been introduced and these have caused signifcant changes to the classifcation o some groups. More details o this are given later, in sub-topic 5 .4.

TOK Wha a fuee he deepme  a e eu? Carl Linnaeuss 1753 book Species Plantarum introduced consistent two-part names (binomials) or all species o the vegetable kingdom then known. Thus the binomial Physalis angulata replaced the obsolete phrase-name, Physalis annua ramosissima, ramis angulosis glabris, foliis dentato-serratis. Linnaeus brought the scientifc nomenclature o plants back to the simplicity and brevity o the vernacular nomenclature out o which it had grown. Folk-names or species rarely exceed three words. In groups o species alike enough to have a vernacular group-name, the species are oten distinguished by a single name attached to the group-name, as in the Ancient Greek    and    (used by Threophrastus), Latin anagallis mas and anagallis emina (used by Pliny), German weiss Seeblumen and geel Seeblumen (used by Fuchs), English wild mynte and water mynte (used by Turner) and Malayan jambu bol and jambu chilli (applied by Malays to dierent species o Eugenia). The International Botanical Congress held in Genoa in 1892 proposed that 1753 be taken as the starting point or both

genera and species. This was incorporated in the American Rochester Code o 1883 and in the code used at the Berlin Botaniches Museum and supported by British Museum o Natural History, Harvard University botanists and a group o Swiss and Belgian botanists. The International Botanical Congress o Vienna in 1905 accepted by 150 votes to 19 the rule that La nomenclature botanique commence avec Linn, Species Plantarum (ann. 1753) pour les groupes de plantes vasculaires. 1 Why was Linnaeuss system or naming plants adopted as the international system, rather than any other system? 2 Why do the international rules o nomenclature state that genus and species names must be in Ancient Greek or Latin? 3 Making decisions by voting is rather unusual in science. Why is it done at International Botanical Congresses? What knowledge issues are associated with this method o decision making?

reviewing classifcation Taxonomists sometimes reclassiy groups o species when new evidence shows that a previous taxon contains species that have evolved rom dierent ancestral species. S ometimes new evidence shows that members o a group do not share a common ancestor, so the group should be split up into two or more taxa. C onversely species classifed in dierent taxa are sometimes ound to be closely related, so two or more taxa are united, or species are moved rom one genus to another or between higher taxa. The classifcation o humans has caused more controversy than any other species. Using standard taxonomic procedures, humans are assigned to the order Primates and the amily Hominidae. There has been much debate about which, i any, o the great apes to include in this amily. O riginally all the great apes were placed in another amily,

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E vo l u t i o n an d b i o d i vE r s i t y the Pongidae, but research has shown that chimpanzees and gorillas are closer to humans than to orang- utans and so should be in the same amily. This would j ust leave orang- utans in the Pongidae. Most evidence suggests that chimpanzees are closer than gorillas to humans, so i humans and chimpanzees are placed in dierent genera, gorillas should also be in a separate genus. A summary o this scheme or human classication is shown in gure 7.

FAMILY

GENUS AND SPECIES  Figure 7

Pongidae

Hominidae

Gorilla gorilla (gorilla)

Homo sapiens (human)

Pan troglodytes (chimpanzee)

Pan paniscus (bonobo)

Pongo pygmaeus (orang-utan)

Classifcation o humans

advntges o nturl clssifction Natural classications help in identication o species and allow the prediction o characteristics shared by species within a group. There is great interest at the moment in the biodiversity o the world. Groups o biologists are surveying areas where little research has been done beore, to nd out what species are present. Even in well-known parts o the world new species are sometimes discovered. Natural classication o species is very helpul in research into biodiversity. It has two specic advantages. 1

Identication o species is easier. I a specimen o an organism is ound and it is not obvious what species it is, the specimen can be identied by assigning it rst to its kingdom, then the phylum within the kingdom, class within the phylum and so on down to species level. D ichotomous keys can be used to help with this process. This process would not work so well with an articial classication. For example, i fowering plants were classied according to fower colour and a white- fowered bluebell Hyacinthoides non-scripta was discovered, it would not be identied correctly as the species normally has blue fowers.

2

B ecause all o the members o a group in a natural classication have evolved rom a common ancestral species, they inherit similar characteristics. This allows prediction o the characteristics o species within a group. For example, i a chemical that is useul as a drug is ound in one plant in a genus, this or related chemicals are likely to be ound in other species in the genus. I a new species o bat was discovered, we could make many predictions about it with reasonable certainty that they are correct: the bat will have hair, mammary glands, a placenta, a our- chambered heart and many other mammalian eatures. None o these predictions could be made i bats were classied articially with all other fying organisms.

 Figure 8 Members o the Hominidae

and Pongidae

Ativity controlling potato blight Phytophthora infestans, the organism that causes the disease potato blight, has hyphae and was classied as a ungus, but molecular biology has shown that it is not a true ungus and should be classied in a dierent kingdom, possibly the Protoctista. Potato blight has proved to be a difcult disease to control using ungicides. Discuss reasons or this.

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dichotomous keys Construction o dichotomous keys or use in identiying specimens D ichotomous keys are oten constructed to use or identiying species within a group. A dichotomy is a division into two; a dichotomous key consists o a numbered series o pairs o descriptions. O ne o these should clearly match the species and the other should clearly be wrong. The eatures that the designer o the key chooses to use in the descriptions should thereore be reliable and easily visible. E ach o the pair o descriptions leads either to another o the numbered pairs o descriptions in the key, or to an identifcation. An example o a key is shown in table 3 . We can use it to identiy the species in fgure 9. In the frst stage o the key, we must decide i hind limbs are visible. They are not, so we are directed to stage 6 o the key. We must now decide i the species has a blowhole. It does not, so it is a dugong or a manatee. A uller key would have another stage to separate dugongs and manatees.

1 Fore and hind limbs visible, can emerge on land ..... 2 Only ore limbs visible, cannot live on land ................ 6 2 Fore and hind limbs have paws ..................................... 3 Fore and hind limbs have fippers ................................. 4 3 Fur is dark ............................................................ sea otters Fur is white ........................................................ polar bears 4 External ear fap visible ........... sea lions and ur seals No external ear fap ........................................................... 5 5 Two long tusks ..................................................... walruses No tusks ............................................................... true seals 6 Mouth breathing, no blowhole ... dugongs and manatees Breathing through blowholes ......................................... 7 7 Two blowholes, no teeth ......................... baleen whales One blowhole, teeth ........ dolphins, porpoises and whales  Table 3

Key to groups of marine mammals

Ay cug dhmu key Keys are usually designed or use in a particular area. All the groups or species that are ound in that area can be identied using the key. There may be a group o organisms in your area or which a key has never been designed. 

You could design a key to the trees in the local orest or on your school campus, using lea descriptions or bark descriptions.



You could design a key to birds that visit bird-eeding stations in your area.



You could design a key to the invertebrates that are associated with one particular plant species.



You could design a key to the ootprints o mammals and birds (gure 10) . They are all right ront ootprints and are not shown to scale.

bear

duck

wolf

rabbit / hare

fox

cat

dog

squirrel

deer

heron

 Figure 10 Footprints of mammals and

 Figure 9

Manatee

birds

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Plants External recognition eatures o bryophytes, licinophytes, conierophytes and angiospermophytes. All plants are classied together in one kingdom. In the lie cycle o every plant, male and emale gametes are ormed and use together. The zygote ormed develops into an embryo. The way in which this embryo develops depends on the type o plant it is. The dierent types o plants are put into phyla. Most plants are in one o our phyla, but there are other smaller phyla. The Ginkgo biloba tree or

Bryophyta Vegetative organs  parts o the plant concerned with growth rather than reproduction



B ryophyta  mosses, liverworts and hornworts



Filicinophyta  erns



C onierophyta  coniers



Angiospermophyta  fowering plants.

The external recognition eatures o these phyla are shown in table 4.

filiinophyta

conierophyta

Angiospermophyta

Rhizoids but no Roots, stems and leaves are usually present true roots. Some with simple stems and leaves; others have only a thallus

No xylem or Vascular tissue  tissues with tubular structures used phloem or transport within the plant

Xylem and phloem are both present

Cambium  cells between xylem and phloem that can produce more o these tissues

No cambium; no true trees and shrubs

Present in coniers and most angiosperms, allowing secondary thickening o stems and roots and development o plants into trees and shrubs

Pollen  small structures containing male gametes that are dispersed

Pollen is not produced

Pollen is produced in male cones

Ovules  contains a emale gamete and develops into a seed ater ertilization

No ovaries or ovules

Ovules are produced Ovules are enclosed in emale cones inside ovaries in fowers

Seeds  dispersible unit consisting o an embryo plant and ood reserves, inside a seed coat

No seeds

Seeds are produced and dispersed

Fruits  seeds together with a ruit wall developed rom the ovary wall

No ruits

 Table 4

266

example is in one o the smaller phyla. The our main plant phyla are:

Pollen is produced by anthers in fowers

Fruits produced or dispersal o seeds by mechanical, wind or animal methods

5 . 3 c l A s s i f i c At i o n o f B i o D i v E r s i t Y

animl phyl Recognition eatures o poriera, cnidaria, platyhelminthes, annelida, mollusca and arthropoda, chordata. Animals are divided up into over 3 0 phyla, based on their characteristics. Six phyla are eatured in table 5 . Two examples o each are shown in fgure 1 1 .

Phyum

Muh/au

Poriera  an sponges, cup sponges, tube sponges, glass sponges

No mouth or anus

None

Internal spicules (sketetal needles)

Many pores over the surace through which water is drawn in or lter eeding. Very varied shapes

Cnidaria  hydras, jellysh, corals, sea anemones

Mouth only

Radial

Sot, but hard corals secrete CaCO 3

Tentacles arranged in rings around the mouth, with stinging cells. Polyps or medusae (jellysh)

Platyhelminthes  fatworms, fukes, tapeworms

Mouth only

Bilateral

Sot, with no skeleton

Flat and thin bodies in the shape o a ribbon. No blood system or system or gas exchange

Mollusca  bivalves, gastropods, snails, chitons, squid, octopus

Mouth and anus

Bilateral

Most have shell made o CaCO 3

A old in the body wall called the mantle secretes the shell. A hard rasping radula is used or eeding

Annelida  marine bristleworms, oligochaetes, leeches

Mouth and anus

Bilateral

Internal cavity with fuid under pressure

Bodies made up o many ringshaped segments, oten with bristles. Blood vessels oten visible

Arthropoda  insects, arachnids, crustaceans, myriapods

Mouth and anus

Bilateral

External skeleton made o plates o chitin

Segmented bodies and legs or other appendages with joints between the sections

 Table 5 Characteristics of six animal

1 2

symmey

skee

ohe exea eg eaue

phyla

Study the organisms shown in fgure 1 1 and assign each one to its phylum.

3 [7]

List the organisms that have: a) j ointed appendages

List the organisms that are:

b) stinging tentacles

a) bilaterally symmetric

c) bristles.

[3 ]

List the organisms that flter eed by pumping water through tubes inside their bodies.

[2 ]

b) radially symmetric c) not symmetrical in their structure.

4 [3 ]

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5

E vo l u t i o n an d b i o d i vE r s i t y

vertebrates Recognition o eatures o birds, mammals, amphibians, reptiles and fsh. Alcyonium glomeratum

Adocia cinerea

Nymphon gracilis

Pycnogonum littorale

Most species o chordate belong to one o fve major classes, each o which contains more than a thousand species. Although the numbers are not certain and new species are still sometimes discovered, there are about 1 0,000 bird species, 9,000 reptiles, 6,000 amphibians and 5 ,700 mammals. All o these classes are outnumbered by the ray-fnned bony fsh, with more than 30,000 species. The recognition eatures o the fve largest classes o chordate are shown in table 6. All o the organisms are vertebrates, because they have a backbone composed o vertebrae.

Bony ayfnned fsh

Corynactis viridis

Polymastia mammiliaris

Amphibians

reptiles

Lepidonotus clara

Sot moist skin permeable to water and gases

Impermeable skin covered in scales o keratin

Skin with Skin has eathers made ollicles with o keratin hair made o keratin

Cyanea capillata

Gills covered by an operculum, with one gill slit

Simple lungs with small olds and moist skin or gas exchange

Lungs with extensive olding to increase the surace area

Lungs with para-bronchial tubes, ventilated using air sacs

No limbs

Tetrapods with pentadactyl limbs

Fins supported by rays

Four legs when adult

Loligo forbesii

Arenicola marina

Eggs and sperm released or external ertilization Remain in water throughout their lie cycle

Larval stage that lives in water and adult that usually lives on land

Prostheceraeus vittatus

Swim bladder Eggs coated containing gas in protective or buoyancy jelly

Caprella linearis

Four legs (in Two legs and most species) two wings

Invertebrate diversity  Table 6

Lungs with alveoli, ventilated using ribs and a diaphragm Four legs in most (or two legs and two wings/arms)

Sperm passed into the emale or internal ertilization Female lays Most give eggs with hard birth to live young and shells all eed young with milk rom mammary glands Beak but no Teeth o Teeth all o one type, with teeth dierent types with a no living parts living core Female lays eggs with sot shells

Do not maintain constant body temperature Gammarus locusta

268

Mammals

Scales which are bony plates in the skin

Procerodes littoralis

 Figure 11

Bids

Maintain constant body temperature

5 . 4 cl AD i s ti cs

5.4 cad udertdig  A clade is a group o organisms that have 



 



evolved rom a common ancestor. Evidence or which species are part o a clade can be obtained rom the base sequences o a gene or the corresponding amino acid sequence o a protein. Sequence dierences accumulate gradually so there is a positive correlation between the number o dierences between two species and the time since they diverged rom a common ancestor. Traits can be analogous or homologous. Cladograms are tree diagrams that show the most probable sequence o divergence in clades. Evidence rom cladistics has shown that classifcations o some groups based on structure did not correspond with the evolutionary origins o a group o species.

applictio  Cladograms including humans and other

primates.  Reclassifcation o the fgwort amily using evidence rom cladistics.

skill  Analysis o cladograms to deduce evolutionary

relationships.

ntre of ciece  Falsifcation o theories with one theory being

superseded by another: plant amilies have been reclassifed as a result o evidence rom cladistics.

Clde A clade is a group o organisms that have evolved rom a common ancestor. S pecies can evolve over time and split to orm new species. This has happened repeatedly with some highly successul species, so that there are now large groups o species all derived rom a common ancestor. These groups o species can be identifed by looking or shared characteristics. A group o organisms evolved rom a common ancestor is called a clade. C lades include all the species alive today, together with the common ancestral species and any species that evolved rom it and then became extinct. They can be very large and include thousands o species, or very small with j ust a ew. For example, birds orm one large clade with about ten thousand living species because they have all evolved rom a common ancestral species. The tree Ginkgo biloba is the only living member o a clade that evolved about 2 70 million years ago. There have been other species in this clade but all are now extinct.

269

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Aciviy the EDGE of Exisence projec The aim o this project is to identiy animal species that have ew or no close relatives and are thereore members o very small clades. The conservation status o these species is then assessed. Lists are prepared o species that are both Evolutionarily Distinct and Globally Endangered, hence the name o the project. Species on these lists can then be targeted or more intense conservation eforts than other species that are either not

threatened or have close relatives. In some cases species are the last members o a clade that has existed or tens or hundreds o millions o years and it would be tragic or them to become extinct as a result o human activities. What species on EDGE lists are in your part o the world and what can you do to help conserve them? http://www.edgeoexistence.org/species/

 Figure 1

Two species on the EDGE list: Loris tardigradus tardigradus (Horton Plains slender loris) rom Sri Lanka and Bradypus pygmaeus (Pygmy three-toed sloth) rom Isla Escudo de Veraguas, a small island of the coast o Panama

Identifying members of a clade Evidence or which species are part o a clade can be obtained rom the base sequences o a gene or the corresponding amino acid sequence o a protein. It is not always obvious which species have evolved from a common ancestor and should therefore be included in a clade. The most obj ective evidence comes from base sequences of genes or amino acid sequences of proteins. S pecies that have a recent common ancestor can be expected to have few differences in base or amino acid sequence. C onversely, species that might look similar in certain respects but diverged from a common ancestor tens of millions of years ago are likely to have many differences.

270

5 . 4 cl AD i s ti cs

Moleculr clocks Sequence diferences accumulate gradually so there is a positive correlation between the number o diferences between two species and the time since they diverged rom a common ancestor. D ierences in the base sequence o D NA and thereore in the amino acid sequence o proteins are the result o mutations. They accumulate gradually over long periods o time. There is evidence that mutations occur at a roughly constant rate so they can be used as a molecular clock. The number o dierences in sequence can be used to deduce how long ago species split rom a common ancestor. For example, mitochondrial D NA rom three humans and our related primates has been completely sequenced. From the dierences in base sequence, a hypothetical ancestry has been constructed. It is shown in fgure 2 . Using dierences in base sequence as a molecular clock, these approximate dates or splits between groups have been deduced:

European Japanese African Common chimpanzee Pygmy chimpanzee (bonobo)



70, 000 years ago, E uropeanJapanese split

Gorilla



1 40, 000 years ago, AricanE uropean/Japanese split

Oran -utan



5 , 000, 000 years ago, humanchimpanzee split

 Figure 2

anlogous nd homologous trits Traits can be analogous or homologous. S imilarities between organisms can either be homologous or analogous. 

Homologous structures are similar because o similar ancestry; or example the chicken wing, human arm and other pentadactyl orelimbs.



Analogous structures are similar because o convergent evolution. The human eye and the octopus eye show similarities in structure and unction but they are analogous because they evolved independently.

Problems in distinguishing between homologous and analogous structures have sometimes led to mistakes in classifcation in the past. For this reason the morphology ( orm and structure) o organisms is now rarely used or identiying members o a clade and evidence rom base or amino acid sequences is trusted more. cornea iris lens retina photoreceptors optic nerve  Figure 3

The human eye (left) and the octopus eye (right) are analogous because they are quite similar yet evolved independently

271

5

birds non-avian dinosaurs crocodiles

lizards

snakes

turtles

E vo l u t i o n an d b i o d i vE r s i t y

ancestral species A ancestral species B ancestral species C  Figure 4 A cladogram

showing the hypothesized relationship between birds and the traditional taxonomic group the reptiles

Activity Figure 5 shows an artists impression o two pterosaurs, which were the rst chordates to develop powered fight. They were neither birds nor dinosaurs. Where might pterosaurs have tted into the cladogram shown in gure 4?

Cladograms Cladograms are tree diagrams that show the most probable sequence o divergence in clades. A cladogram is a tree diagram based on similarities and dierences between the species in a clade. C ladograms are almost always now based on base or amino acid sequences. C omputer programs have been developed that calculate how species in a clade could have evolved with the smallest number o changes o base or amino acid sequence. This is known as the principle o parsimony and although it does not prove how a clade actually evolved, it can indicate the most probable sequence o divergence in clades. The branching points on cladograms are called nodes. Usually two clades branch o at a node but sometimes there are three or more. The node represents a hypothetical ancestral species that split to orm two or more species. O ption B includes instructions or constructing cladograms rom base sequences using computer sotware. Figure 4 is an example o a cladogram or birds and reptiles. It has been based on morphology, so that extinct groups can be included. 

B irds, non- avian dinosaurs and ancestral species A orm a clade called dinosauria.



B irds, non- avian dinosaurs, crocodiles and ancestral species B are part o a clade called archosaurs.



Lizards, snakes and ancestral species C orm a clade called squamates.

This cladogram suggests either that birds should be regarded as reptiles or that reptiles should be divided into two or more groups, as some reptiles are more closely related to birds than to other reptiles.

 Figure 5 Two pterosaurs in

fight

Primate cladograms Cladograms including humans and other primates. The closest relatives o humans are chimpanzees and bonobos. The entire genome o these three species has been sequenced giving very strong evidence or the construction o a cladogram ( fgure 6) . The numbers on the cladogram are estimates o population sizes and dates when splits occurred. These are based on a molecular clock with a mutation rate o 1 0 9 yr 1 . Figure 7 is a cladogram or primates and the most closely related other groups o mammal. Primates are an order o mammals that have adaptations or climbing trees. Humans, monkeys, baboons, gibbons and lemurs are primates.

272

45,000 4.5 Myr ago

27,000 1 Myr ago 12,000 Bonobo  Figure 6

Chimpanzee

Human

5 . 4 cl AD i s ti cs Cavies and Coypu

anlysis of cldogrms

Porcupines

Analysis o cladograms to deduce evolutionary relationships.

Mice and Rats

The pattern o branching in a cladogram is assumed to match the evolutionary origins o each species. The sequence o splits at nodes is thereore a hypothetical sequence in which ancestors o existing clades diverged. I two clades on a cladogram are linked at a node, they are relatively closely related. I two species are only connected via a series o nodes, they are less closely related.

Rabbits

S ome cladograms include numbers to indicate numbers o dierences in base or amino acid sequence or in genes. B ecause genetic changes are assumed to occur at a relatively constant rate, these numbers can be used to estimate how long ago two clades diverged. This method o estimating times is called a molecular clock. S ome cladograms are drawn to scale according to estimates o how long ago each split occurred. Although cladograms can provide strong evidence or the evolutionary history o a group, they cannot be regarded as proo. C ladograms are constructed on the assumption that the smallest possible number o mutations occurred to account or current base or amino acid sequence dierences. S ometimes this assumption is incorrect and pathways o evolution were more convoluted. It is thereore important to be cautious in analysis o cladograms and where possible compare several versions that have been produced independently using dierent genes.

Beavers Chipmunks

Primates Treeshrews  Figure 7

Avy A adogram for he grea ape The great apes are a amily o primates. The taxonomic name is Hominidae. There are fve species on Earth today, all o which are decreasing in number apart rom humans. Figure 6 is a cladogram or three o the species. Use this inormation to expand the cladogram to include all the great apes: the split between humans and gorillas occurred about 10 million years ago and the split between humans and orangutans about 15 million years ago.

Daa-baed queon: Origins of turtles and lizards C ladograms based on morphology suggest that turtles and lizards are not a clade. To test this hypothesis, microRNA genes have been compared or nine species o chordate. The results were used to construct the cladogram in fgure 8. The numbers on the cladogram show which microRNA genes are shared by members o a clade but not members o other clades. For example, there are six microRNA genes ound in humans and short-tailed opossums but not in any o the other chordates on the cladogram. 1

the short- tailed opossum or to the duck-billed platypus. [2 ] 2

C alculate how many microRNA genes are ound in the mammal clade on the cladogram but not in the other clades. [2 ]

3

D iscuss whether the evidence in the cladogram supports the hypothesis that turtles and lizards are not a clade. [3 ]

4

Evaluate the traditional classifcation o tetrapod chordates into amphibians, reptiles, birds and mammals using evidence rom the cladogram. [3 ]

D educe, using evidence rom the cladogram, whether humans are more closely related to

273

5

E vo l u t i o n an d b i o d i vE r s i t y

African clawed frog

6 Human 340

671

761

885

1251

1397

3

Short-tailed opossum

186

590

873

Duck-billed platypus

19 Zebra nch 1451

1460

1467

1559

1641

1567

1669

1729

1743

1744

1756

1759

1781

1784

1789

1803

2131

2954

1791

1

2964

490

1397

1

Chicken

Alligator

1677

4 Painted turtle 5390

5391

5392

5393

Lizard

 Figure 8

Cladograms and reclassifcation Evidence rom cladistics has shown that classifcations o some groups based on structure did not correspond with the evolutionary origins o a group o species. The construction o cladograms based on base and amino acid sequences only became possible towards the end o the 2 0th century. B eore that the sequence data was not available and computer sotware had not been developed to do the analysis. The construction o cladograms and identifcation o clades is known as cladistics. C ladistics has caused some revolutions in plant and animal classifcation. It is now clear rom cladograms that traditional classifcation based on morphology does not always match the evolutionary origins o groups o species. As a result some groups have been reclassifed. S ome groups have been merged, others have been divided and in some cases species have been transerred rom one group to another. Reclassifcation o groups o organisms is time- consuming and potentially disruptive or biologists, but it is certainly worthwhile. The new classifcations based on cladistics are likely to be much closer to a truly natural classifcation so their predictive value will be higher. They have revealed some unnoticed similarities between groups and also some signifcant dierences between species previously assumed to be similar.

274

5 . 4 cl AD i s ti cs

Cladograms and alsifcation Falsifcation o theories with one theory being superseded by another: plant amilies have been reclassifed as a result o evidence rom cladistics. The reclassifcation o plants on the basis o discoveries in cladistics is a good example o an important process in science: the testing o theories and o replacement o theories ound to be alse with new theories. The classifcation o angiospermophytes into amilies based on their morphology was begun by the French botanist Antoine Laurent de Jussieu in Genera plantarum, published in 1 789 and revised repeatedly during the 1 9th century.

Classifcation o the fgwort amily Reclassifcation o the fgwort amily using evidence rom cladistics. There are more than 400 amilies o angiosperms. Until recently the eighth largest was the S crophulariaceae, commonly known as the fgwort amily. It was one o the original amilies proposed by de Jussieu in 1 789. He gave it the name S crophulariae and included sixteen genera, based on similarities in their morphology. As more plants were discovered, the amily grew until there were over 2 75 genera, with more than 5 , 000 species.

Taxonomists recently investigated the evolutionary origins o the fgwort amily using cladistics. O ne important research proj ect compared the base sequences o three chloroplast genes in a large number o species in genera traditionally assigned to the S crophulariaceae and genera in closely related amilies. It was ound that species in the fgwort amily were not a true clade and that fve clades had incorrectly been combined into one amily.

Two small families were merged with the gwort family: the buddleja family, Buddlejaceae and the myoporum family, Myoporaceae

Two genera were moved to a newly-created family, the calceolaria family, Calceolariaceae

The gwort family Scrophulariaceae

Thirteen genera have been transferred to a newly-created family, the lindernia family, Linderniaceae

Nearly fty genera have been moved to the plantain family, Plantaginaceae

About twelve genera of parasitic plants have been moved to the broomrape family, Orobanchaceae

 Figure 9

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E vo l u t i o n an d b i o d i vE r s i t y

A major reclassifcation has now been carried out. Less than hal o the species have been retained in the amily, which is now only the thirty-sixth largest among the angiosperms. A summary o the

 Figure 10

Antirrhinum majus has been transerred rom the fgwort amily to the plantain amily

276

changes is shown in fgure 9. This reclassifcation has been welcomed as it was widely appreciated beore that the Scrophulariaceae had been a rag-bag o species rather than a natural group.

 Figure 11

Scrophularia peregrina has remained in the fgwort amily

QuEstion s

Questions The bar charts in fgure 1 2 show the growth o three populations o an alga, Ectocarpus siliculosus, at dierent copper concentrations. O ne population came rom an unpolluted environment at Rhosneigr in Wales. The other two came rom the undersides o ships that had been painted with a copper- containing anti- ouling paint. % increase in algal volume

500

4

Which o the ollowing processes are required or copper tolerance to develop in a population? ( i)

variation in copper tolerance

( ii)

inheritance o copper tolerance

( iii) ailure o algae with lower copper tolerance to survive or reproduce. a)

Rhosneigr

i) only

b) i) and ii) only 0

c)

M.V. San Nicholas

i) and iii) only

d) i) , ii) and iii) .

500 0 M.V. Amama 500 0 0.0 0.01 0.05 0.1 0.5 1.0 5.0 10.0 concentration of copper (mg dm -3 )

5

In fgure 1 3 , each number represents a species. The closer that two numbers are on the diagram the more similar the two species. The circles represent taxonomic groups. For example, the diagram shows that 2 , 3 , 4 and 5 are in the same genus.

Figure 12 1

2

How much higher was the maximum copper concentration tolerated by the algae rom ships than the algae rom an unpolluted environment? a)

0.09 times higher

b) 0.1 1 times higher

c)

1 .0 times higher

d) 1 0 times higher.

1

8 9 10 19 20 21 22 23

What is the reason or results lower than zero on the bar charts? a)

Increases in volume were less than 1 00% .

d) Results were too small to measure accurately.

3

What was the reason or the dierence in copper tolerance between the algae? a)

The algae on the ships absorbed copper.

b) The algae can develop copper tolerance and pass it on to their ospring. c)

1112 13 14 15 16 17 18

34 67

24 25 26 27 28 29 30 31 32 33

The volume o algae decreased.

b) The algae all died. c)

23 45

The copper in the paint caused mutations.

d) The copper in the paint caused natural selection or higher levels o copper tolerance.

Figure 13 a)

S tate one species that is in a genus with no other species.

b) S tate the species that are in a amily with two genera. c)

S tate the species that are in an order with two amilies.

[1 ] [2 ] [2 ]

d) State the species that are in a class with three orders. [2 ] e)

D educe whether species 8 is more closely related to species 1 6 or species 6.

f)

Explain why three concentric circles have been drawn around species 3 4 on the diagram. [2 ]

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The map in gure 1 4 shows the distribution in the 1 95 0s o two orms o Biston betularia in B ritain and Ireland. Biston betularia is a species o moth that fies at night. It spends the daytime roosting on the bark o trees. The non-melanic orm has white wings, peppered with black spots. The melanic orm has black wings. B eore the industrial revolution, the melanic orm was very rare. The prevailing wind direction is rom the Atlantic O cean, to the west. a)

S tate the maximum and minimum percentages o the melanic orm.

Key Non-melanic Melanic

[2 ]

b) O utline the trends in the distribution o the two orms o Biston betularia, shown in gure 1 4. [2 ] c)

Explain how natural selection can cause moths such as Biston betularia to develop camoufaged wing markings. [4]

d) S uggest reasons or the distribution o the two orms.

278

[2 ]

Figure 14

6 H U m A N p H yS I o l o g y Intrductin Research into human physiology is the foundation of modern medicine. B ody functions are carried out by specialized organ systems. The structure of the wall of the small intestine allows it to move, digest and absorb food. The blood system continuously transports substances to cells and simultaneously collects waste

products. The skin and immune system resist the continuous threat of invasion by pathogens. The lungs are actively ventilated to ensure that gas exchange can occur passively. Neurons transmit the message, synapses modulate the message. Hormones are used when signals need to be widely distributed.

6.1 Digestion and absorption Understandin  The contraction o circular and longitudinal

    

muscle layers o the small intestine mixes the ood with enzymes and moves it along the gut. The pancreas secretes enzymes into the lumen o the small intestine. Enzymes digest most macromolecules in ood into monomers in the small intestine. Villi increase the surace area o epithelium over which absorption is carried out. Villi absorb monomers ormed by digestion as well as mineral ions and vitamins. Diferent methods o membrane transport are required to absorb diferent nutrients.

Aicatins  Processes occurring in the small intestine that

result in the digestion o starch and transport o the products o digestion to the liver.  Use o dialysis tubing to model absorption o digested ood in the intestine.

Skis  Production o an annotated diagram o the

digestive system.  Identication o tissue layers in transverse sections o the small intestine viewed with a microscope or in a micrograph.

Nature f science  Use models as representations o the real

world: dialysis tubing can be used to model absorption in the intestine.

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Structure of the digestive system Production of an annotated diagram of the digestive system. The part of the human body used for digestion can be described in simple terms as a tube through which food passes from the mouth to the anus. The role of the digestive system is to break down the diverse mixture of large carbon compounds in food, to yield ions and smaller compounds that can be absorbed. For proteins, lipids and polysaccharides digestion involves several stages that occur in different parts of the gut. D igestion requires surfactants to break up lipid droplets and enzymes to catalyse reactions. Glandular cells in the lining of the stomach and intestines produce some of the enzymes.

S urfactants and other enzymes are secreted by accessory glands that have ducts leading to the digestive system. C ontrolled, selective absorption of the nutrients released by digestion takes place in the small intestine and colon, but some small molecules, notably alcohol, diffuse through the stomach lining before reaching the small intestine. Figure 1 is a diagram of the human digestive system. The part of the esophagus that passes through the thorax has been omitted. This diagram can be annotated to indicate the functions of different parts. A summary of functions is given in table 1 below.

Structure mouth

Mouth

Voluntary control of eating and swallowing. Mechanical digestion of food by chewing and mixing with saliva, which contains lubricants and enzymes that start starch digestion

Esophagus

Movement of food by peristalsis from the mouth to the stomach

Stomach

Churning and mixing with secreted water and acid which kills foreign bacteria and other pathogens in food, plus initial stages of protein digestion

Small intestine

Final stages of digestion of lipids, carbohydrates, proteins and nucleic acids, neutralizing stomach acid, plus absorption of nutrients

Pancreas

Secretion of lipase, amylase and protease

Liver

Secretion of surfactants in bile to break up lipid droplets

Gall bladder

Storage and regulated release of bile

Large intestine

Re-absorption of water, further digestion especially of carbohydrates by symbiotic bacteria, plus formation and storage of feces

esophagus

gall bladder liver stomach pancreas small intestine

large intestine anus  Figure 1

280

The human digestive system

Function

 Table 1

6 .1 D i g e S ti o n an D ab S o rpti o n

Structure of the wall of the small intestine Identifcation o tissue layers in transverse sections o the small intestine viewed with a microscope or in a micrograph. The wall o the small intestine is made o layers o living tissues, which are usually quite easy to distinguish in sections o the wall. From the outside o the wall going inwards there are our layers: 

serosa  an outer coat



muscle layers  longitudinal muscle and inside it circular muscle



sub-mucosa  a tissue layer containing blood and lymph vessels



mucosa  the lining o the small intestine, with the epithelium that absorbs nutrients on its inner surace.

 Figure 2

Longitudinal section through the wall o the small intestine. Folds are visible on the inner surace and on these olds are fnger-like projections called villi. All o the our main tissue layers are visible, including both circular and longitudinal parts o the muscle layer. The mucosa is stained darker than the sub-mucosa

peristalsis The contraction o circular and longitudinal muscle layers o the small intestine mixes the ood with enzymes and moves it along the gut. The circular and longitudinal muscle in the wall o the gut is smooth muscle rather than striated muscle. It consists o relatively short cells, not elongated fbres. It oten exerts continuous moderate orce, interspersed with short periods o more vigorous contraction, rather than remaining relaxed unless stimulated to contract. Waves o muscle contraction, called peristalsis, pass along the intestine. C ontraction o circular muscles behind the ood constricts the gut to prevent it rom being pushed back towards the mouth. C ontraction o longitudinal muscle where the ood is located moves it on along the gut. The contractions are controlled unconsciously not by the brain but by the enteric nervous system, which is extensive and complex.

acvy tssu l dms f h s wll To practice your skill at identiying tissue layers, draw a plan diagram o the tissues in the longitudinal section o the intestine wall in fgure 2. To test your skill urther, draw a plan diagram to predict how the tissues o the small intestine would appear in a transverse section.

S wallowed ood moves quickly down the esophagus to the stomach in one continuous peristaltic wave. Peristalsis only occurs in one direction, away rom the mouth. When ood is returned to the mouth rom the stomach during vomiting, abdominal muscles are used rather than the circular and longitudinal muscle in the gut wall. In the intestines the ood is moved only a ew centimetres at a time so the overall progression through the intestine is much slower, allowing time or digestion. The main unction o peristalsis in the intestine is churning o the semi- digested ood to mix it with enzymes and thus speed up the process o digestion.

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pancreatic juice The pancreas secretes enzymes into the lumen of the small intestine. The pancreas contains two types o gland tissue. Small groups o cells secrete the hormones insulin and glucagon into the blood. The remainder o the pancreas synthesizes and secretes digestive enzymes into the gut in response to eating a meal. This is mediated by hormones synthesized and secreted by the stomach and also by the enteric nervous system. The structure o the tissue is shown in fgure 4. Small groups o gland cells cluster round the ends o tubes called ducts, into which the enzymes are secreted.

 Figure 3

Three-dimensional image showing the wave of muscle contraction (brown) in the esophagus during swallowing. Green indicates when the muscle is exerting less force. Time is shown left to right. At the top the sphincter between the mouth and the esophagus is shown permanently constricted apart from a brief opening when swallowing starts

The digestive enzymes are synthesized in pancreatic gland cells on ribosomes on the rough endoplasmic reticulum. They are then processed in the Golgi apparatus and secreted by exocytosis. Ducts within the pancreas merge into larger ducts, fnally orming one pancreatic duct, through which about a litre o pancreatic juice is secreted per day into the lumen o the small intestine. Pancreatic j uice contains enzymes that digest all the three main types o macromolecule ound in ood: 

amylase to digest starch



lipases to digest triglycerides, phospholipids



proteases to digest proteins and peptides.

Digestion in the small intestine

secretory vesicles

one acinus

Enzymes digest most macromolecules in food into monomers in the small intestine. The enzymes secreted by the pancreas into the lumen o the small intestine carry out these hydrolysis reactions:

basement membrane

secretory cells wall of duct



starch is digested to maltose by amylase



triglycerides are digested to atty acids and glycerol or atty acids and monoglycerides by lipase



phospholipids are digested to atty acids, glycerol and phosphate by phospholipase



proteins and polypeptides are digested to shorter peptides by protease.

lumen of duct  Figure 4 Arrangement of cells and

ducts in a part of the pancreas that secretes digestive enzymes

This does not complete the process o digestion into molecules small enough to be absorbed. The wall o the small intestine produces a variety o other enzymes, which digest more substances. S ome enzymes produced by gland cells in the intestine wall may be secreted in intestinal j uice but most remain immobilized in the plasma membrane o epithelium cells lining the intestine. They are active there and continue to be active when the epithelium cells are abraded o the lining and mixed with the semi- digested ood.

282



Nucleases digest D NA and RNA into nucleotides.



Maltase digests maltose into glucose.

6 .1 D i g e S ti o n an D ab S o rpti o n



Lactase digests lactose into glucose and galactose.



Sucrase digests sucrose into glucose and ructose.



Exopeptidases are proteases that digest peptides by removing single amino acids either rom the carboxy or amino terminal o the chain until only a dipeptide is let.



D ipeptidases digest dipeptides into amino acids.

B ecause o the great length o the small intestine, ood takes hours to pass through, allowing time or digestion o most macromolecules to be completed. Some substances remain largely undigested, because humans cannot synthesize the necessary enzymes. C ellulose or example is not digested and passes on to the large intestine as one o the main components o dietary fbre.

Villi and the surface area for digestion

 Figure 5 Cystic fbrosis causes the pancreatic

duct to become blocked by mucus. Pills containing synthetic enzymes help digestion in the small intestine. The photograph shows one days supply or a person with cystic fbrosis

Villi increase the surface area of epithelium over which absorption is carried out. The process o taking substances into cells and the blood is called absorption. In the human digestive system nutrients are absorbed epithelium principally in the small intestine. The rate o absorption depends on the surace area o the epithelium that carries out the process. The small intestine in adults is approximately seven metres long and layer of microvilli 2 5 3 0 millimetres wide and there are olds on its inner surace, giving on surface of epithelium a large surace area. This area is increased by the presence o villi. Villi are small fnger-like proj ections o the mucosa on the inside o the intestine wall. A villus is between 0. 5 and 1 . 5 mm long and there can be as many as 40 o them per square millimetre o small intestine wall. They increase the surace area by a actor o about 1 0.

lacteal (a branch of the lymphatic system)

blood capillary

goblet cells (secrete mucus)

Absorption by villi Villi absorb monomers formed by digestion as well as mineral ions and vitamins. The epithelium that covers the villi must orm a barrier to harmul substances, while at the same time being permeable enough to allow useul nutrients to pass through.

 Figure 6 Structure o an

intestinal villus

Villus cells absorb these products o digestion o macromolecules in ood: 

glucose, ructose, galactose and other monosaccharides



any o the twenty amino acids used to make proteins



atty acids, monoglycerides and glycerol



bases rom digestion o nucleotides.

They also absorb substances required by the body and present in oods but not needing digestion: 

mineral ions such as calcium, potassium and sodium



vitamins such as ascorbic acid ( vitamin C ) .

 Figure 7

Scanning electron micrograph o villi in the small intestine

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Hum C Ean LLpBHI ys O LO i oGlo Y gy S ome harmul substances pass through the epithelium and are subsequently removed rom the blood and detoxied by the liver. S ome harmless but unwanted substances are also absorbed, including many o those that give ood its colour and favour. These pass out in urine. S mall numbers o bacteria pass through the epithelium but are quickly removed rom the blood by phagocytic cells in the liver.

methods of absorption Diferent methods o membrane transport are required to absorb diferent nutrients. To be absorbed into the body, nutrients must pass rom the lumen o the small intestine to the capillaries or lacteals in the villi. The nutrients must rst be absorbed into epithelium cells through the exposed part o the plasma membrane that has its surace area enlarged with microvilli. The nutrients must then pass out o this cell through the plasma membrane where it aces inwards towards the lacteal and blood capillaries o the villus. Many dierent mechanisms move nutrients into and out o the villus epithelium cells: simple diusion, acilitated diusion, active transport and exocytosis. These methods can be illustrated using two dierent examples o absorption: triglycerides and glucose. 

Triglycerides must be digested beore they can be absorbed. The products o digestion are atty acids and monoglycerides, which can be absorbed into villus epithelium cells by simple diusion as they can pass between phospholipids in the plasma membrane.



Fatty acids are also absorbed by acilitated diusion as there are atty acid transporters, which are proteins in the membrane o the microvilli.



O nce inside the epithelium cells, atty acids are combined with monoglycerides to produce triglycerides, which cannot diuse back out into the lumen.

lumen of small intestine

interior of villus

villus epithelium

Na +

3Na + low Na + concentration

glucose

blood capillary

2K+ glucose

fatty acids and monoglycerides lacteal

triglyceride  Figure 8

284

Methods of absorption in the small intestine

lipoprotein

6 .1 D i g e S ti o n an D ab S o rpti o n



Triglycerides coalesce with cholesterol to orm droplets with a diameter o about 0. 2 m, which become coated in phospholipids and protein.



These lipoprotein particles are released by exocytosis through the plasma membrane on the inner side o the villus epithelium cells. They then either enter the lacteal and are carried away in the lymph, or enter the blood capillaries in the villi.



Glucose cannot pass through the plasma membrane by simple diusion because it is polar and thereore hydrophilic.



Sodiumpotassium pumps in the inwards- acing part o the plasma membrane pump sodium ions by active transport rom the cytoplasm to the interstitial spaces inside the villus and potassium ions in the opposite direction. This creates a low concentration o sodium ions inside villus epithelium cells.



Sodiumglucose co-transporter proteins in the microvilli transer a sodium ion and a glucose molecule together rom the intestinal lumen to the cytoplasm o the epithelium cells. This type o acilitated diusion is passive but it depends on the concentration gradient o sodium ions created by active transport.



Glucose channels allow the glucose to move by acilitated diusion rom the cytoplasm to the interstitial spaces inside the villus and on into blood capillaries in the villus.

Starch digestion in the small intestine Processes occurring in the small intestine that result in the digestion of starch and transport of the products of digestion to the liver. S tarch digestion illustrates some important processes including catalysis, enzyme specifcity and membrane permeability. S tarch is a macromolecule, composed o many -glucose monomers linked together in plants by condensation reactions. It is a maj or constituent o plant- based oods such as bread, potatoes and pasta. S tarch molecules cannot pass through membranes so must be digested in the small intestine to allow absorption. All o the reactions involved in the digestion o starch are exothermic, but without a catalyst they happen at very slow rates. There are two types o molecule in starch: 

amylose has unbranched chains o - glucose linked by 1 , 4 bonds;



amylopectin has chains o -glucose linked by 1 , 4 bonds, with some 1 , 6 bonds that make the molecule branched.

CH 2 OH O OH OH

CH 2 OH O OH O

O OH

OH CH 2 OH O OH

OH

CH 2 OH O

OH

CH 2 OH O OH

O O OH

CH 2 OH O OH O

OH

O OH

 Figure 9

Small portion of an amylopectin molecule showing six -glucose molecules, all linked bv 1,4 bonds apart from one 1,6 bond that creates a branch

The enzyme that begins the digestion o both orms o starch is amylase. S aliva contains amylase but most starch digestion occurs in the small intestine, catalysed by pancreatic amylase. Any 1 , 4 bond in starch molecules can be broken by this enzyme, as long as there is a chain o at least our glucose monomers. Amylose is thereore

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digested into a mixture o two- and three- glucose ragments called maltose and maltotriose. B ecause o the specicity o its active site, amylase cannot break 1 , 6 bonds in amylopectin. Fragments o the amylopectin molecule containing a 1 , 6 bond that amylase cannot digest are called dextrins. D igestion o starch is completed by three enzymes in the membranes o microvilli on villus epithelium cells. Maltase, glucosidase and dextrinase digest maltose, maltotriose and dextrins into glucose. Glucose is absorbed into villus epithelium cells by co- transport with sodium ions. It then moves by acilitated diusion into the fuid in interstitial spaces inside the villus. The dense network o

capillaries close to the epithelium ensures that glucose only has to travel a short distance to enter the blood system. C apillary walls consist o a single layer o thin cells, with pores between adj acent cells, but these capillaries have larger pores than usual, aiding the entry o glucose. B lood carrying glucose and other products o digestion fows though villus capillaries to venules in the sub- mucosa o the wall o the small intestine. The blood in these venules is carried via the hepatic portal vein to the liver, where excess glucose can be absorbed by liver cells and converted to glycogen or storage. Glycogen is similar in structure to amylopectin, but with more 1 , 6 bonds and thereore more extensive branching.

modelling physiological processes Use models as representations of the real world: dialysis tubing can be used to model absorption in the intestine. Living systems are complex and when experiments are done on them, many actors can infuence the results. It can be very dicult to control all o the variables and analysis o results becomes dicult. S ometimes it is better to carry out experiments using only parts o systems. For example, much research in physiology has been carried out using clones o cells in tissue culture rather than whole organisms. Another approach is to use a model to represent part o a living system. B ecause it is much simpler, a model can be used to investigate specic aspects o a process. A recent example is the D ynamic Gastric Model, a computer- controlled model o the human stomach that carries out mechanical and chemical digestion o real ood samples. It can be used to investigate the eects o diet, drugs, alcohol and other actors on digestion. A simpler example is the use o dialysis tubing made rom cellulose. Pores in the tubing allow water and small molecules or ions to pass through reely, but not large molecules. These properties

286

 Figure 10

The Dynamic Gastric Model with its inventor, Richard Faulks, adjusting the antrum mechanism

mimic the wall o the gut, which is also more permeable to small rather than large particles. D ialysis tubing can be used to model absorption by passive diusion and by osmosis. It cannot model active transport and other processes that occur in living cells

6 .1 D i g e S ti o n an D ab S o rpti o n

modelling the sall intestine Use of dialysis tubing to model absorption of digested food in the intestine. To make a model o the small intestine, cut a length o dialysis tubing and seal one end by tying a knot in the tubing or tying with a piece o cotton thread. Pour in a suitable mixture o oods and seal the open end by tying with a piece o cotton thread. Two experiments using model intestines made in this way are suggested here:

1 Investigating the need for digestion using a model of the small intestine S et up the apparatus shown in gure 1 1 and leave it or one hour.

Results To obtain the results or the experiment, take the bags out o each tube, open them and pour the solutions rom them into separate test tubes rom the liquids in the tubes. You should now have our samples o fuid. D ivide each o these samples into two halves and test one hal or starch and the other hal or sugars.

10 ml of 1% starch solution and 1 ml of 1% amylase solution

10 ml of 1% starch solution and 1 ml of water

water maintained at 40C

water

 Figure 11

bags made of dialysis (Visking) tubing

water

S uggest improvements to the method, or suggest an entirely dierent method o investigating the need or digestion.

2 Investigating membrane permeability using a model of the small intestine C ola drinks contain a mixture o substances with dierent particle sizes. They can be used to represent ood in the small intestine. D ialysis tubing is semi- permeable so can be used to model the wall o the small intestine.

Predictions C ola contains glucose, phosphoric acid and caramel, a complex carbohydrate added to produce a brown colour. Predict which o these substances will diuse out o the bag, with reasons or your predictions. Predict whether the bag will gain or lose mass during the experiment.

Instructions 1

Make the model intestine with cola inside.

2

Rinse the outside o the bag to wash o any traces o cola and then dry the bag.

tube top of bag sealed with cotton thread cola, left to go at before being put into the tube

dialysis tubing

pure water  minimum volume to surround the bag

base of bag knotted to prevent leaks

Apparatus for showing the need for digestion

Record all the results in the way that you think is most appropriate.

spotting tile

Conclusions and evaluation S tate careully all the conclusions that you can make rom your results. D iscuss the strengths and weaknesses o this method o investigating the need or digestion.

pH indicator  Figure 12

Apparatus for membrane permeability experiment

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3

Find the mass o the bag using an electronic balance.

4

When you are ready to start the experiment, place the bag in pure water in a test tube.

5

Test the water around the bag at suitable time intervals. A suggested range is 1 , 2 , 4, 8 and 1 6 minutes. At each time lit the bag up and down a ew times to mix the water in the tube, then do these tests: 

Look careully at the water to see whether it is still clear or has become brown.



Use a dropping pipette to remove a ew drops o the water and test them in a spotting tile with a narrow- range pH indicator. Use a colour chart to work out the pH.



D ip a glucose test strip into the water and record the colour that it turns. Instructions

vary or these test strips. Follow the instructions and work out the glucose concentration o the water. 6

Ater testing the water or the last time, remove the bag, dry it and fnd its mass again with the electronic balance.

Conclusions a) Explain the conclusions that you can draw about the permeability o the dialysis tubing rom the tests o the water and rom the change in mass o the bag. [5 ] b) C ompare and contrast the dialysis tubing and the plasma membranes that carry out absorption in villus epithelium cells in the wall o the intestine. [5 ] c) Use the results o your experiment to predict the direction o movement o water by osmosis across villus epithelium cells. [5 ]

TOK What are some o the variables that afect perspectives as to what is normal? In some adult humans, levels o lactase are too low to digest lactose in milk adequately. Instead, lactose passes through the small intestine into the large intestine, where bacteria eed on it, producing carbon dioxide, hydrogen and methane. These gases cause some unpleasant symptoms, discouraging consumption o milk. The condition is known as lactose intolerance. It has sometimes in the past been regarded as an abnormal condition, or even as a disease, but it could be argued that lactose intolerance is the normal human condition. The rst argument or this view is a biological one. Female mammals produce milk to eed their young ofspring. When a young mammal is weaned, solid oods replace milk and lactase secretion declines. Humans who

288

continue to consume milk into adulthood are thereore unusual. Inability to consume milk because o lactose intolerance should not thereore be regarded as abnormal. The second argument is a simple mathematical one: a high proportion o humans are lactose intolerant. The third argument is evolutionary. Our ancestors were almost certainly all lactose intolerant, so this is the natural or normal state. Lactose tolerance appears to have evolved separately in at least three centres: Northern Europe, parts o Arabia, the Sahara and eastern Sudan, and parts o East Arica inhabited by the Tutsi and Maasai peoples. Elsewhere, tolerance is probably due to migration rom these centres.

6 . 2 t h e b l o o D S yS t e m

6.2 t d ss Understanding  Arteries convey blood at high pressure rom the  





  

 





ventricles to the tissues o the body. Arteries have muscle and elastic bres in their walls. The muscle and elastic bres assist in maintaining blood pressure between pump cycles. Blood fows through tissues in capillaries with permeable walls that allow exchange o materials between cells in the tissue and the blood in the capillary. Veins collect blood at low pressure rom the tissues o the body and return it to the atria o the heart. Valves in veins and the heart ensure circulation o blood by preventing backfow. There is a separate circulation or the lungs. The heartbeat is initiated by a group o specialized muscle cells in the right atrium called the sinoatrial node. The sinoatrial node acts as a pacemaker. The sinoatrial node sends out an electrical signal that stimulates contraction as it is propagated through the walls o the atria and then the walls o the ventricles. The heart rate can be increased or decreased by impulses brought to the heart through two nerves rom the medulla o the brain. Epinephrine increases the heart rate to prepare or vigorous physical activity.

Applications  William Harveys discovery o the circulation o

the blood with the heart acting as the pump.  Causes and consequences o occlusion o the coronary arteries.  Pressure changes in the let atrium, let ventricle and aorta during the cardiac cycle.

Skills  Identication o blood vessels as arteries,

capillaries or veins rom the structure o their walls.  Recognition o the chambers and valves o the heart and the blood vessels connected to it in dissected hearts or in diagrams o heart structure.

Nature of science  Theories are regarded as uncertain: William

Harvey overturned theories developed by the ancient Greek philosopher Galen on movement o blood in the body.

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William Harvey and the circulatin f bld William Harveys discovery of the circulation of the blood with the heart acting as the pump. William Harvey is usually credited with the discovery o the circulation o the blood as he combined earlier discoveries with his own research ndings to produce a convincing overall theory or blood fow in the body. He overcame widespread opposition by publishing his results and also by touring Europe to demonstrate experiments that alsied previous theories and provided evidence or his theory. As a result his theory became generally accepted.

published his theory about the circulation o blood in 1 62 8. It was not until 1 660, ater his death, that blood was seen fowing rom arteries to veins though capillaries as he had predicted.

Harvey demonstrated that blood fow through the larger vessels is unidirectional, with valves to prevent backfow. He also showed that the rate o fow through maj or vessels was ar too high or blood to be consumed in the body ater being pumped out by the heart, as earlier theories proposed. It must thereore return to the heart and be recycled. Harvey showed that the heart pumps blood out in the arteries and it returns in veins. He predicted the presence o numerous ne vessels too small to be seen with contemporary equipment that linked arteries to veins in the tissues o the body. B lood capillaries are too narrow to be seen with the naked eye or with a hand lens. Microscopes had not been invented by the time that Harvey

 Figure 1

Harveys experiment to demonstrate that blood fow in the veins o the arm is unidirectional

overturning ancient theries in science Theories are regarded as uncertain: William Harvey overturned theories developed by the ancient Greek philosopher Galen on movement of blood in the body. D uring the Renaissance, interest was reawakened in the classical writings o Greece and Rome. This stimulated literature and the arts, but in some ways it hampered progress in science. It became almost impossible to question the doctrines o such writers as Aristotle, Hippocrates, Ptolemy and Galen. According to Galen, blood is ormed in the liver and is pumped to and ro between the liver and the right ventricle o the heart. A little blood passes into the let ventricle, where it meets air rom the lungs and becomes vital spirits. The

290

vital spirits are distributed to the body by the arteries. S ome o the vital spirits fow to the brain, to be converted into animal spirits, which are then distributed by the nerves to the body. William Harvey was unwilling to accept these doctrines without evidence. He made careul observations and did experiments, rom which he deduced that blood circulates through the pulmonary and systemic circulations. He predicted the existence o capillaries, linking arteries and veins, even though the lenses o the time were not powerul enough or him to see them.

6 . 2 t h e b l o o D S yS t e m

The ollowing extract is rom Harveys book On the Generation of Animals, published in 1 65 1 when he was 73 . And hence it is that without the due admonition of the senses, without frequent observation and reiterated experiment, our mind goes astray after phantoms and appearances. Diligent observation is therefore requisite in every science, and the senses are frequently to be appealed to. We are, I say, to strive after personal experience, not to rely of the experience of

others: without which no one can properly become a student of any branch of natural science. I would not have you therefore, gentle reader, to take anything on trust from me concerning the Generation of Animals: I appeal to your own eyes as my witness and judge. The method of pursuing truth commonly pursued at this time therefore is to be held erroneous and almost foolish, in which so many enquire what things others have said, and omit to ask whether the things themselves be actually so or not.

Arteries Arteries convey blood at high pressure rom the ventricles to the tissues o the body. Arteries are vessels that convey blood rom the heart to the tissues o the body. The main pumping chambers o the heart are the ventricles. They have thick strong muscle in their walls that pumps blood into the arteries, reaching a high pressure at the peak o each pumping cycle. The artery walls work with the heart to acilitate and control blood fow. E lastic and muscle tissue in the walls are used to do this. E lastic tissue contains elastin bres, which store the energy that stretches them at the peak o each pumping cycle. Their recoil helps propel the blood on down the artery. C ontraction o smooth muscle in the artery wall determines the diameter o the lumen and to some extent the rigidity o the arteries, thus controlling the overall fow through them. Both the elastic and muscular tissues contribute to the toughness o the walls, which have to be strong to withstand the constantly changing and intermittently high blood pressure without bulging outwards (aneurysm) or bursting. The bloods progress along major arteries is thus pulsatile, not continuous. The pulse refects each heartbeat and can easily be elt in arteries that pass near the body surace, including those in the wrist and the neck. E ach organ o the body is supplied with blood by one or more arteries. For example, each kidney is supplied by a renal artery and the liver by the hepatic artery. The powerul, continuously active muscles o the heart itsel are supplied with blood by coronary arteries.

Artery walls Arteries have muscle and elastic fbres in their walls. The wall o the artery is composed o several layers: 

tunica externa  a tough outer layer o connective tissue



tunica media  a thick layer containing smooth muscle and elastic bres made o the protein elastin



tunica intima  a smooth endothelium orming the lining o the artery.

acivi Discussin qusins n Wii hrvs ds 1 William Harvey reused to accept doctrines without evidence. Are there academic contexts where it is reasonable to accept doctrines on the basis o authority rather than evidence gathered rom primary sources? 2 Harvey welcomed questions and criticisms o his theories when teaching anatomy classes. Suggest why he might have done this. 3 Can you think o examples o the phantoms and appearances that Harvey reers to? 4 Why does Harvey recommend reiteration o experiments? 5 Harvey practised as a doctor, but ater the publication in 1628 o his work on the circulation o the blood, ar ewer patients consulted him. Why might this have been?

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Hum C Ean LLpBHI ys O LO i oGlo Y gy tunica externa

tunica media

tunica intima (endothelium)

lumen  Figure 3

Structure of an artery

activity mesuring blood pressures Because arteries are distensible, blood pressure in those that pass near the body surace can be measured relatively easily. A common method is to infate an arm cu until it squeezes the tissues (skin, supercial at as well as the vessels themselves) enough to stop blood fow. The pressure is then released slowly until fow resumes and the operator or instrument can hear the pulse again. The pressures at which blood fow stops and resumes are the systolic and diastolic pressures. They are measured with a pressure monitor. According to the American Heart Association the desired blood pressures or adults o 18 years or older measured in this way are: systolic 90-119 mmHg diastolic 60-79 mmHg

 Figure 4 Blood pressure monitor

292

 Figure 2

The cardiovascular system. The main artery that supplies oxygenated blood to the tissues of the body is the aorta, shown as the red vessel that emerges from the heart and forms an arch with branches carrying blood to the arms and head. The aorta continues through the thorax and abdomen, with branches serving the liver, kidneys, intestines and other organs

Arterial blood pressure The muscle and elastic bres assist in maintaining blood pressure between pump cycles. The blood entering an artery rom the heart is at high pressure. The peak pressure reached in an artery is called the systolic pressure. It pushes the wall o the artery outwards, widening the lumen and stretching elastic bres in the wall, thus storing potential energy. At the end o each heartbeat the pressure in the arteries alls suciently or the stretched elastic bres to squeeze the blood in the lumen. This mechanism saves energy and prevents the minimum pressure inside the artery, called the diastolic pressure, rom becoming too low. B ecause it is relatively high, blood fow in the arteries is relatively steady and continuous although driven by a pulsating heart. The circular muscles in the wall o the artery orm a ring so when they contract, in a process called vasoconstriction, the circumerence is reduced and the lumen is narrowed. Vasoconstriction increases blood pressure in the arteries. B ranches o arteries called arterioles have a particularly high density o muscle cells that respond to various hormone and neural signals to control blood fow to downstream tissues. Vasoconstriction o arterioles restricts blood fow to the part o the body that they supply and the opposite process, called vasodilation, increases it.

6 . 2 t h e b l o o D S yS t e m

Capillaries Blood fows through tissues in capillaries with permeable walls that allow exchange o materials between cells in the tissue and the blood in the capillary. C apillaries are the narrowest blood vessels with diameter o about 1 0 m. They branch and rej oin repeatedly to orm a capillary network with a huge total length. C apillaries transport blood through almost all tissues in the body. Two exceptions are the tissues o the lens and the cornea in the eye which must be transparent so cannot contain any blood vessels. The density o capillary networks varies in other tissues but all active cells in the body are close to a capillary. The capillary wall consists o one layer o very thin endothelium cells, coated by a lter- like protein gel, with pores between the cells. The wall is thus very permeable and allows part o the plasma to leak out and orm tissue fuid. Plasma is the fuid in which the blood cells are suspended. Tissue fuid contains oxygen, glucose and all other substances in blood plasma apart rom large protein molecules, which cannot pass through the capillary wall. The fuid fows between the cells in a tissue, allowing the cells to absorb useul substances and excrete waste products. The tissue fuid then re- enters the capillary network.

acivi bruiss Bruises are caused by damage to capillary walls and leakage o plasma and blood cells into spaces between cells in a tissue. The capillaries are quickly repaired, hemoglobin is broken down to green and yellow bile pigments which are transported away and phagocytes remove the remains o the blood cells by endocytosis. When you next have a bruise, make observations over the days ater the injury to ollow the healing process and the rate at which hemoglobin is removed.

The permeabilities o capillary walls dier between tissues, enabling particular proteins and other large particles to reach certain tissues but not others. Permeabilities can also change over time and capillaries repair and remodel themselves continually in response to the needs o tissues that they peruse.

Veins Veins collect blood at low pressure rom the tissues o the body and return it to the atria o the heart. Veins transport blood rom capillary networks back to the atria o the heart. B y now the blood is at much lower pressure than it was in the arteries. Veins do not thereore need to have as thick a wall as arteries and the wall contains ar ewer muscle and elastic bres. They can thereore dilate to become much wider and thus hold more blood than arteries. Around 80% o a sedentary persons blood is in the veins though this proportion alls during vigorous exercise. B lood fow in veins is assisted by gravity and by pressures exerted on them by other tissues especially skeletal muscles. C ontraction makes a muscle shorter and wider so it squeezes on adjacent veins like a pump. Walking, sitting or even just dgeting greatly improves venous blood fow. E ach part o the body is served by one or more veins. For example blood is carried rom the arms in the subclavian veins and rom the head in the j ugular veins. The hepatic portal vein is unusual because it does not carry blood back to the heart. It carries blood rom the stomach and intestines to the liver. It is regarded as a portal vein rather than an artery because the blood it carries is at low pressure so it is relatively thin.

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activity Stnding on your hed Pocket valves and vein walls become less efcient with age, causing poor venous return to the heart. Have you ever perormed gymnastic moves such as headstands or handstands, or experienced very high g-orces on a ride at an amusement park? Young people can mostly do any o these activities easily but older people may not be able to. What is the explanation?

Valves in veins Valves in veins and the heart ensure circulation o blood by preventing backow. B lood pressure in veins is sometimes so low that there is a danger o backfow towards the capillaries and insucient return o blood to the heart. To maintain circulation, veins contain pocket valves, consisting o three cup-shaped faps o tissue. 

I blood starts to fow backwards, it gets caught in the faps o the pocket valve, which ll with blood, blocking the lumen o the vein.



When blood fows towards the heart, it pushes the faps to the sides o the vein. The pocket valve thereore opens and blood can fow reely.

These valves allow blood to fow in one direction only and make ecient use o the intermittent and oten transient pressures provided by muscular and postural changes. They ensure that blood circulates in the body rather than fowing to and ro.

Identifying blood vessels Identication o blood vessels as arteries, capillaries or veins rom the structure o their walls. B lood vessels can be identied as arteries, capillaries or veins by looking at their structure. Table 1 below gives dierences that may be useul.

artery

 Figure 5 Which

veins in this gymnast will need valves to help with venous return?

and vein in transverse section. The tunica externa and tunica intima are stained more darkly than the tunica media. Clotted blood is visible in both vessels

Vein

Diameter

Larger than 10 m

Around 10 m

Variable but much larger than 10 m

Relative thickness o wall and diameter o lumen

Relatively thick wall and narrow lumen

Extremely thin wall

Relatively thin wall with variable but oten wide lumen

Number o layers in wall

Three layers, tunica externa, media and intima. These layers may be sub-divided to orm more layers

Only one layer  the tunica intima which is an endothelium consisting o a single layer o very thin cells

Three layers  tunica externa, media and intima

Muscle and elastic bres in the wall

Abundant

None

Small amounts

Valves

None

None

Present in many veins

 Figure 6 Artery

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Cpillry

 Table 1

6 . 2 t h e b l o o D S yS t e m

The double circulation lungs

There is a separate circulation for the lungs.

pulmonary circulation

There are valves in the veins and heart that ensure a one- way fow, so blood circulates through arteries, capillaries and veins. Fish have a single circulation. B lood is pumped at high pressure to their gills to be oxygenated. Ater fowing through the gills the blood still has enough pressure to fow directly, but relatively slowly, to other organs o the body and then back to the heart. In contrast, the lungs used by mammals or gas exchange are supplied with blood by a separate circulation. B lood capillaries in lungs cannot withstand high pressures so blood is pumped to them at relatively low pressure. Ater passing through the capillaries o the lungs the pressure o the blood is low, so it must return to the heart to be pumped again beore it goes to other organs. Humans thereore have two separate circulations:

heart

systemic circulation



the pulmonary circulation, to and rom the lungs



the systemic circulation, to and rom all other organs, including the heart muscles.

other organs  Figure 7

Figure 7 shows the double circulation in a simplied orm. The pulmonary circulation receives deoxygenated blood that has returned rom the systemic circulation, and the systemic circulation receives blood that has been oxygenated by the pulmonary circulation. It is thereore essential that blood fowing to and rom these two circulations is not mixed. The heart is thereore a double pump, delivering blood under dierent pressures separately to the two circulations. semilunar valve

Heart structure Recognition of the chambers and valves of the heart and the blood vessels connected to it in dissected hearts or in diagrams of heart structure. 

aorta pulmonary artery

vena cavae

pulmonary veins

The heart has two sides, let and right, that pump blood to the systemic and pulmonary circulations.



Each side o the heart has two chambers, a ventricle that pumps blood out into the arteries and an atrium that collects blood rom the veins and passes it to the ventricle.



Each side o the heart has two valves, an atrioventricular valve between the atrium and the ventricle and a semilunar valve between the ventricle and the artery.



The double circulation

O xygenated blood fows into the let side o the heart through the pulmonary veins rom the lungs and out through the aorta.

semilunar valve

atrioventricular valve

right atrium

left ventricle

right ventricle septum  Figure 8 Structure of the heart

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

D eoxygenated blood fows into the let side o the heart through the vena cava and out in the pulmonary arteries.

The heart is a complicated three- dimensional structure. The best way to learn about its structure is by doing a dissection. A resh specimen o a mammalian heart, with blood vessels still attached, a dissecting dish or board and dissecting instruments are needed.

1 Arteries and veins Tidy up the blood vessels attached to the heart by removing membranes and other tissue rom around them. Identiy the thick- walled arteries and the thin-walled veins.

4 Left ventricle Identiy the let ventricle. It has a smooth wall, with a tree- like pattern o blood vessels. Using a sharp scalpel, make an incision as shown by the dashed line X in gure 9. This should open up the let ventricle. Look at the thick muscular wall that you have cut through.

5 Atrioventricular valve Extend the incision urther towards the atrium i necessary until you can see the two thin faps o the atrioventricular valve. Tendons attached to the sides o the let ventricle prevent the valve inverting into the atrium.

6 Left atrium and pulmonary vein

2 Pulmonary artery and aorta Push a glass rod or other blunt- ended instrument into the heart through the arteries and eel through the wall o the heart to where the end o the rod has reached. Identiy the pulmonary artery, through which you will reach the thinner- walled right ventricle, and the aorta, through which you will reach the thicker- walled let ventricle.

Identiy the let atrium. It will look surprisingly small as there is no blood inside it. The outer surace o its wall has a wrinkled appearance. Extend the incision that you have already made, either with the scalpel or with scissors, to cut through the wall o the let atrium as ar as the pulmonary vein. Look at the thin wall o the atrium and the opening o the pulmonary vein or veins ( there may be two) .

3 Dorsal and ventral sides

7 Aorta

Lay the heart so that the aorta is behind the pulmonary artery, as in gure 9. The ventral side is now uppermost and the dorsal side underneath. The dorsal side o an animal is its back.

Find the aorta again and measure the diameter o its lumen, in millimetres. Using scissors, cut through the wall o the aorta, starting at its end and working towards the let ventricle. Look at the smooth inner surace o the aorta and try stretching the wall to see how tough it is.

8 Semilunar valve

aorta pulmonary artery

right artrium

left atrium

Where the aorta exits the let ventricle, there will be three cup- shaped faps in the wall. These orm the semilunar valve. Try pushing a blunt instrument into the faps to see how blood fowing backwards pushes the faps together, closing the valve.

X

9 Coronary artery

coronary artery Y

 Figure 9

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Ventral view of the exterior of the heart

Look careully at the inner surace o the aorta, near the semilunar valve. A small hole should be visible, which is the opening to the coronary arteries. Measure the diameter o the lumen o this artery. The coronary arteries supply the wall o the heart with oxygen and nutrients.

6 . 2 t h e b l o o D S yS t e m

10 Septum

right ventricle  Figure 10

septum

left ventricle

Make a transverse section through the heart near the base o the ventricles, along the dotted line marked Y in gure 9. Measure the thickness in millimetres o the walls o the let and right ventricles and o the septum between them ( gure 1 0) . The septum contains conducting bres, which help to stimulate the ventricles to contract.

Transverse section through the ventricles

Atherosclerosis Causes and consequences of occlusion of the coronary arteries. One o the commonest current health problems is atherosclerosis, the development o atty tissue called atheroma in the artery wall adjacent to the endothelium. Low density lipoproteins (LD L) containing ats and cholesterol accumulate and phagocytes are then attracted by signals rom endothelium cells and smooth muscle. The phagocytes engul the ats and cholesterol by endocytosis and grow very large. Smooth muscle cells migrate to orm a tough cap over the atheroma. The artery wall bulges into the lumen narrowing it and thus impeding blood fow. S mall traces o atheroma are normally visible in childrens arteries by the age o ten, but do not aect health. In some older people atherosclerosis becomes much more advanced but oten goes unnoticed until a maj or artery becomes so blocked that the tissues it supplies become compromised. C oronary occlusion is a narrowing o the arteries that supply blood containing oxygen and nutrients to the heart muscle. Lack o oxygen (anoxia) causes pain, known as angina, and impairs the muscles ability to contract, so the heart beats aster as it tries to maintain blood circulation with some o its muscle out o action. The brous cap covering atheromas sometimes ruptures, which stimulates the ormation o blood clots that can block arteries supplying blood to the heart and cause acute heart problems. This is described in sub-topic 6.3. The causes o atherosclerosis are not yet ully understood. Various actors have been shown to be associated with an increased risk o atheroma but are not the sole causes o the condition: 

high blood concentrations o LD L ( low density lipoprotein)



chronic high blood glucose concentrations, due to overeating, obesity or diabetes

acivi Srucur nd funcin f  r Discuss the answers to these questions. 1 Why are the walls of the atria thinner than the walls of the ventricles? 2 What prevents the atrioventricular valve from being pushed into the atrium when the ventricle contracts? 3 Why is the left ventricle wall thicker than the right ventricle wall? 4 Does the left side of the heart pump oxygenated or deoxygenated blood? 5 Why does the wall of the heart need its own supply of blood, brought by the coronary arteries? 6 Does the right side of the heart pump a greater volume of blood per minute, a smaller volume, or the same volume as the left?

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activity Crnitine nd coronry occusion A chemical called carnitine that is ound in certain oods is converted into TMAO by bacteria in the gut. Find out what oods contain the highest concentrations o carnitine and discuss whether this nding should infuence dietary advice.



chronic high blood pressure due to smoking, stress or any other cause



consumption o trans ats, which damage the endothelium o the artery.

There are also some more recent theories that include microbes: 

inection o the artery wall with Chlamydia pneumoniae



production o trimethylamine N- oxide ( TMAO ) by microbes in the intestine.

 Figure 11

A normal artery (left) has a much wider lumen than an artery that is occluded by atheroma (right)

The sinoatrial node The heartbeat is initiated by a group o specialized muscle cells in the right atrium called the sinoatrial node. The heart is unique in the body as its muscles can contract without stimulation rom motor neurons. The contraction is called myogenic, meaning that it is generated in the muscle itsel. The membrane o a heart muscle cell depolarizes when the cell contracts and this activates adj acent cells, so they also contract. A group o cells thereore contracts almost simultaneously at the rate o the astest.

 Figure 12

298

The sinoatrial node

The region o the heart with the astest rate o spontaneous beating is a small group o special muscle cells in the wall o the right atrium, called the sinoatrial node. These cells have ew o the proteins that cause contraction in other muscle cells, but they have extensive membranes. The sinoatrial node thereore initiates each heartbeat, because the membranes o its cells are the frst to depolarize in each cardiac cycle.

6 . 2 t h e b l o o D S yS t e m

Initiating the heartbeat The sinoatrial node acts as a pacemaker. B ecause the sinoatrial node initiates each heartbeat, it sets the pace or the beating o the heart and is oten called the pacemaker. I it becomes deective, its output may be regulated or even replaced entirely by an artifcial pacemaker. This is an electronic device, placed under the skin with electrodes implanted in the wall o the heart that initiate each heartbeat in place o the sinoatrial node.

Atrial and ventricular contraction The sinoatrial node sends out an electrical signal that stimulates contraction as it is propagated through the walls o the atria and then the walls o the ventricles. The sinoatrial node initiates a heartbeat by contracting and simultaneously sends out an electrical signal that spreads throughout the walls o the atria. This can happen because there are interconnections between adjacent fbres across which the electrical signal can be propagated. Also the fbres are branched so each fbre passes the signal on to several others. It takes less than a tenth o a second or all cells in the atria to receive the signal. This propagation o the electrical signal causes the whole o both let and right atria to contract. Ater a time delay o about 0. 1 seconds, the electrical signal is conveyed to the ventricles. The time delay allows time or the atria to pump the blood that they are holding into the ventricles. The signal is then propagated throughout the walls o the ventricles, stimulating them to contract and pump blood out into the arteries. D etails o the electrical stimulation o the heartbeat are included in O ption D .

 Figure 13

Heart monitor displaying the heart rate, the electrical activity of the heart and the percentage saturation with oxygen of the blood

TOK Wa ars r in ica dcisin aking: inn r cnsquncs? There are some circumstances in which prolonging the lie o an individual who is sufering brings in to question the role o the physician. Sometimes, an active pacemaker may be involved in prolonging the lie o a patient and the physician receives a request to deactivate the device. This will accelerate the pace o the patients death. Euthanasia involves taking active steps to end the lie o a patient and it is illegal in many jurisdictions. However, there is a widely accepted practice o withdrawing lie-sustaining interventions such as dialysis, mechanical ventilation, or tube eeding rom terminally ill patients. This is oten a decision o the amily o the patient. The withdrawal o lie support is seen as distinct rom euthanasia because the patient dies o their condition rather than the active steps to end the patients lie in the case o euthanasia. However, the distinction can be subtle. The consequence is the same: the death o the patient. The intent can be the same: to end the patients sufering. Yet in many jurisdictions, one action is illegal and the other is not.

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The cardiac cycle Pressure changes in the left atrium, left ventricle and aorta during the cardiac cycle. 0.1 5  0.4 seconds  The pressure in the ventricles rises above the pressure in the arteries so the semilunar valves open and blood is pumped rom the ventricles into the arteries, transiently maximizing the arterial blood pressure.

The pressure changes in the atrium and ventricle o the heart and the aorta during a cardiac cycle are shown in gure 1 5 . To understand them it is necessary to appreciate what occurs at each stage o the cycle. Figure 1 4 below summarizes the events, with timings assuming a heart rate o 7 5 beats per minute. Typical volumes o blood are shown and also an indication o the direction o blood fow to or rom a chamber o the heart.



0.0  0.1 seconds  The atria contract causing a rapid but relatively small pressure increase, which pumps blood rom the atria to the ventricles, through the open atrioventricular valves. 

0.4  0.45 seconds The contraction o the ventricular muscles wanes and pressure inside the ventricles rapidly drops below the pressure in the arteries, causing the semilunar valves to close.



The semilunar valves are closed and blood pressure in the arteries gradually drops to its minimum as blood continues to fow along them but no more is pumped in.



The atrioventricular valves remain closed.

0.45  0.8 seconds Pressure in the ventricles drops below the pressure in the atria so the atrioventricular valves open.



0.1  0.1 5 seconds  The ventricles contract, with a rapid pressure build up that causes the atrioventricular valves to close. 

Pressure slowly rises in the atria as blood drains into them rom the veins and they ll.



The semilunar valves remain closed.

B lood rom the veins drains into the atria and rom there into the ventricles, causing a slow increase in pressure.

vein 25 ml atrium relaxing

atrium contracts 25 ml atrioventricular valve valve open atrium

atrioventricular valve closed ventricle contracting 70 ml

ventricle relaxing

ventricle semilunar valve artery

atrium relaxing

45 ml

atrioventricular valve open ventricle relaxing

valve closed

valve open

semilunar valve closed

diastolic

systolic

diastolic

tissues of the body 0

0.1 0.15

0.4 0.45 time (seconds)

0.8

 Figure 14 One cardiac cycle is represented on the diagram, starting on the let with contraction o the atrium. Vertical

arrows show fows o blood to and rom the atrium and ventricle

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6 . 2 t h e b l o o D S yS t e m

D-sd qusins: Heart action and blood pressures

1

D educe when blood is being pumped rom the atrium to the ventricle. Give both the start and the end times. [2 ]

2

Deduce when the ventricle starts to contract. [1 ]

3

The atrioventricular valve is the valve between the atrium and the ventricle. S tate when the atrioventricular valve closes. [1 ]

4

The semilunar valve is the valve between the ventricle and the artery. S tate when the semilunar valve opens.

[1 ]

5

D educe when the semilunar valve closes.

[1 ]

6

D educe when blood is being pumped rom the ventricle to the artery. Give both the start and the end times.

pressure / mm Hg

Figure 1 5 shows the pressures in the atrium, ventricle and artery on one side o the heart, during one second in the lie o the heart. ventricle

120

artery

100

80

60

40

20

7

atrium

[2 ]

D educe when the volume o blood in the ventricle is:

0

20 0

a) at a maximum

[1 ]

b) at a minimum.

[1 ]

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

time / s  Figure 15 Pressure changes during the cardiac cycle

Changing the heart rate

acivi

The heart rate can be increased or decreased by impulses brought to the heart through two nerves rom the medulla o the brain.

lisning  r sunds

The sinoatrial node that sets the rhythm or the beating o the heart responds to signals rom outside the heart. These include signals rom branches o two nerves originating in a region in the medulla o the brain called the cardiovascular centre. S ignals rom one o the nerves cause the pacemaker to increase the requency o heartbeats. In healthy young people the rate can increase to three times the resting rate. S ignals rom the other nerve decrease the rate. These two nerve branches act rather like the throttle and brake o a car.

Sounds produced by blood fow can be heard with a simple tube or stethoscope placed on the chest near the heart. The consequences o this whole cardiac cycle or the fow o blood out o the heart can be elt as the pulse in a peripheral artery. (a)

The cardiovascular centre receives inputs rom receptors that monitor blood pressure and its pH and oxygen concentration. The pH o the blood refects its carbon dioxide concentration. 

Low blood pressure, low oxygen concentration and low pH all suggest that the heart rate needs to speed up, to increase the fow rate o blood to the tissues, deliver more oxygen and remove more carbon dioxide.



High blood pressure, high oxygen concentration and high pH are all indicators that the heart rate may need to slow down.

(b)

 Figure 16 Taking the pulse: (a)

radial pulse (b) carotid pulse

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Epinephrine Epinephrine increases the heart rate to prepare or vigorous physical activity. The sinoatrial node also responds to epinephrine in the blood, by increasing the heart rate. This hormone is also sometimes called adrenalin and is produced by the adrenal glands. The secretion o epinephrine is controlled by the brain and rises when vigorous physical activity may be necessary because o a threat or opportunity.  S o epinephrine has the nickname  ght or fight hormone .

 Figure 17

Adventure sports such as rock climbing cause epinephrine secretion

In the past when humans were hunter- gatherers rather than armers, epinephrine would have been secreted when humans were hunting or prey or when threatened by a predator. In the modern world athletes oten use pre- race routines to stimulate adrenalin secretion so that their heart rate is already increased when vigorous physical activity begins.

6.3 Defence against infectious disease Understanding  The skin and mucous membranes orm a

   



  

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primary deence against pathogens that cause inectious disease. Cuts in the skin are sealed by blood clotting. Clotting actors are released rom platelets. The cascade results in the rapid conversion o brinogen to brin by thrombin. Ingestion o pathogens by phagocytic white blood cells gives non-specic immunity to diseases. Production o antibodies by lymphocytes in response to particular pathogens gives specic immunity. Antibiotics block processes that occur in prokaryotic cells but not in eukaryotic cells. Viral diseases cannot be treated using antibiotics because they lack a metabolism. Some strains o bacteria have evolved with genes which coner resistance to antibiotics and some strains o bacteria have multiple resistance.

Applications  Causes and consequences o blood clot

ormation in coronary arteries.  Efects o HIV on the immune system and methods o transmission.  Florey and Chains experiments to test penicillin on bacterial inections in mice.

Nature of science  Risks associated with scientic research:

Florey and Chains tests on the saety o penicillin would not be compliant with current protocols on testing.

6 . 3 D e Fe n Ce ag ai n S t i n Fe Cti o u S D i S e aS e

Skin as a barrier to infection The skin and mucous membranes orm a primary deence against pathogens that cause inectious disease. There are many different microbes in the environment that can grow inside the human body and cause a disease. S ome microorganisms are opportunistic and although they can invade the body they also commonly live outside it. O thers are specialized and can only survive inside a human body. Microbes that cause disease are called pathogens. The primary defence of the body against pathogens is the skin. Its outermost layer is tough and provides a physical barrier against the entry of pathogens and protection against physical and chemical damage. S ebaceous glands are associated with hair follicles and they secrete a chemical called sebum, which maintains skin moisture and slightly lowers skin pH. The lower pH inhibits the growth of bacteria and fungi. Mucous membranes are a thinner and softer type of skin that is found in areas such as the nasal passages and other airways, the head of the penis and foreskin and the vagina. The mucus that these areas of skin secrete is a sticky solution of glycoproteins. Mucus acts as a physical barrier; pathogens and harmful particles are trapped in it and either swallowed or expelled. It also has antiseptic properties because of the presence of the anti-bacterial enzyme lysozyme.

Cuts and clots

Figure 1 Scanning electron micrograph of bacteria on the surface of teeth. Mucous membranes in the mouth prevent these and other microbes from invading body tissues

acvy im hm sk A digital microscope can be used to produce images o the diferent types o skin covering the human body. Figure 2 shows our images produced in this way.

Cuts in the skin are sealed by blood clotting. When the skin is cut, blood vessels in it are severed and start to bleed. The bleeding usually stops after a short time because of a process called clotting. The blood emerging from a cut changes from being a liquid to a semi- solid gel. This seals up the wound and prevents further loss of blood and blood pressure. C lotting is also important because cuts breach the barrier to infection provided by the skin. C lots prevent entry of pathogens until new tissue has grown to heal the cut.

platelets and blood clotting Clotting actors are released rom platelets. B lood clotting involves a cascade of reactions, each of which produces a catalyst for the next reaction. As a result blood clots very rapidly. It is important that clotting is under strict control, because if it occurs inside blood vessels the resulting clots can cause blockages. The process of clotting only occurs if platelets release clotting factors. Platelets are cellular fragments that circulate in the blood. They are smaller than either red or white blood cells. When a cut or other inj ury involving damage to blood vessels occurs, platelets aggregate at the site forming a temporary plug. They then release the clotting factors that trigger off the clotting process. 

Figure 2

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Fibrin production The cascade results in the rapid conversion o fbrinogen to fbrin by thrombin. platelets

lymphocyte

red blood cell

phagocyte

The cascade o reactions that occurs ater the release o clotting actors rom platelets quickly results in the production o an enzyme called thrombin. Thrombin in turn converts the soluble protein fbrinogen into the insoluble fbrin. The fbrin orms a mesh in cuts that traps more platelets and also blood cells. The resulting clot is initially a gel, but i exposed to the air it dries to orm a hard scab. Figure 4 shows red blood cells trapped in this fbrous mesh.

Figure 3 Cells and cell ragments rom blood. Lymphocytes and phagocytes are types o white blood cell

Coronary thrombosis Causes and consequences o blood clot ormation in coronary arteries. In patients with coronary heart disease, blood clots sometimes orm in the coronary arteries. These arteries branch o rom the aorta close to the semilunar valve. They carry blood to the wall o the heart, supplying the oxygen and glucose needed by cardiac muscle fbres or cell respiration. The medical name or a blood clot is a thrombus. Coronary thrombosis is the ormation o blood clots in the coronary arteries.

Figure 4 Scanning electron micrograph o clotted blood with fbrin and trapped blood cells

I the coronary arteries become blocked by a blood clot, part o the heart is deprived o oxygen and nutrients. C ardiac muscle cells are then unable to produce sufcient ATP by aerobic respiration and their contractions become irregular and uncoordinated. The wall o the heart makes quivering movements called fbrillation that do not pump blood eectively. This condition can prove atal unless it resolves naturally or through medical intervention. Atherosclerosis causes occlusion in the coronary arteries. Where atheroma develops the endothelium o the arteries tends to become damaged and roughened; especially, the artery wall is hardened by deposition o calcium salts. Patches o atheroma sometimes rupture causing a lesion. C oronary occlusion, damage to the capillary epithelium, hardening o arteries and rupture o atheroma all increase the risk o coronary thrombosis. There are some well-known actors that are correlated with an increased risk o coronary thrombosis and heart attacks:

Figure 5 Early intervention during a heart attack can save the patients lie so it is important to know what to do by being trained

304



smoking



high blood cholesterol concentration



high blood pressure



diabetes



obesity



lack o exercise.

O  course correlation does not prove causation, but doctors nonetheless advise patients to avoid these risk actors i possible.

6 . 3 D e Fe n Ce ag ai n S t i n Fe Cti o u S D i S e aS e

phagocytes Ingestion o pathogens by phagocytic white blood cells gives non-specifc immunity to diseases. I microorganisms get past the physical barriers o skin and mucous membranes and enter the body, white blood cells provide the next line o deence. There are many dierent types o white blood cell. Some are phagocytes that squeeze out through pores in the walls o capillaries and move to sites o inection. There they engul pathogens by endocytosis and digest them with enzymes rom lysosomes. When wounds become inected, large numbers o phagocytes are attracted, resulting in the ormation o a white liquid called pus.

Antibody roduction Production o antibodies by lymphocytes in response to particular pathogens gives specifc immunity. I microorganisms get past the physical barriers o the skin and invade the body, proteins and other molecules on the surace o pathogens are recognized as oreign by the body and they stimulate a specifc immune response. Any chemical that stimulates an immune response is reerred to as an antigen. The specifc immune response is the production o antibodies in response to a particular pathogen. The antibodies bind to an antigen on that pathogen. Antibodies are produced by types o white blood cell called lymphocytes. Each lymphocyte produces j ust one type o antibody, but our bodies can produce a vast array o dierent antibodies. This is because we have small numbers o lymphocytes or producing each o the many types o antibody. There are thereore too ew lymphocytes initially to produce enough antibodies to control a pathogen that has not previously inected the body. However, antigens on the pathogen stimulate cell division o the small group o lymphocytes that produce the appropriate type o antibody. A large clone o lymphocytes called plasma cells are produced within a ew days and they secrete large enough quantities o the antibody to control the pathogen and clear the inection. Antibodies are large proteins that have two unctional regions: a hypervariable region that binds to a specifc antigen and another region that helps the body to fght the pathogen in one o a number o ways, including these: 

making a pathogen more recognizable to phagocytes so they are more readily enguled



preventing viruses rom docking to host cells so that they cannot enter the cells.

Antibodies only persist in the body or a ew weeks or months and the plasma cells that produce them are also gradually lost ater the inection has been overcome and the antigens associated with it are no longer present. However, some o the lymphocytes produced during an inection are not active plasma cells but instead become memory cells

Figure 6 Avian infuenza viruses. In this electron micrograph o a virus in transverse section, alse colour has been used to distinguish the protein coat that is recognized as antigens by the immune system (purple) rom the DNA o the virus (green)

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Hum C Ean LLpBHI ys O LO i oGlo Y gy that are very long-lived. These memory cells remain inactive unless the same pathogen inects the body again, in which case they become active and divide to produce plasma cells very rapidly. Immunity to an inectious disease involves either having antibodies against the pathogen, or memory cells that allow rapid production o the antibody.

Human immunodefciency virus Efects o HIV on the immune system and methods o transmission. The production o antibodies by the immune system is a complex process and includes dierent types o lymphocyte, including helper T- cells. The human immunodefciency virus ( HIV) invades and destroys helper T-cells. The consequence is a progressive loss o the capacity to produce antibodies. In the early stages o inection, the immune system makes antibodies against HIV. I these can be detected in a persons body, they are said to be HIV- positive. HIV is a retrovirus that has genes made o RNA and uses reverse transcriptase to make D NA copies o its genes once it has entered a host cell. The rate at which helper T-cells are destroyed by HIV varies considerably and can be slowed down by using anti-retroviral drugs. In most HIV- positive patients antibody production eventually becomes so ineective that a group o opportunistic inections strike, which would be easily ought o by a healthy immune system. S everal o these are normally so rare that they are marker

diseases or the latter stages o HIV inection, or example Kaposis sarcoma. A collection o several diseases or conditions existing together is called a syndrome. When the syndrome o conditions due to HIV is present, the person is said to have acquired immune defciency syndrome ( AID S ) . AID S spreads by HIV inection. The virus only survives outside the body or a short time and inection normally only occurs i there is blood to blood contact between inected and uninected people. There are various ways in which this can occur: 

sexual intercourse, during which abrasions to the mucous membranes o the penis and vagina can cause minor bleeding



transusion o inected blood, or blood products such as Factor VIII



sharing o hypodermic needles by intravenous drug users.

Antibiotics Antibiotics block processes that occur in prokaryotic cells but not in eukaryotic cells. An antibiotic is a chemical that inhibits the growth o microorganisms. Most antibiotics are antibacterial. They block processes that occur in prokaryotes but not in eukaryotes and can thereore be used to kill bacteria inside the body without causing harm to human cells. The processes targeted by antibiotics are bacterial D NA replication, transcription, translation, ribosome unction and cell wall ormation.

Figure 7 Fleming's petri dish which frst showed the inhibition o bacterial growth by penicillin rom a mycelium o Penicillium

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Many antibacterial antibiotics were discovered in saprotrophic ungi. These ungi compete with saprotrophic bacteria or the dead organic matter on which they both eed. B y secreting antibacterial antibiotics, saprotrophic ungi inhibit the growth o their bacterial competitors. An example is penicillin. It is produced by some strains o the Penicillium ungus, but only when nutrients are scarce and competition with bacteria would be harmul.

6 . 3 D e Fe n Ce ag ai n S t i n Fe Cti o u S D i S e aS e

Testing penicillin Florey and Chains experiments to test penicillin on bacterial inections in mice. Howard Florey and Ernst C hain ormed a research team in O xord in the late 1 93 0s that investigated the use o chemical substances to control bacterial inections. The most promising o these was penicillin, discovered by Alexander Fleming in 1 92 8. Florey and C hains team developed a method o growing the ungus Penicillium in liquid culture in conditions that stimulated it to secrete penicillin. They also developed methods or producing reasonably pure samples o penicillin rom the cultures.

acvy Wrld aiDS Dy The red AIDS awareness ribbon is an international symbol o awareness and support or those living with HIV. It is worn on World AIDS Day each year  December 1st. Are you aware how many people in your area are afected and what can be done to support them?

The penicillin killed bacteria on agar plates, but they needed to test whether it would control bacterial inections in humans. They frst tested it on mice. E ight mice were deliberately inected with Streptococcus bacteria that cause death rom pneumonia. Four o the inected mice were given inj ections with penicillin. Within 2 4 hours all the untreated mice were dead but the our given penicillin were healthy. Florey and C hain decided that they should next do tests on human patients, which required much larger quantities. When enough penicillin had been produced, a 43 - year-old policeman was chosen or the frst human test. He had an acute and liethreatening bacterial inection caused by a scratch on the ace rom a thorn on a rose bush. He was given penicillin or our days and his condition improved considerably, but supplies o penicillin ran out and he suered a relapse and died rom the inection. Larger quantities o penicillin were produced and fve more patients with acute inections were tested. All were cured o their inections, but sadly one o them died. He was a small child who had an inection behind the eye. This had weakened the wall o the artery carrying blood to the brain and although cured o the inection, the child died suddenly o brain hemorrhage when the artery burst. Pharmaceutical companies in the United S tates then began to produce penicillin in much larger quantities, allowing more extensive testing, which confrmed that it was a highly eective treatment or many previously incurable bacterial inections.

Figure 8 Penicillin  the green ball represents a variable part of the molecule

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penicillin and drug testing Risks associated with scientifc research: Florey and Chains tests on the saety o penicillin would not be compliant with current protocols on testing. When any new drug is introduced there are risks that it will prove to be ineffective in some or all patients or that it will cause harmful side effects. These risks are minimized by strict protocols that pharmaceutical companies must follow. Initial tests are performed on animals and then on small numbers of healthy humans. O nly if a drug passes these tests is it tested on patients with the disease that the drug is intended to treat. The last tests involve very large numbers of patients to test whether the drug is effective in all patients and to check that there are no severe or common side effects. There are some famous cases of drugs causing problems during testing or after release. 

Thalidomide was introduced in the 1 95 0s as a treatment for various mild conditions but when it was found to relieve morning sickness in pregnant women it was prescribed for that purpose. The side effects of the drug on the fetus had not been tested and more than 1 0, 000 children were born with birth deformities before the problem was recognized.



In 2 006 six healthy volunteers were given TGN1 41 2 , a new protein developed for treatment of autoimmune diseases and leukemia. All six rapidly became very ill and suffered multiple organ failure. Although the volunteers recovered, they may have suffered long- term damage to their immune systems.

It is very unlikely that Florey and C hain would have been allowed to carry out tests on a new

drug today with the methods that they used for penicillin. They tested the drug on human patients after only a very brief period of animal testing. Penicillin was a new type of drug and there could easily have been severe side effects. Also the samples that they were using were not pure and there could have been side effects from the impurities. On the other hand, the patients that they used were all on the point of death and several were cured of their infections as a result of the experimental treatment. B ecause of expeditious testing with greater risk-taking than would now be allowed, penicillin was introduced far more quickly than would be possible today. D uring the D -day landings in June 1 944 penicillin was used to treat wounded soldiers and the number of deaths from bacterial infection was greatly reduced.

Figure 9 Wounded US troops on Omaha beach 6 June 1944

Viruses and antibiotics Viral diseases cannot be treated using antibiotics because they lack a metabolism. Viruses are non- living and can only reproduce when they are inside living cells. They use the chemical processes of a living host cell, instead of having a metabolism of their own. They do not have their own means of transcription or protein synthesis and they rely on the

308

6 . 3 D e Fe n Ce ag ai n S t i n Fe Cti o u S D i S e aS e host cells enzymes or ATP synthesis and other metabolic pathways. These processes cannot be targeted by drugs as the host cell would also be damaged. All o the commonly used antibiotics such as penicillin, streptomycin, chloramphenicol and tetracycline control bacterial inections and are not eective against viruses. Not only is it inappropriate or doctors to prescribe them or a viral inection, but it contributes to the overuse o antibiotics and increases in antibiotic resistance in bacteria. There are a ew viral enzymes which can be used as targets or drugs to control viruses without harming the host cell. O nly a ew drugs have been discovered or developed to control viruses in this way. These are known as antivirals rather than antibiotics.

acvy Dssh bw bcrl d vrl fcs How can a doctor distinguish between bacterial and viral infections, without prescribing an antibiotic and seeing if it cures the infection?

Resistance to antibiotics Some strains of bacteria have evolved with genes which confer resistance to antibiotics and some strains of bacteria have multiple resistance. In 2 01 3 the governments chie medical ofcer or England, S ally D avies, said this: The danger posed by growing resistance to antibiotics should be ranked along with terrorism on a list of threats to the nation. If we dont take action, then we may all be back in an almost 1 9th-century environment where infections kill us as a result of routine operations. We wont be able to do a lot of our cancer treatments or organ transplants.

Figure 10 Many viruses cause a common cold. Children lack immunity to most of them so frequently catch a cold. Antibiotics do not cure them

The development o resistance to antibiotics by natural selection is described in sub- topic 5 .2 . S trains o bacteria with resistance are usually discovered soon ater the introduction o an antibiotic. This is not o huge concern unless a strain develops multiple resistance, or example methicillin-resistant Staphylococcus aureus ( MRS A) which has inected the blood or surgical wounds o hospital patients and resists all commonly used antibiotics. Another example o this problem is multidrug-resistant tuberculosis ( MD R-TB ) . The WHO has reported more than 3 00, 000 cases worldwide per year with the disease reaching epidemic proportions in some areas. Antibiotic resistance is an avoidable problem. These measures are required: 

doctors prescribing antibiotics only or serious bacterial inections



patients completing courses o antibiotics to eliminate inections completely



hospital sta maintaining high standards o hygiene to prevent crossinection



armers not using antibiotics in animal eeds to stimulate growth



pharmaceutical companies developing new types o antibiotic  no new types have been introduced since the 1 980s.

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Data-based questions: Antibiotic resistance

1

a) D escribe the pattern o erythromycin resistance over the period rom 1 992 to 2 002 . [3]

[2 ]

Evaluate the claim that reduction in the use o erythromycin has led to a reduction in the incidence o antibiotic resistance in S. pyogenes. [3 ] 20 15 10

2002

2001

2000

1999

1997

1998

1995

0

1996

5 1993

3

C alculate the percentage dierence in antibiotic resistance between 2002 and 1 992.

1994

The data in fgure 1 1 shows the incidence in Finland, over a 1 0-year period, o Streptococcus pyogenes strains that are resistant to the antibiotic erythromycin. S. pyogenes is responsible or the condition known as strep throat.

2

1992

In the early 1 990s, Finnish public health authorities began discouraging the use o the antibiotic erythromycin or URIs in response to rising bacterial resistance to the antibiotic, and the national erythromycin consumption per capita dropped by 43 per cent.

b) Suggest a reason or the pattern shown. [2 ]

% antibiotic resistance

B acterial resistance to antibiotics is a direct consequence o the overuse o these drugs. In the US A, currently more than hal o the doctor visits or upper respiratory tract inections ( URIs) are prescribed antibiotics, despite knowledge that most URIs are caused by viruses.

year

Figure 11 The incidence of Streptococcus pyogenes strains that are resistant to the antibiotic erythromycin over a 10-year period in Finland

6.4 gas exchane Understanding  Ventilation maintains concentration gradients

 

 



310

o oxygen and carbon dioxide between air in alveoli and blood fowing in adjacent capillaries. Type I pneumocytes are extremely thin alveolar cells that are adapted to carry out gas exchange. Type II pneumocytes secrete a solution containing suractant that creates a moist surace inside the alveoli to prevent the sides o the alveolus adhering to each other by reducing surace tension. Air is carried to the lungs in the trachea and bronchi and then to the alveoli in bronchioles. Muscle contractions cause the pressure changes inside the thorax that orce air in and out o the lungs to ventilate them. Dierent muscles are required or inspiration and expiration because muscles only do work when they contract.

Applications  External and internal intercostal muscles,

and diaphragm and abdominal muscles as examples o antagonistic muscle action.  Causes and consequences o lung cancer.  Causes and consequences o emphysema.

Skills  Monitoring o ventilation in humans at rest and

ater mild and vigorous exercise. (Practical 6)

Nature of science  Obtain evidence or theories: epidemiological

studies have contributed to our understanding o the causes o lung cancer.

6 . 4 g aS e xCh an g e

Ventilation Ventilation maintains concentration gradients o oxygen and carbon dioxide between air in alveoli and blood fowing in adjacent capillaries. All organisms absorb one gas rom the environment and release a dierent one. This process is called gas exchange. Leaves absorb carbon dioxide to use in photosynthesis and release the oxygen produced by this process. Humans absorb oxygen or use in cell respiration and release the carbon dioxide produced by this process. Terrestrial organisms exchange gases with the air. In humans gas exchange occurs in small air sacs called alveoli inside the lungs ( gure 1 ) .

type I pneumocytes in alveolus wall

phagocyte 10

network of blood capillaries

0

m

type II pneumocytes in alveolus wall

Figure 1 Gas exchange happens by diusion between air in the alveoli and blood fowing in the adj acent capillaries. The gases only diuse because there is a concentration gradient: the air in the alveolus has a higher concentration o oxygen and a lower concentration o carbon dioxide than the blood in the capillary. To maintain these concentration gradients resh air must be pumped into the alveoli and stale air must be removed. This process is called ventilation.

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Data-based questions: Concentration gradients Figure 2 shows the typical composition o atmospheric air, air in the alveoli and gases dissolved in air returning to the lungs in the pulmonary arteries. oxygen carbon dioxide nitrogen

1

E xplain why the oxygen concentration in the alveoli is not as high as in resh air that is inhaled. [2 ]

2

a)

C alculate the dierence in oxygen concentration between air in the alveolus and blood arriving at the alveolus. [1 ]

b)

D educe the process caused by this concentration dierence.

700 598

partial pressure / mm Hg

600

570

570

565

c)

500 400 300 200

3 atmospheric air that is inhaled

40 air in alveoli

40 45

[2 ]

d) D espite the high concentration o nitrogen in air in alveoli, little or none diuses rom the air to the blood. S uggest reasons or this. [2 ]

120

105

100 0

(i) C alculate the dierence in carbon dioxide concentration between air inhaled and air exhaled. [1 ] (ii) Explain this dierence.

159

[1 ]

27

blood travelling air exhaled to alveoli

Figure 2 Partial pressures of gases in the pulmonary system

Ventilation experiments Monitoring of ventilation in humans at rest and after mild and vigorous exercise. (Practical 6) In an investigation o the eect o exercise on ventilation, the type or intensity o exercise is the independent variable and the ventilation parameter that is measured is the dependent variable. 

A simple approach or the independent variable is to choose levels o activity ranging rom inactive to very active, such as lying down, sitting and standing, walking, j ogging and sprinting. A more quantitative approach is to do the same activity at dierent measured rates, or example running at dierent speeds on a treadmill. This allows the ventilation parameters to be correlated with work rate in j oules per minute during exercise.

Ventilation o the lungs is carried out by drawing some resh air into the lungs and then expelling some o the stale air rom the lungs. The volume o air drawn in and expelled is the tidal volume. The number o times that air is drawn in or expelled per minute is the ventilation rate.

312

Either or both o these can be the dependent variable in an investigation o the eect o exercise on ventilation rate. They should be measured ater carrying on an activity or long enough to reach a constant rate. The example methods given below include a simple and a more advanced technique that could be used or the investigation. 1

Ventilation rate The most straightorward way to measure ventilation rate is by simple observation. C ount the number o times air is exhaled or inhaled in a minute. B reathing should be maintained at a natural rate, which is as slow as possible without getting out o breath.





Ventilation rate can also be measured by data logging. An infatable chest belt is placed around the thorax and air is pumped in with a bladder. A dierential pressure sensor is then used to measure

6 . 4 g aS e xCh an g e

pressure variations inside the belt due to chest expansions. The rate o ventilations can be deduced and the relative size o ventilations may also be recorded. 2

Tidal volume 

S imple apparatus is shown in gure 3 . O ne normal breath is exhaled through the delivery tube into a vessel and the volume is measured. It is not sae to use this apparatus or repeatedly inhaling and exhaling air as the C O 2 concentration will rise too high.

To ensure that the experimental design is rigorous, all variables apart rom the independent and dependent variables should be kept constant. Ventilation parameters should be measured several times at all levels o exercise with each person in the trial. As many dierent people as possible should be tested.

bell jar with graduations delivery tube



S pecially designed spirometers are available or use with data logging. They measure fow rate into and out o the lungs and rom these measurements lung volumes can be deduced.

pneumatic trough

Figure 3

Type I pneumocytes

bronchiole

Type I pneumocytes are extremely thin alveolar cells that are adapted to carry out gas exchange. The lungs contain huge numbers o alveoli with a very large total surace area or diusion. The wall o each alveolus consists o a single layer o cells, called the epithelium. Most o the cells in this epithelium are Type I pneumocytes. They are fattened cells, with the thickness o only about 0.1 5 m o cytoplasm. The wall o the adj acent capillaries also consists o a single layer o very thin cells. The air in the alveolus and the blood in the alveolar capillaries are thereore less than 0. 5 m apart. The distance over which oxygen and carbon dioxide has to diuse is thereore very small, which is an adaptation to increase the rate o gas exchange.

0.25 mm

alveolus

Type II pneumocytes

epithelium of alveolus wall nucleus of epithelium cell

Type II pneumocytes secrete a solution containing surfactant that creates a moist surface inside the alveoli to prevent the sides of the alveolus adhering to each other by reducing surface tension. Type II pneumocytes are rounded cells that occupy about 5 % o the alveolar surace area. They secrete a fuid which coats the inner surace o the alveoli. This lm o moisture allows oxygen in the alveolus to dissolve and then diuse to the blood in the alveolar capillaries. It also provides an area rom which carbon dioxide can evaporate into the air and be exhaled.

basement membrane endothelium of capillary alveolus blood plasma erythrocyte 1 m

Figure 4 Structure of alveoli

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water surface

monolayer of surfactant

Figure 5 Pulmonary suractant molecules on the surace o the flm o moisture lining the alveoli

trachea

intercostal muscle

The fuid secreted by the Type II pneumocytes contains a pulmonary suractant. Its molecules have a structure similar to that o phospholipids in cell membranes. They orm a monolayer on the surace o the moisture lining the alveoli, with the hydrophilic heads acing the water and the hydrophobic tails acing the air. This reduces the surace tension and prevents the water rom causing the sides o the alveoli to adhere when air is exhaled rom the lungs. This helps to prevent collapse o the lung. Premature babies are oten born with insucient pulmonary suractant and can suer rom inant respiratory distress syndrome. Treatment involves giving the baby oxygen and also one or more doses o suractant, extracted rom animal lungs.

right bronchus

Airways for ventilation right lung diaphragm

Air is carried to the lungs in the trachea and bronchi and then to the alveoli in bronchioles.

bronchioles ribs

Air enters the ventilation system through the nose or mouth and then passes down the trachea. This has rings o cartilage in its wall to keep it open even when air pressure inside is low or pressure in surrounding tissues is high. The trachea divides to orm two bronchi, also with walls strengthened with cartilage. O ne bronchus leads to each lung.

Figure 6 The ventilation system

(a) inspiration

ribs

Inside the lungs the bronchi divide repeatedly to orm a tree-like structure o narrower airways, called bronchioles. The bronchioles have smooth muscle bres in their walls, allowing the width o these airways to vary. At the end o the narrowest bronchioles are groups o alveoli, where gas exchange occurs.

vertebral column ribs

diaphragm

pressure changes during ventilation

(b) expiration

Muscle contractions cause the pressure changes inside the thorax that force air in and out of the lungs to ventilate them.

air movement ribcage movement diaphragm movement

Figure 7 Ventilation o the lungs

314

Ventilation o the lungs involves some basic physics. I particles o gas spread out to occupy a larger volume, the pressure o the gas becomes lower. C onversely, i a gas is compressed to occupy a smaller volume, the pressure rises. I gas is ree to move, it will always fow rom regions o higher pressure to regions o lower pressure.

6 . 4 g aS e xCh an g e D uring ventilation, muscle contractions cause the pressure inside the thorax to drop below atmospheric pressure. As a consequence, air is drawn into the lungs rom the atmosphere ( inspiration) until the lung pressure has risen to atmospheric pressure. Muscle contractions then cause pressure inside the thorax to rise above atmospheric, so air is orced out rom the lungs to the atmosphere ( expiration) .

Antagonistic muscles Dierent muscles are required or inspiration and expiration because muscles only do work when they contract. Muscles can be in two states: contracting and relaxing. 

Muscles do work when they contract by exerting a pulling orce ( tension) that causes a particular movement. They become shorter when they do this.



Muscles lengthen while they are relaxing, but this happens passively  they do not lengthen themselves. Most muscles are pulled into an elongated state by the contraction o another muscle. They do not exert a pushing orce (compression) while relaxing so do no work at this time.

Muscles thereore can only cause movement in one direction. I movement in opposite directions is needed at dierent times, at least two muscles will be required. When one muscle contracts and causes a movement, the second muscle relaxes and is elongated by the frst. The opposite movement is caused by the second muscle contracting while the frst relaxes. When muscles work together in this way they are known as an antagonistic pair o muscles.

Figure 8 Diferent muscles are used or bending the leg at the knee and or the opposite movement o straightening it

Inspiration and expiration involve opposite movements, so dierent muscles are required, working as antagonistic pairs.

Antagonistic muscle action in ventilation External and internal intercostal muscles, and diaphragm and abdominal muscles as examples o antagonistic muscle action. Ventilation involves two pairs o opposite movements that change the volume and thereore the pressure inside the thorax:

Diaphragm

isprto Moves downwards and fattens

eprto Moves upwards and becomes more domed

Ribcage

Moves upwards and outwards

Moves downwards and inwards

Antagonistic pairs o muscles are needed to cause these movements.

Volume and pressure changes

isprto The volume inside the thorax increases and consequently the pressure decreases

eprto The volume inside the thorax decreases and consequently the pressure increases

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Movement of the diaphragm

Movement of the ribcage

Diaphragm

The diaphragm contracts and so it moves downwards and pushes the abdomen wall out

The diaphragm relaxes so it can be pushed upwards into a more domed shape

Abdomen wall muscles

Muscles in the abdomen wall relax allowing pressure from the diaphragm to push it out

Muscles in the abdomen wall contract pushing the abdominal organs and diaphragm upwards

External intercostal muscles

The external intercostal muscles contract, pulling the ribcage upwards and outwards

The external intercostal muscles relax and are pulled back into their elongated state.

Internal intercostal muscles

The internal intercostal muscles relax and are pulled back into their elongated state

The internal intercostal muscles contract, pulling the ribcage inwards and downwards

Epidemiology Obtain evidence for theories: epidemiological studies have contributed to our understanding of the causes of lung cancer. Epidemiology is the study o the incidence and causes o disease. Most epidemiological studies are observational rather than experimental because it is rarely possible to investigate the causes o disease in human populations by carrying out experiments. As in other felds o scientifc research, theories about the causes o a disease are proposed. To obtain evidence or or against a theory, survey data is collected that allows the association between the disease and its theoretical cause to be tested. For example, to test the theory that smoking causes lung cancer, the smoking habits o people who have developed lung cancer and people who have not are needed. Examples o very large epidemiological surveys that provided strong evidence or a link between smoking and lung cancer are included in sub-topic 1 .6. A correlation between a risk actor and a disease does not prove that the actor causes the disease. There are usually conounding actors which

also have an eect on the incidence. They can cause spurious associations between a disease and a actor that does not cause it. For example, an association has repeatedly been ound by epidemiologists between leanness and an increased risk o lung cancer. C areul analysis showed that among smokers leanness is not signifcantly associated with an increased risk. Smoking reduces appetite and so is associated with leanness and o course smoking is a cause o lung cancer. This explains the spurious association between leanness and lung cancer. To try to compensate or conounding actors it is usually necessary to collect data on many actors apart rom the one being investigated. This allows statistical procedures to be carried out to take account o conounding actors and try to isolate the eect o single actors. Age and sex are almost always recorded and sometimes epidemiological surveys include only males or emales or only people in a specifc age range.

Causes of lung cancer Causes and consequences of lung cancer. Lung cancer is the most common cancer in the world, both in terms o the number o cases and the number o deaths due to the disease. The

316

general causes o cancer are described in subtopic 1 .6. The specifc causes o lung cancer are considered here.

6 . 4 g aS e xCh an g e

and smoke rom burning coal, wood or other organic matter.

Figure 9 A large tumour (red) is visible in the right lung. The tumour is a bronchial carcinoma 

Smoking causes about 87% o cases. Tobacco smoke contains many mutagenic chemicals. As every cigarette carries a risk, the incidence o lung cancer increases with the number smoked per day and the number o years o smoking.



Passive smoking causes about 3% o cases. This happens when non-smokers inhale tobacco smoke exhaled by smokers. The number o cases will decline in countries where smoking is banned indoors and in public places.



Air pollution probably causes about 5 % o lung cancers. The sources o air pollution that are most signifcant are diesel exhaust umes, nitrogen oxides rom all vehicle exhaust umes



Radon gas causes signifcant numbers o cases in some parts o the world. It is a radioactive gas that leaks out o certain rocks such as granite. It accumulates in badly ventilated buildings and people then inhale it.



Asbestos, silica and some other solids can cause lung cancer i dust or other particles o them are inhaled. This usually happens on construction sites or in quarries, mines or actories.

The consequences o lung cancer are oten very severe. Some o them can be used to help diagnose the disease: difculties with breathing, persistent coughing, coughing up blood, chest pain, loss o appetite, weight loss and general atigue. In many patients the tumour is already large when it is discovered and may also have metastasized, with secondary tumours in the brain or elsewhere. Mortality rates are high. O nly 1 5 % o patients with lung cancer survive or more than 5 years. I a tumour is discovered early enough, all or part o the aected lung may be removed surgically. This is usually combined with one or more courses o chemotherapy. O ther patients are treated with radiotherapy. The minority o patients who are cured o lung cancer, but have lost some o their lung tissue, are likely to continue to have pain, breathing difculties, atigue and also anxiety about the possible return o the disease.

Emphysema Causes and consequences of emphysema. In healthy lung tissue each bronchiole leads to a group o small thin-walled alveoli. In a patient with emphysema these are replaced by a smaller number o larger air sacs with much thicker walls. The total surace area or gas exchange is considerably reduced and the distance over which diusion o gases occurs is increased, and so gas exchange is thereore much less eective. The lungs also become less elastic, so ventilation is more difcult. The molecular mechanisms involved are not ully understood, though there is some evidence or these theories:



Phagocytes inside alveoli normally prevent lung inections by engulfng bacteria and produce elastase, a protein-digesting enzyme, to kill them inside the vesicles ormed by endocytosis.



An enzyme inhibitor called alpha 1 -antitrypsin (A1 AT) usually prevents elastase and other proteases rom digesting lung tissue. In smokers, the number o phagocytes in the lungs increases and they produce more elastase.



Genetic actors aect the quantity and eectiveness o A1 AT produced in the lungs.

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In about 3 0% o smokers digestion o proteins in the alveolus wall by the increased quantity o proteases is not prevented and alveolus walls are weakened and eventually destroyed. Emphysema is a chronic disease because the damage to alveoli is usually irreversible. It causes low oxygen saturation in the blood and higher

than normal carbon dioxide concentrations. As a result the patient lacks energy and may eventually fnd even tasks such as climbing stairs too onerous. In mild cases there is shortness o breath during vigorous exercise but eventually even mild activity causes it. Ventilation is laboured and tends to be more rapid than normal.

Data-based questions: Emphysema and gas exchange Figure 1 0 shows healthy lung tissue and tissue rom a lung with emphysema, at the same magnifcation. S moking usually causes emphysema. B reathing polluted air makes the disease worse. 1

2 3

Figure 10 Healthy lung tissue (top) and lung tissue showing emphysema (bottom)

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a)

Place a ruler across each micrograph and count how many times the edge o the ruler crosses a gas exchange surace. Repeat this several times or each micrograph, in such a way that the results are comparable. S tate your results using suitable units. [3 ]

b) Explain the conclusions that you draw rom the results.

[3 ]

Explain why people who have emphysema eel tired all the time.

[3 ]

S uggest why people with emphysema oten have an enlarged and strained right side o the heart. [1 ]

6 . 5 n e u r o n S an D S yn apS e S

6.5 ns d sss Understanding

Applications

 Neurons transmit electrical impulses.

 Secretion and reabsorption o acetylcholine by

 The myelination o nerve bres allows or

neurons at synapses.  Blocking o synaptic transmission at cholinergic synapses in insects by binding o neonicotinoid pesticides to acetylcholine receptors.



  

 



saltatory conduction. Neurons pump sodium and potassium ions across their membranes to generate a resting potential. An action potential consists o depolarization and repolarization o the neuron. Nerve impulses are action potentials propagated along the axons o neurons. Propagation o nerve impulses is the result o local currents that cause each successive part o the axon to reach the threshold potential. Synapses are junctions between neurons and between neurons and receptor or efector cells. When pre-synaptic neurons are depolarized they release a neurotransmitter into the synapse. A nerve impulse is only initiated i the threshold potential is reached.

Skills  Analysis o oscilloscope traces showing resting

potentials and action potentials.

Nature of science  Cooperation and collaboration between groups

o scientists: biologists are contributing to research into memory and learning.

Neurons Neurons transmit electrical impulses. Two systems o the body are used or internal communication: the endocrine system and the nervous system. The endocrine system consists o glands that release hormones. The nervous system consists o nerve cells called neurons. There are about 8 5 billion neurons in the human nervous system. Neurons help with internal communication by transmitting nerve impulses. A nerve impulse is an electrical signal. Neurons have a cell body with cytoplasm and a nucleus but they also have narrow outgrowths called nerve fbres along which nerve impulses travel. 

D endrites are short branched nerve fbres, or examples those used to transmit impulses between neurons in one part o the brain or spinal cord.



Axons are very elongated nerve fbres, or example those that transmit impulses rom the tips o the toes or the fngers to the spinal cord.

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cell body axon

skeletal muscle (eector)

dendrites  Figure 1

Neuron with dendrites that transmit impulses to the cell body and an axon that transmits impulses a considerable distance to muscle fbres

myelinated nerve fbres The myelination o nerve fbres allows or saltatory conduction. The basic structure o a nerve fbre along which a nerve impulse is transmitted is very simple: the fbre is cylindrical in shape, with a plasma membrane enclosing a narrow region o cytoplasm. The diameter in most cases is about 1 m, though some nerve fbres are wider than this. A nerve fbre with this simple structure conducts nerve impulses at a speed o about 1 metre per second.  Figure 2

Nerve fbres (axons) transmitting electrical impulses to and rom the central nervous system are grouped into bundles

myelin nucleus of node of sheath Schwann cell Ranvier

axon

S ome nerve fbres are coated along most o their length by a material called myelin. It consists o many layers o phospholipid bilayer. S pecial cells called S chwann cells deposit the myelin by growing round and round the nerve fbre. E ach time they grow around the nerve fbre a double layer o phospholipid bilayer is deposited. There may be 2 0 or more layers when the S chwann cell stops growing. There is a gap between the myelin deposited by adj acent S chwann cells. The gap is called a node o Ranvier. In myelinated nerve fbres the nerve impulse can j ump rom one node o Ranvier to the next. This is called saltatory conduction. It is much quicker than continuous transmission along a nerve fbre so myelinated nerve fbres transmit nerve impulses much more rapidly than unmyelinated nerve fbres. The speed can be as much as 1 00 metres per second.

 Figure 3

Detail o a myelinated nerve fbre showing the gaps between adjacent Schwann cells (nodes o Ranvier)

 Figure 4 Transverse section

o axon showing the myelin sheath ormed by the Schwann cell's membrane wrapped round the axon many times (red)

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Resting potentials Neurons pump sodium and potassium ions across their membranes to generate a resting potential.

uid outside neuron Na + Na+ channel closed Na +





Sodiumpotassium pumps transer sodium (Na + ) and potassium (K + ) ions across the membrane. Na+ ions are pumped out and K+ ions are pumped in. The numbers o ions pumped is unequal  when three Na+ ions are pumped out, only two K+ ions are pumped in, creating concentration gradients or both ions. Also the membrane is about 5 0 times more permeable to K + ions than Na + ions, so K + ions leak back across the membrane aster than Na + ions. As a result, the Na + concentration gradient across the membrane is steeper than the K + gradient, creating a charge imbalance.

K+ Na +

Na +

A neuron that is not transmitting a signal has a potential dierence or voltage across its membrane that is called the resting potential. This potential is due to an imbalance o positive and negative charges across the membrane. 

Na + Na +

K+

Na + Na +

Na +

Na+ /K+ pump

K+

-

K+ channel closed

K+

-

K+

K+

K+ -

-

K+ K+

-

-

Na+

K+

K+

K+

protein

K+

K+

K+

K+

K+ K+

K+

cytoplasm  Figure 5 The resting potential

is generated by the sodiumpotassium pump

In addition to this, there are proteins inside the nerve fbre that are negatively charged ( organic anions) , which increases the charge imbalance.

These actors together give the neuron a resting membrane potential o about - 70 mV.

Action potentials An action potential consists of depolarization and repolarization of the neuron. An action potential is a rapid change in membrane potential, consisting o two phases: 

depolarization  a change rom negative to positive



repolarization  a change back rom positive to negative.

D epolarization is due to the opening o sodium channels in the membrane, allowing Na + ions to diuse into the neuron down the concentration gradient. The entry o Na + ions reverses the charge imbalance across the membrane, so the inside is positive relative to the outside. This raises the membrane potential to a positive value o about + 3 0 mV.

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Hum C Ean LLpBHI ys O LO i oGlo Y gy uid outside neuron

uid outside neuron Na+

Na +

Na+ channel open Na +

K+

Na+

K+

Na+

Na + channel closed

Na+

Na+

K+

K+

Na+

K+ K

K+

Na +

-

-

K+

+

K

-

K+

Na + Na

+

-

K+ Na +

K+

protein K

K+

K+

Na+ +

K+ channel closed

Na+

Na +

K+

- -

K+

K+

K+

Na+ /K+ pump

Na + /K+ pump

-

K+

K+ K+

+

+

Na+

Na +

Na+

Na

K+

K+

K+

K+ Na +

-

K+

K+

Na+

Na + K+

Na +

K

K+

-

Na+

K+ +

K+

- K+

protein

Na +

Na+

-

K+ K+

Na +

Na+

+

-K K+

-

K+

K+

cytoplasm

cytoplasm  Figure 6 Neuron

depolarizing

impulse movement + + + + + + + + +

A

        

B

+        

cell membrane cytoplasm

 + + + + + + + +

 Figure 7

Neuron repolarizing

Repolarization happens rapidly after depolarization and is due to the closing of the sodium channels and opening of potassium channels in the membrane. This allows potassium ions to diffuse out of the neuron, down their concentration gradient, which makes the inside of the cell negative again relative to the outside. The potassium channels remain open until the membrane has fallen to a potential close to - 7 0 mV. The diffusion of potassium repolarizes the neuron, but it does not restore the resting potential as the concentration gradients of sodium and potassium ions have not yet been re- established. This takes a few milliseconds and the neuron can then transmit another nerve impulse.

Na Na++

proagation of action otentials   + + + + + + +

C

+ +       

++ NaNa

K+

+    + + + + +

D

 + + +      Na + Na +

K+

+ + +    + + +

E

   + + +    Na + Na +

 Figure 8 Action

along axons

322

potentials are propagated

K+ channel open

Nerve impulses are action potentials propagated along the axons of neurons. A nerve impulse is an action potential that starts at one end of a neuron and is then propagated along the axon to the other end of the neuron. The propagation of the action potential happens because the ion movements that depolarize one part of the neuron trigger depolarization in the neighbouring part of the neuron. Nerve impulses always move in one direction along neurons in humans and other vertebrates. This is because an impulse can only be initiated at one terminal of a neuron and can only be passed on to other neurons or

Na +

6 . 5 n e u r o n S an D S yn apS e S dierent cell types at the other terminal. Also, there is a reractive period ater a depolarization that prevents propagation o an action potential backwards along an axon.

loca currents Propagation o nerve impulses is the result o local currents that cause each successive part o the axon to reach the threshold potential. The propagation o an action potential along an axon is due to movements o sodium ions. D epolarization o part o the axon is due to diusion o sodium ions into the axon through sodium channels. This reduces the concentration o sodium ions outside the axon and increases it inside. The depolarized part o the axon thereore has dierent sodium ion concentrations to the neighbouring part o the axon that has not yet depolarized. As a result, sodium ions diuse between these regions both inside and outside the axon. Inside the axon there is a higher sodium ion concentration in the depolarized part o the axon so sodium ions diuse along inside the axon to the neighbouring part that is still polarized. O utside the axon the concentration gradient is in the opposite direction so sodium ions diuse rom the polarized part back to the part that has j ust depolarized. These movements are shown in fgure 1 0. They are called local currents. Local currents reduce the concentration gradient in the part o the neuron that has not yet depolarized. This makes the membrane potential rise rom the resting potential o - 70mV to about - 5 0 mV. Sodium channels in the axon membrane are voltage-gated and open when a membrane potential o - 5 0mV is reached. This is thereore known as the threshold potential. Opening o the sodium channels causes depolarization.

activit ns i  s m d  mfsh Anemonesh have a nervous system similar to ours, with a central nervous system and neurons that transmit nerve impulses in one direction only. Sea anemones have no central nervous system. Their neurons orm a simple network and will transmit impulses in either direction along their nerve bres. They both protect each other rom predators more efectively than they can themselves. Explain how they do this.

 Figure 9 Anemonefsh among

the tentacles o a sea anemone

Thus local currents cause a wave o depolarization and then repolarization to be propagated along the axon at a rate o between one and a hundred ( or more) metres per second.

impulse movement

+ N a d i u s i o n

outside inside N a + d i u s i o n

part that has just depolarized (action potential)  Figure 10

membrane

part that has not yet depolarized (resting potential)

Local currents

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Hum C Ean LLpBHI ys O LO i oGlo Y gy action potential peak

Analysing oscilloscope traces

0

re po la riz at io n

de po la riz at io n

potential dierence across membrane (mV)

+35

50 70 undershoot

Analysis o oscilloscope traces showing resting potentials and action potentials. threshold potential resting potential

0 1 2 3 45 6 7 time/ms stimulus  Figure 11

Changes in membrane polarity during an action potential

Membrane potentials in neurons can be measured by placing electrodes on each side o the membrane. The potentials can be displayed using an oscilloscope. The display is similar to a graph with time on the x- axis and the membrane potential on the y- axis. I there is a resting potential, a horizontal line appears on the oscilloscope screen at a level o - 7 0 mV, assuming that this is the resting potential o the neuron. I an action potential occurs, a narrow spike is seen, with the rising and alling phases showing the depolarization and repolarization. The oscilloscope trace may also show the potential rising beore the depolarization until the threshold potential is reached. The repolarization does not usually return the membrane potential to - 70 mV immediately and there is a phase in which the potential changes gradually until the resting potential is reached.

Data-based questions: Analysing an oscilloscope trace The oscilloscope trace in gure 1 2 was taken rom a digital oscilloscope. It shows an action potential in a mouse hippocampal pyramidal neuron that happened ater the neuron was stimulated with a pulse o current.

1

2

membrane voltage (mV)

3 50

50 time (ms)

D educe with a reason the threshold potential needed to open voltage- gated sodium channels in this neuron.

[2 ]

Estimate the time taken or the depolarization, and the repolarization.

[2 ]

Predict the time taken rom the end o the depolarization or the resting potential to be regained. [2 ]

5

D iscuss how many action potentials could be stimulated per second in this neuron.

[2 ]

S uggest a reason or the membrane potential rising briefy at the end o the repolarization.

[1 ]

50 0

[1 ]

4 0 resting potential

S tate the resting potential o the mouse hippocampal pyramidal neuron.

100

6

 Figure 12

Synapses Synapses are junctions between neurons and between neurons and receptor or efector cells. Synapses are junctions between cells in the nervous system. In sense organs there are synapses between sensory receptor cells and neurons. In both the brain and spinal cord there are immense numbers o synapses between neurons. In muscles and glands there are synapses between neurons and

324

6 . 5 n e u r o n S an D S yn apS e S muscle bres or secretory cells. Muscles and glands are sometimes called eectors, because they eect (carry out) a response to a stimulus. C hemicals called neurotransmitters are used to send signals across synapses. This system is used at all synapses where the pre- synaptic and post- synaptic cells are separated by a fuid-lled gap, so electrical impulses cannot pass across. This gap is called the synaptic clet and is only about 2 0 nm wide.

Synaptic transmission When pre-synaptic neurons are depolarized they release a neurotransmitter into the synapse. S ynaptic transmission occurs very rapidly as a result o these events: 

A nerve impulse is propagated along the pre-synaptic neuron until it reaches the end o the neuron and the pre-synaptic membrane.



D epolarization o the pre-synaptic membrane causes calcium ions ( C a 2+ ) to diuse through channels in the membrane into the neuron.



Infux o calcium causes vesicles containing neurotransmitter to move to the pre-synaptic membrane and use with it. Neurotransmitter is released into the synaptic clet by exocytosis.



The neurotransmitter diuses across the synaptic clet and binds to receptors on the post- synaptic membrane.

pre-synaptic cell

nerve impulse

pre-synaptic membrane neurotransmitter (e.g. acetylcholine)



Sodium ions diuse down their concentration gradient into the post- synaptic neuron, causing the postsynaptic membrane to reach the threshold potential.

The neurotransmitter is rapidly broken down and removed rom the synaptic clet.

synaptic cleft 20nm approximately

neurotransmitter activates receptors

The binding o the neurotransmitter to the receptors causes adj acent sodium ion channels to open.

An action potential is triggered in the post- synaptic membrane and is propagated on along the neuron.

synaptic knob synaptic vesicles





Electron micrograph o a synapse. False colour has been used to indicate the pre-synaptic neuron (purple) with vesicles o neurotransmitter (blue) and the post-synaptic neuron (pink) . The narrowness o the synaptic clet is visible

Ca 2+ diuses into knob





 Figure 13

ion channel opened post-synaptic membrane post-synaptic cell

 Figure 14 A nerve impulse is propagated

across a synapse by the release, difusion and post-synaptic binding o neurotransmitter

Dt-bsd qstis: Parkinsons disease D opamine is one o the many neurotransmitters that are used at synapses in the brain. In Parkinsons disease, there is a loss o dopaminesecreting neurons, which causes slowness in initiating movement, muscular rigidity and in many cases shaking. Figure 1 5 shows the

metabolic pathways involved in the ormation and breakdown o dopamine. 1

Explain how symptoms o Parkinsons disease are relieved by giving the ollowing drugs: a) L- D O PA

[1 ]

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2

b) selegeline, which is an inhibitor o monoamine oxidase- B ( MAO - B )

[1 ]

c) tolcapone, which is an inhibitor o catechol- O - methyl transerase ( C O MT)

[1 ]

tyrosine COOH tyrosine hydroxylase HO CH 2 CH NH 2 (FOOD) COMT

HO

COOH CH 2 CH NH 2

HO

CH 3 O

d) ropinirole, which is an agonist o dopamine

[1 ]

e) safnamide, which inhibits reuptake o dopamine by pre-synaptic neurons.

[1 ]

HO

L-DOPA

CH 2 CH 2 NH 2 HO

HO

D iscuss how a cure or Parkinsons disease might in the uture be developed by: a) stem cell therapy

[3 ]

b) gene therapy.

[2 ]

HO CH 3 O

CH 2 COOH

COMT

HO

COOH

CH 2 CH NH 2 HO dopa dopamine decarboxylase

HO

MAO-B O CH 2 C H aldehyde dehydrogenase CH 2 COOH

HO

 Figure 15 The formation and

breakdown of L-DOPA and dopamine. The enzymes catalysing each step are shown in red

Acetylcholine Secretion and reabsorption of acetylcholine by neurons at synapses. Acetylcholine is used as the neurotransmitter in many synapses, including synapses between neurons and muscle fbres. It is produced in the pre- synaptic neuron by combining choline, absorbed rom the diet, with an acetyl group produced during aerobic respiration. The acetylcholine is loaded into vesicles and then released into the synaptic clet during synaptic transmission. choline acetyl group  Figure 16 Acetylcholine

The receptors or acetylcholine in the post- synaptic membrane have a binding site to which acetylcholine will bind. The acetylcholine only remains bound to the receptor or a short time, during which only one action potential is initiated in the post- synaptic neuron. This is because the enzyme acetylcholinesterase is present in the synaptic clet and rapidly breaks acetylcholine down into choline and acetate. The choline is reabsorbed into the pre- synaptic neuron, where it is converted back into active neurotransmitter by recombining it with an acetyl group.

Neonicotinoids Blocking of synaptic transmission at cholinergic synapses in insects by binding of neonicotinoid pesticides to acetylcholine receptors. Neonicotinoids are synthetic compounds similar to nicotine. They bind to the acetylcholine receptor in cholinergic synapses in the central nervous system o insects. Acetylcholinesterase does not

326

6 . 5 n e u r o n S an D S yn apS e S

break down neonicotinoids, so the binding is irreversible. The receptors are blocked, so acetylcholine is unable to bind and synaptic transmission is prevented. The consequence in insects is paralysis and death. Neonicotinoids are thereore very eective insecticides. O ne o the advantages o neonicotinoids as pesticides is that they are not highly toxic to humans and other mammals. This is because a much greater proportion o synapses in the central nervous system are cholinergic in insects than in mammals and also because neonicotinoids bind much less strongly to acetylcholine receptors in mammals than insects. Neonicotinoid pesticides are now used on huge areas o crops. In particular one neonicotinoid, imidacloprid, is the most widely used insecticide in the world. However, concerns have been raised about the eects o these insecticides on honeybees and other benefcial insects. There has been considerable controversy over this and the evidence o harm is disputed by the manuacturers and some government agencies.

Threshold potentials A nerve impulse is only initiated i the threshold potential is reached.

activit rsch dts  ictiids There are currently intense research eforts to try to discover whether neonicotinoids are to blame or collapses in honeybee colonies. What are the most recent research ndings and do they suggest that these insecticides should be banned?

 Figure 17 Research has

shown that the neonicotinoid pesticide imidacloprid reduces growth of bumblebee colonies

Nerve impulses ollow an all-or-nothing principle. An action potential is only initiated i the threshold potential is reached, because only at this potential do voltage-gated sodium channels start to open, causing depolarization. The opening o some sodium channels and the inward diusion o sodium ions increases the membrane potential causing more sodium channels to open  there is a positive eedback eect. I the threshold potential is reached there will thereore always be a ull depolarization. At a synapse, the amount o neurotransmitter secreted ollowing depolarization o the pre-synaptic membrane may not be enough to cause the threshold potential to be reached in the post-synaptic membrane. The post-synaptic membrane does not then depolarize. The sodium ions that have entered the post-synaptic neuron are pumped out by sodium potassium pumps and the post-synaptic membrane returns to the resting potential. A typical post- synaptic neuron in the brain or spinal cord has synapses not j ust with one but with many pre- synaptic neurons. It may be necessary or several o these to release neurotransmitter at the same time or the threshold potential to be reached and a nerve impulse to be initiated in the post- synaptic neuron. This type o mechanism can be used to process inormation rom dierent sources in the body to help in decision- making.

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Research into memory and learning Cooperation and collaboration between groups of scientists: biologists are contributing to research into memory and learning. Higher unctions o the brain including memory and learning are only partly understood at present and are being researched very actively. They have traditionally been investigated by psychologists but increasingly the techniques o molecular biology and biochemistry are being used to unravel the mechanisms at work. O ther branches o science are also making important contributions, including biophysics, medicine, pharmacology and computer science.  Figure 18 Many synapses are visible in this

scanning electron micrograph between the cell body o one post-synaptic neuron and a large number o diferent pre-synaptic neurons (blue)

The C entre or Neural C ircuits and B ehaviour at O xord University is an excellent example o collaboration between scientists with dierent areas o expertise. The our group leaders o the research team and the area o science that they originally studied are: 

Proessor Gero Miesenbck  medicine and physiology



D r Martin B ooth  engineering and optical microscopy



D r Korneel Hens  chemistry and biochemistry



Proessor S cott Waddell  genetics, molecular biology and neurobiology.

The centre specializes in research techniques known as optogenetics. Neurons are genetically engineered to emit light during synaptic transmission or an action potential, making activity in specifc neurons in brain tissue visible. They are also engineered so specifc neurons in brain tissue respond to a light signal with an action potential. This allows patterns o activity in the neurons o living brain tissue to be studied.

 Figure 19

Memory and learning are unctions o the cerebrumthe olded upper part o the brain

328

There are many research groups in universities throughout the world that are investigating memory, learning and other brain unctions. Although there is sometimes competition between scientists to be the frst group to make a discovery, there is also a strongly collaborative element to scientifc research. This extends across scientifc disciplines and national boundaries. Success in understanding how the brain works will undoubtedly be the achievement o many groups o scientists in many countries throughout the world.

6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n

6.6 hs, sss d dc Understanding  Insulin and glucagon are secreted by  and





 







 cells in the pancreas to control blood glucose concentration. Thyroxin is secreted by the thyroid gland to regulate the metabolic rate and help control body temperature. Leptin is secreted by cells in adipose tissue and acts on the hypothalamus o the brain to inhibit appetite. Melatonin is secreted by the pineal gland to control circadian rhythms. A gene on the Y chromosome causes embryonic gonads to develop as testes and secrete testosterone. Testosterone causes prenatal development o male genitalia and both sperm production and development o male secondary sexual characteristics during puberty. Estrogen and progesterone cause prenatal development o emale reproductive organs and emale secondary sexual characteristics during puberty. The menstrual cycle is controlled by negative and positive eedback mechanisms involving ovarian and pituitary hormones.

Applications  Causes and treatment o type I and type II 

 



diabetes. Testing o leptin on patients with clinical obesity and reasons or the ailure to control the disease. Causes o jet lag and use o melatonin to alleviate it. The use in IVF o drugs to suspend the normal secretion o hormones, ollowed by the use o artifcial doses o hormones to induce superovulation and establish a pregnancy. William Harveys investigation o sexual reproduction in deer.

Skills  Annotate diagrams o the male and emale

reproductive system to show names o structures and their unctions.

Nature of science  Developments in scientifc research ollow

improvements in apparatus: William Harvey was hampered in his observational research into reproduction by lack o equipment. The microscope was invented 17 years ater his death.

Control of blood glucose concentration Insulin and glucagon are secreted by  and  cells in the pancreas to control blood glucose concentration. C ells in the pancreas respond to changes in blood glucose levels. If the glucose concentration deviates substantially from the set point of about 5 mmol L - 1 , homeostatic mechanisms mediated by the pancreatic hormones insulin and glucagon are initiated.

329

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Hum C Ean LLpBHI ys O LO i oGlo Y gy The pancreas is eectively two glands in one organ. Most o the pancreas is exocrine glandular tissue that secretes digestive enzymes into ducts leading to the small intestine. There are small regions o endocrine tissue called islets o Langerhans dotted through the pancreas that secrete hormones directly into the blood stream. The two cell types in the islets o Langerhans secrete dierent hormones.

 Figure 1

Fluorescent light micrograph of the pancreas showing two islets of Langerhans surrounded by exocrine gland tissue. Alpha cells in the islets are stained yellow and beta cells are stained red



Alpha cells (  cells) synthesize and secrete glucagon i the blood glucose level alls below the set point. This hormone stimulates breakdown o glycogen into glucose in liver cells and its release into the blood, increasing the concentration.



B eta cells (  cells) synthesize insulin and secrete it when the blood glucose concentration rises above the set point. This hormone stimulates uptake o glucose by various tissues, particularly skeletal muscle and liver, in which it also stimulates the conversion o glucose to glycogen. Insulin thereore reduces blood glucose concentration. Like most hormones, insulin is broken down by the cells it acts upon, so its secretion must be ongoing. S ecretion begins within minutes o eating and may continue or several hours ater a meal.

Diabetes Causes and treatment of type I and type II diabetes. Diabetes is the condition where a person has consistently elevated blood glucose levels even during prolonged asting, leading to the presence o glucose in the urine. C ontinuously elevated glucose damages tissues, particularly their proteins. It also impairs water reabsorption rom urine while it is orming in the kidney, resulting in an increase in the volume o urine and body dehydration. I a person needs to urinate more requently, is constantly thirsty, eels tired and craves sugary drinks, they should test or glucose in the urine to check whether they have developed diabetes. There are two main types o this disease: 



330

Type I diabetes, or early- onset diabetes, is characterized by an inability to produce sufcient quantities o insulin. It is an autoimmune disease arising rom the destruction o beta cells in the islets o Langerhans by the bodys own immune system. In children and young people the more severe and obvious symptoms o the disease usually start rather suddenly. The causes o this and other autoimmune diseases are still being researched. Type II diabetes, sometimes called late- onset diabetes, is characterized by an inability to

process or respond to insulin because o a defciency o insulin receptors or glucose transporters on target cells. O nset is slow and the disease may go unnoticed or many years. Until the last ew decades, this orm o diabetes was very rare in people under 5 0 and common only in the over 65 s. The causes o this orm o diabetes are not well understood but the main risk actors are sugary, atty diets, prolonged obesity due to habitual overeating and lack o exercise, together with genetic actors that aect energy metabolism. The treatment o the two types o diabetes is dierent: 

Type I diabetes is treated by testing the blood glucose concentration regularly and inj ecting insulin when it is too high or likely to become too high. Inj ections are oten done beore a meal to prevent a peak o blood glucose as the ood is digested and absorbed. Timing is very important because insulin molecules do not last long in the blood. B etter treatments are being developed using implanted devices that can release exogenous insulin into the blood as and when it is necessary. A permanent cure may be achievable by coaxing stem cells to become ully unctional replacement beta cells.

6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n

Type II diabetes is treated by adj usting the diet to reduce the peaks and troughs o blood glucose. S mall amounts o ood should be eaten requently rather than inrequent large meals. Foods with high sugar content should be avoided. S tarchy ood should only be eaten



i it has a low glycemic index, indicating that it is digested slowly. High- fbre oods should be included to slow the digestion o other oods. S trenuous exercise and weight loss are benefcial as they improve insulin uptake and action.

acvy

The glucose tolerance test is a method used to diagnose diabetes. In this test, the patient drinks a concentrated glucose solution. The blood glucose concentration is monitored to determine the length o time required or excess glucose to be cleared rom the blood.

Fds f y ii dbcs

concentration / mg 100 cm 3

D-bsd qss: The glucose tolerance test

400 350 300 250 200 150 100 50 0

Discuss which o the oods in fgure 2 are suitable or a person with type II diabetes. They should be oods with a low glycemic index.

diabetic unaected

0

0.5

1

2 3 4 time after glucose ingestion / h

5

 Figure 3

A person with diabetes and an unafected person give very diferent responses to the glucose tolerance test

With reerence to fgure 3 , compare the person with normal glucose metabolism to the person with diabetes with respect to: a) The concentration o glucose at time zero, i.e. beore the consumption o the glucose drink. b) The length o time required to return to the level at time zero. c) The maximum glucose level reached.

 Figure 2

d) The time beore glucose levels start to all.

tyx Thyroxin is secreted by the thyroid gland to regulate the metabolic rate and help control body temperature. The hormone thyroxin is secreted by the thyroid gland in the neck. Its chemical structure is unusual as the thyroxin molecule contains our atoms o iodine. Prolonged defciency o iodine in the diet thereore prevents the synthesis o thyroxin. This hormone is also unusual as almost all cells in the body are targets. Thyroxin regulates the bodys metabolic rate, so all cells need to respond but the most metabolically active, such as liver, muscle and brain are the main targets. Higher metabolic rate supports more protein synthesis and growth and it increases the generation o body heat. In a person with normal physiology, cooling triggers increased thyroxin secretion by the thyroid gland, which stimulates heat production so body temperature rises.

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Hum C Ean LLpBHI ys O LO i oGlo Y gy Thyroxin thus regulates the metabolic rate and also helps to control body temperature. The importance o thyroxin is revealed by the eects o thyroxin defciency ( hypothyroidism) :

 Figure 4 Structure of thyroxin

with atoms of



lack o energy and eeling tired all the time



orgetulness and depression



weight gain despite loss o appetite as less glucose and at are being broken down to release energy by cell respiration



eeling cold all the time because less heat is being generated



constipation because contractions o muscle in the wall o the gut slow down.



impaired brain development in children.

iodine shown purple

leptin Leptin is secreted by cells in adipose tissue and acts on the hypothalamus of the brain to inhibit appetite. Leptin is a protein hormone secreted by adipose cells ( at storage cells) . The concentration o leptin in the blood is controlled by ood intake and the amount o adipose tissue in the body. The target o this hormone is groups o cells in the hypothalamus o the brain that contribute to the control o appetite. Leptin binds to receptors in the membrane o these cells. I adipose tissue increases, blood leptin concentrations rise, causing long- term appetite inhibition and reduced ood intake.

 Figure 5 Mouse with obesity

due to lack of leptin and a mouse with normal body mass

The importance o this system was demonstrated by research with a strain o mice discovered in the 1 95 0s that eed ravenously, become inactive and gain body weight, mainly through increased adipose tissue. They grow to a body weight o about 1 00 grams, compared with wild type mice o 2 02 5 grams. B reeding experiments showed that the obese mice had two copies o a recessive allele, ob. In the early 1 990s it was shown that the wild- type allele o this gene supported the synthesis o a new hormone that was named leptin. Adipose cells in mice that have two recessive ob alleles cannot produce leptin. When ob/ob mice were inj ected with leptin their appetite declined, energy expenditure increased and body mass dropped by 3 0% in a month.

leptin and obesity Testing of leptin on patients with clinical obesity and reasons for the failure to control the disease. The discovery that obesity in mice could be caused by a lack o leptin and cured by leptin injections soon led to attempts to treat obesity in humans in this way. Amgen, a biotechnology company based in C aliornia, paid $2 0 million or the commercial rights to leptin and a large clinical trial was carried

332

out. Seventy-three obese volunteers injected themselves either with one o several leptin doses or with a placebo. A double blind procedure was used, so neither the researchers nor the volunteers knew who was injecting leptin until the results were analysed.

6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n

The leptin inj ections induced skin irritation and swelling and only 47 patients completed the trial. The eight patients receiving the highest dose lost 7.1 kg o body mass on average compared with a loss o 1 .3 kg in the 1 2 volunteers who were inj ecting the placebo. However, in the group receiving the highest dose the results varied very widely rom a loss o 1 5 kg to a gain o 5 kg. Also any body mass lost during the trial was usually regained rapidly aterwards. S uch disappointing outcomes are requent in drug research  the physiology o humans is dierent in many ways rom mice and other rodents. In contrast to ob/ob mice, most obese humans have exceptionally high blood leptin concentrations. The target cells in the hypothalamus may have become resistant to leptin so ail to respond to it, even at high concentrations. Appetite is thereore not inhibited and ood intake is excessive. More adipose

tissue develops, causing a rise in blood leptin concentration but the leptin resistance prevents inhibition o appetite. Inj ection o extra leptin inevitably ails to control obesity i the cause is leptin resistance, j ust as insulin inj ections alone are ineective with early- stage type II diabetes. A very small proportion o cases o obesity in humans are due to mutations in the genes or leptin synthesis or its various receptors on target cells. Trials in people with such obesity have shown signifcant weight loss while the leptin injections are continuing. However leptin is a short-lived protein and has to be injected several times a day and consequently most o those oered this treatment have reused it. Also leptin has been shown to aect the development and unctioning o the reproductive system, so injections are not suitable in children and young adults. All in all leptin has not ulflled its early promise as a means o solving the human obesity problem.

melatonin Melatonin is secreted by the pineal gland to control circadian rhythms. Humans are adapted to live in a 2 4- hour cycle and have rhythms in behaviour that ft this cycle. These are known as circadian rhythms. They can continue even i a person is placed experimentally in continuous light or darkness because an internal system is used to control the rhythm. C ircadian rhythms in humans depend on two groups o cells in the hypothalamus called the suprachiasmatic nuclei ( S C N) . These cells set a daily rhythm even i grown in culture with no external cues about the time o day. In the brain they control the secretion o the hormone melatonin by the pineal gland. Melatonin secretion increases in the evening and drops to a low level at dawn and as the hormone is rapidly removed rom the blood by the liver, blood concentrations rise and all rapidly in response to these changes in secretion. The most obvious eect o melatonin is the sleep- wake cycle. High melatonin levels cause eelings o drowsiness and promote sleep through the night. Falling melatonin levels encourage waking at the end o the night. Experiments have shown that melatonin contributes to the nighttime drop in core body temperature, as blocking the rise in melatonin levels reduces it and giving melatonin artifcially during the day causes a drop in core temperature. Melatonin receptors have been discovered in the kidney, suggesting that decreased urine production at night may be another eect o this hormone. When humans are placed experimentally in an environment without light cues indicating the time o day, the S C N and pineal gland usually

 Figure 6 Until

a baby is about three months old it does not develop a regular day-night rhythm o melatonin secretion so sleep patterns do not ft those o the babys parents

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Hum C Ean LLpBHI ys O LO i oGlo Y gy maintain a rhythm o slightly longer than 2 4 hours. This indicates that timing o the rhythm is normally adj usted by a ew minutes or so each day. A special type o ganglion cell in the retina o the eye detects light o wavelength 460480 nm and passes impulses to cells in the S C N. This indicates to the SC N the timing o dusk and dawn and allows it to adj ust melatonin secretion so that it corresponds to the day- night cycle.

Jet lag and melatonin Causes of jet lag and use of melatonin to alleviate it. Jet lag is a common experience or someone who has crossed three or more time zones during air travel. The symptoms are diculty in remaining awake during daylight hours and diculty sleeping through the night, atigue, irritability, headaches and indigestion. The causes are easy to understand: the S C N and pineal gland are continuing to set a circadian rhythm to suit the timing o day and night at the point o departure rather than the destination.

Jet lag only lasts or a ew days, during which impulses sent by ganglion cells in the retina to the SC N when they detect light help the body to adj ust to the new regime. Melatonin is sometimes used to try to prevent or reduce j et lag. It is taken orally at the time when sleep should ideally be commencing. Most trials o melatonin have shown that it is eective at promoting sleep and helping to reduce j et lag, especially i fying eastwards and crossing ve or more time zones.

Sex determination in males A gene on the Y chromosome causes embryonic gonads to develop as testes and secrete testosterone. Human reproduction involves the usion o a sperm rom a male with an egg rom a emale. Initially the development o the embryo is the same in all embryos and embryonic gonads develop that could either become ovaries or testes. The developmental pathway o the embryonic gonads and thereby the whole baby depends on the presence or absence o one gene. 

I the gene SRY is present, the embryonic gonads develop into testes. This gene is located on the Y chromosome, so is only present in 5 0% o embryos. S RY codes or a D NA- binding protein called TD F ( testis determining actor) . TD F stimulates the expression o other genes that cause testis development.



5 0% o embryos have two X chromosomes and no Y so they do not have a copy o the S RY gene. TD F is thereore not produced and the embryonic gonads develop as ovaries.

Testosterone Testosterone causes prenatal development of male genitalia and both sperm production and development of male secondary sexual characteristics during puberty.

 Figure 7

334

X and Y chromosomes

The testes develop rom the embryonic gonads in about the eighth week o pregnancy, at the time when the embryo is becoming a etus and is about 3 0mm long. The testes develop testosterone-secreting cells at an early stage and these produce testosterone until about the teenth week

6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n o pregnancy. D uring the weeks o secretion, testosterone causes male genitalia to develop, which are shown in fgure 8. At puberty the secretion o testosterone increases. This stimulates sperm production in the testes, which is the primary sexual characteristic o males. Testosterone also causes the development o secondary sexual characteristics during puberty such as enlargement o the penis, growth o pubic hair and deepening o the voice due to growth o the larynx.

Sex deterination in feales Estrogen and progesterone cause prenatal development of female reproductive organs and female secondary sexual characteristics during puberty. I the gene S RY is not present in an embryo because there is no Y chromosome, the embryonic gonads develop as ovaries. Testosterone is thereore not secreted, but the two emale hormones, estrogen and progesterone, are always present in pregnancy. At frst they are secreted by the mothers ovaries and later by the placenta. In the absence o etal testosterone and the presence o maternal estrogen and progesterone, emale reproductive organs develop which are shown in fgure 9. During puberty the secretion o estrogen and progesterone increases, causing the development o emale secondary sexual characteristics. These include enlargement o the breasts and growth o pubic and underarm hair.

male and feale reproductive systes Annotate diagrams of the male and female reproductive system to show names of structures and their functions. The tables on the next page indicate unctions that should be included when diagrams o male and emale reproductive systems are annotated. seminal vesicle bladder bladder sperm duct sperm duct

prostate gland

seminal vesicle erectile tissue

penis

prostate gland

penis epididymis

testis epididymis

urethra

urethra scrotum

testis scrotum

 Figure 8

foreskin

Male reproductive system in front and side view

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Hum C Ean LLpBHI ys O LO i oGlo Y gy

oviduct

ovary opening to oviduct

oviduct ovary

uterus

uterus

cervix

bladder

vagina

urethra

large intestine vulva

vagina

cervix

labia (vulva)

 Figure 9

Female reproductive system in front and side view

male reproductive syste

Feale reproductive syste

Testis

Produce sperm and testosterone

Ovary

Produce eggs, estrogen and progesterone

Scrotum

Hold testes at lower than core body temperature

Oviduct

Epididymis

Store sperm until ejaculation

Collect eggs at ovulation, provide a site or ertilization then move the embryo to the uterus

Sperm duct

Transer sperm during ejaculation

Uterus

Provide or the needs o the embryo and then etus during pregnancy

Seminal vesicle and prostate gland

Secrete fuid containing alkali, proteins and ructose that is added to sperm to make semen

Cervix

Protect the etus during pregnancy and then dilate to provide a birth canal

Urethra

Transer semen during ejaculation and urine during urination

Vagina

Stimulate penis to cause ejaculation and provide a birth canal

Penis

Penetrate the vagina or ejaculation o semen near the cervix

Vulva

Protect internal parts o the emale reproductive system

menstrual cycle The menstrual cycle is controlled by negative and positive eedback mechanisms involving ovarian and pituitary hormones. The menstrual cycle occurs in most women rom puberty until the menopause, apart rom during pregnancies. E ach time the cycle occurs it gives the chance o a pregnancy. The frst hal o the menstrual cycle is called the ollicular phase because a group o ollicles is developing in the ovary. In each ollicle an egg is stimulated to grow. At the same time the lining o the uterus ( endometrium) is repaired and starts to thicken. The most developed ollicle breaks open, releasing its egg into the oviduct. The other ollicles degenerate. The second hal o the cycle is called the luteal phase because the wall o the ollicle that released an egg becomes a body called the corpus luteum. C ontinued development o the endometrium prepares

336

6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n it or the implantation o an embryo. I ertilization does not occur the corpus luteum in the ovary breaks down. The thickening o the endometrium in the uterus also breaks down and is shed during menstruation.

TOK

Figure 1 0 shows hormone levels in a woman over a 3 6- day period, including one complete menstrual cycle. The pattern o changes is typical or a woman who is not pregnant. The hormone levels are measured in mass per millilitre. The actual masses are very small, so progesterone, FS H and LH are measured in nanograms ( ng) and estrogen is measured in picograms ( pg) . Figure 1 0 also shows the state o the ovary and o the endometrium.

Human eggs can be obtained by using FSH to stimulate the ovaries, then collecting eggs rom the ovaries using a micropipette. Women have sometimes undergone this procedure to produce eggs or donation to another woman who is unable to produce eggs hersel.

t w x d vs  w jdgg  ly f  c?

The our hormones in fgure 1 0 all help to control the menstrual cycle by both negative and positive eedback. FS H and LH are protein hormones produced by the pituitary gland that bind to FS H and LH receptors in the membranes o ollicle cells. E strogen and progesterone are ovarian hormones, produced by the wall o the ollicle and corpus

LH FSH

800 600 400 200

menstruation

menstruation

hormone level /ng ml 1

1000

follicle starting to develop

400

corpus luteum

follicle nearly mature

8

progesterone estrogen

300

6

200

4

100

2

0 26 28 2 4 5 8 days of menstrual cycle thickness of endometrium

ovulation

28  Figure 10

10 12 14 16 18 20 22 24 26 28

7

14

21

2

4

progesterone level/ng ml 1

estrogen level/pg ml 1

0

Recently stem-cell researchers have used eggs in therapeutic cloning experiments. The nucleus o an egg is removed and replaced with a nucleus rom an adult. I the resulting cell developed as an embryo, stem cells could be removed rom it and cloned. It might then be possible to produce tissues or organs or transplanting to the adult who donated the nucleus. There would be no danger o tissue rejection because the stem cells would be genetically identical to the recipient. There is a shortage o eggs both or donation to other women and or research. In 2006, scientists in England got permission to ofer women cut-price IVF treatment, i they were willing to donate some eggs or research. In Sweden only travel and other direct expenses can be paid to egg donors, and in Japan egg donation is banned altogether. 1 Is there a distinction to be drawn between donating eggs or therapeutic cloning experiments and donating eggs to a woman who is unable to produce eggs hersel, or example because her ovaries have been removed? Can the same act be judged diferently depending on motives?

28

The menstrual cycle

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Hum C Ean LLpBHI ys O LO i oGlo Y gy luteum. They are absorbed by many cells in the emale body, where they infuence gene expression and thereore development. 

FS H rises to a peak towards the end o the menstrual cycle and stimulates the development o ollicles, each containing an oocyte and ollicular fuid. FS H also stimulates secretion o estrogen by the ollicle wall.



E strogen rises to a peak towards the end o the ollicular phase. It stimulates the repair and thickening o the endometrium ater menstruation and an increase in FS H receptors that make the ollicles more receptive to FS H, boosting estrogen production ( positive eedback) . When it reaches high levels estrogen inhibits the secretion o FS H ( negative eedback) and stimulates LH secretion.



LH rises to a sudden and sharp peak towards the end o the ollicular phase. It stimulates the completion o meiosis in the oocyte and partial digestion o the ollicle wall allowing it to burst open at ovulation. LH also promotes the development o the wall o the ollicle ater ovulation into the corpus luteum which secretes estrogen ( positive eedback) and progesterone.



Progesterone levels rise at the start o the luteal phase, reach a peak and then drop back to a low level by the end o this phase. Progesterone promotes the thickening and maintenance o the endometrium. It also inhibits FSH and LH secretion by the pituitary gland ( negative eedback) .

Data-based questions: The female athlete triad

1

a) O utline the relationship between number o menstrual cycles per year and bone density. [3 ] b) Compare the results or the neck o the emur with the results or the trochanter. [3]

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2

3

Explain the reasons or some o the runners having:

a) higher bone density than the mean

[2 ]

b) lower bone density than the mean.

[4]

a) S uggest reasons or emale athletes having ew or no menstrual cycles.

[2 ]

b) Suggest one reason or eating disorders and low body weight in emale athletes. [1 ] t-score (SD)

The emale athlete triad is a syndrome consisting o three interrelated disorders that can aect emale athletes: osteoporosis, disordered eating and menstrual disorders. O steoporosis is reduced bone mineral density. It can be caused by a diet low in calcium, vitamin D or energy, or by low estrogen levels. Figure 1 1 shows the bone mineral density in two parts o the emur or emale runners who had dierent numbers o menstrual cycles per year. The t- score is the number o standard deviations above or below mean peak bone mass or young women.

1 neck of femur 0.5

trochanter of femur

0 0.5 1 menstrual cycles per year 03 410 1113

 Figure 11

Bone mass in women grouped by number of menstrual cycles

6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n

In vitro fertilization The use in IVF o drugs to suspend the normal secretion o hormones, ollowed by the use o artifcial doses o hormones to induce superovulation and establish a pregnancy. The natural method o ertilization in humans is in vivo, meaning that it occurs inside the living tissues o the body. Fertilization can also happen outside the body in careully controlled laboratory conditions. This is called in vitro ertilization, almost always abbreviated to IVF. This procedure has been used extensively to overcome ertility problems in either the male or emale parent. There are several dierent protocols or IVF, but the rst stage is usually down- regulation. The woman takes a drug each day, usually as a nasal spray, to stop her pituitary gland secreting FSH or LH. S ecretion o estrogen and progesterone thereore also stops. This suspends the normal menstrual cycle and allows doctors to control the timing and amount o egg production in the womans ovaries. Intramuscular inj ections o FS H and LH are then given daily or about ten days, to stimulate ollicles to develop. The FS H inj ections give a much higher concentration o this hormone than during a normal menstrual cycle and as

a consequence ar more ollicles develop than usual. Twelve is not unusual and there can be as many twenty ollicles. This stage o IVF is thereore called superovulation. When the ollicles are 1 8 mm in diameter they are stimulated to mature by an inj ection o HC G, another hormone that is normally secreted by the embryo. A micropipette mounted on an ultrasound scanner is passed through the uterus wall to wash eggs out o the ollicles. Each egg is mixed with 5 0, 000 to 1 00, 000 sperm cells in sterile conditions in a shallow dish, which is then incubated at 3 7 C until the next day. I ertilization is successul then one or more embryos are placed in the uterus when they are about 48 hours old. Because the woman has not gone through a normal menstrual cycle extra progesterone is usually given as a tablet placed in the vagina, to ensure that the uterus lining is maintained. I the embryos implant and continue to grow then the pregnancy that ollows is no dierent rom a pregnancy that began by natural conception.

William Harvey and sexual reproduction William Harveys investigation o sexual reproduction in deer. William Harvey is chiefy remembered or his discovery o the circulation o the blood, but he also had a lielong obsession with how lie is transmitted rom generation to generation and pioneered research into sexual reproduction. He was taught the seed and soil theory o Aristotle, according to which the male produces a seed, which orms an egg when it mixes with menstrual blood. The egg develops into a etus inside the mother. William Harvey tested Aristotles theory using a natural experiment. Deer are seasonal breeders and only become sexually active during the autumn. Harvey examined the uterus o emale deer during the mating season by slaughtering and dissecting them. He expected to nd eggs developing in the uterus immediately ater mating, but only ound signs o anything developing in emales two or more months ater the start o the mating season.

 Figure 12

IVF allows the earliest stages in a human life to be seen. This micrograph shows a zygote formed by fertilization. The nuclei of the egg and sperm are visible in the centre of the zygote. There is a protective layer of gel around the zygote called the fertilization membrane

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Hum C Ean LLpBHI ys O LO i oGlo Y gy

He regarded his experiments with deer as proo that Aristotles theory o reproduction was alse and concluded the etus doth neither proceed rom the seed o male or emale in coition, nor yet rom any commixture o that seed. Although Aristotles seed and soil theory was alse, Harveys conclusion that the etus did not result rom events during coitus ( sexual intercourse) was also alse. Harvey was well aware that he had not discovered the basis o sexual reproduction: neither the philosophers nor the physicians o yesterday or today have satisactorily explained, or solved the problem o Aristotle.  Figure 13

William Harveys book on the reproduction of animals Exercitationes de Generatione Animalium published in 1651

Improvements in apparatus and research breakthroughs Developments in scientifc research ollow improvements in apparatus: William Harvey was hampered in his observational research into reproduction by lack o equipment. The microscope was invented seventeen years ater his death. Harvey was understandably reluctant to publish his research into sexual reproduction, but he did eventually do so in 1 65 1 when he was 73 years old in his work Exercitationes de Generatione Animalium. He knew that he had not solved the mystery o sexual reproduction: When I plainly see nothing at all doth remain in the uterus ater coition, ... no more than remains in the braine ater sensation, ... I have invented this Fable. Let the learned and ingenious ock o men consider o it; let the supercilious reject it: and or the scofng ticklish generation, let them laugh their swinge. Because I say, there is no sensible thing in the uterus ater coition; and yet there is a necessity, that something should be there, which may render the animal ruitul.

340

William Harvey ailed to solve the mystery because eective microscopes were not available when he was working, so usion o gametes and subsequent embryo development remained undiscovered. He was unlucky with his choice o experimental animal because embryos in the deer that he used remain microscopically small or an unusually long period. Microscopes were invented seventeen years ater Harveys death, allowing the discovery o sperm, eggs and early stage embryos. Scientifc research has oten been hampered or a time by defciencies in apparatus, with discoveries only being made ollowing improvements. This will continue into the uture and we can look orward to urther transormations in our understanding o the natural world as new techniques and technology are invented.

QueStion S

Questions 1

Using the data in table 1 : a)

outline the relationship between the age of the mother and the success rate of IVF

[3 ]

b) outline the relationship between the number of embryos transferred and the chance of having a baby as a result of IVF c)

accidents during the daytime as a result of disrupted sleep and tiredness. Figure 1 5 shows the percentage oxygen saturation of arterial blood during a night of sleep in a patient with severe obstructive sleep apnea. 100 70

2

100 70

3

100 70

4

100 70

5

100 70

6

100 70

7

100 70

8

100 70

[3 ]

discuss how many embryos fertility centres should be allowed to transfer. [4]

hours

prcg f rgcs r iVF cycl ag f ccrdg  h mbr f mbrys rsfrrd mhr 1 2 3 single single twins single twins triplets < 30 10.4 20.1 9.0 17.5 3.6 0.4 3034 13.4 21.8 7.9 18.2 7.8 0.6 3539 19.1 19.1 5.0 17.4 5.6 0.6 > 39 4.1 12.5 3.5 12.7 1.7 0.1 Table 1

2

1

Figure 1 4 shows variations in liver glycogen over the course of one day.

O2%

0

a)

E xplain the variation in liver glycogen.

[3 ]

a)

liver glycogen level

an evening snack

8:00

12:00 16:00

20:00 24:00 time of da

breakfast 4:00

8:00

S ometimes the ventilation of the lungs stops. This is called apnea. O ne possible cause is the blockage of the airways by the soft palate during sleep. This is called obstructive sleep apnea. It has some potentially harmful consequences, including an increased risk of

50

60

Explain the causes of falls in saturation. [2]

( ii) E xplain the causes of rises in saturation.

[2 ]

( iii) C alculate how long each cycle of falling and rising saturation takes.

[2 ]

b) Estimate the minimum oxygen saturation that the patient experienced during the night, and when it occurred. [2 ]

Figure 14

3

30 40 minutes

Hour 8 shows a typical pattern due to obstructive sleep apnea. (i)

dinner

20

Figure 15

b) E valuate the contribution of glycogen to blood sugar homeostasis. [2 ]

lunch

10

c)

4

D educe the sleep patterns of the patient during the night when the trace was taken. [2 ]

The action potential of a squid axon was recorded, with the axon in normal sea water. The axon was then placed in water with a Na + concentration of one- third of that of sea water.

341

61

h u m an p h yS i o lo g y The action potential was recorded again. Figure 1 6 shows these recordings.

a) Using only the data in gure 1 7, outline the eect o reduced Na + concentration on:

membrabe potential (mV)

( i) +40

( ii) the duration o the action potential.

sea water

+20 -20

33%

c) D iscuss the eect o reduced Na concentration on the time taken to return to the resting potential. [2 ]

-40 -60 -80 2

Geneticists discovered a mutant variety o ruit fy that shakes vigorously when anaesthetized with ether. Studies have shown that the shaker mutant has K + channels that do not unction properly. Figure 1 7 shows action potentials in normal ruit fies and in shaker mutants. 40

wild-type drosophila normal action potential

0 -40 4

8

40

12

16

shaker mutant abnormal action potential

0 -40 4

Figure 17

[3 ]

+

Figure 16

membrabe potential/mV

[2 ]

b) Explain the eects o reduced Na concentration on the action potential.

0

time (ms)

342

[2 ]

+

1

5

the magnitude o depolarization

8

12 time (ms)

16

d) C ompare the action potentials o shaker and normal ruit fies. [3 ] e) E xplain the dierences between the action potentials.

7

N U CLE I C ACI D S ( AH L)

Introduction The discovery of the structure of D NA revolutionized biology. Information stored in a coded form in D NA is copied onto mRNA. The

structure of D NA is ideally suited to its function. Information transferred from D NA to mRNA is translated into an amino acid sequence.

7.1 DNA structure and replication Understanding  DNA structure suggested a mechanism or DNA     

replication. Nucleosomes help to supercoil the DNA. DNA replication is continuous on the leading strand and discontinuous on the lagging strand. DNA replication is carried out by a complex system o enzymes. DNA polymerases can only add nucleotides to the 3 end o a primer. Some regions o DNA do not code or proteins but have other important unctions.

Nature of science  Making careul observations: Rosalind

Franklins X-ray diraction provided crucial evidence that DNA is a double helix.

Applications  Rosalind Franklins and Maurice Wilkins

investigation o DNA structure by X-ray diraction.  Tandem repeats are used in DNA profling.  Use o nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation o samples or base sequencing.

Skills  Analysis o results o the Hershey and Chase

experiment providing evidence that DNA is the genetic material.  Utilization o molecular visualization sotware to analyse the association between protein and DNA within a nucleosome.

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The HersheyChase experiment Analysis of the results of the HersheyChase experiment providing evidence that DNA is the genetic material. From the late 1 800s, scientists were convinced that chromosomes played a role in heredity and that the hereditary material had a chemical nature. Aware that chromosomes were composed o both protein and nucleic acid, both molecules were contenders to be the genetic material. Until the 1 940s, the view that protein was the hereditary material was avoured, as it was a class o macromolecules that had great variety due to twenty naturally occurring sub-units as opposed to our nucleotide sub- units. Further, many specifc unctions had been identifed or proteins. Variety and specifcity o unction were two properties that were expected to be essential requirements or the hereditary material.

Alred Hershey and Martha C hase wanted to ascertain whether the genetic material o viruses was protein or D NA. In the 1 95 0s, it was known that viruses are inectious particles which transorm cells into virus-producing actories by becoming bound to host cells and inj ecting their genetic material. The non-genetic portion o the virus remains outside the cell. An inected cell then manuactures large numbers o new viruses and bursts, releasing them to the environment ( see fgure 1 ) . Viruses are oten specifc to a certain cell type. The virus they chose to work with was the T2 bacteriophage because o its very simple structure. It has a coat composed entirely o protein while D NA is ound inside the coat.

DNA protein

 Figure 1 Coloured transmission electron micrograph (TEM)

o T2 viruses (blue) bound to an Escherichia coli bacterium. Each virus consists o a large DNA-containing head and a tail composed o a central sheath with several fbres. The fbres attach to the host cell surace, and the virus DNA is injected into the cell through the sheath. It instructs the host to build copies o the virus (blue, in cell)

 Figure 2

Diagram illustrating the structure o the T2 virus

Data-based questions: The HersheyChase experiment Alred Hershey and Martha C hase were two scientists who worked to resolve the debate over the chemical nature o the genetic material. In their experiment, they took advantage o the act that D NA contains phosphorus but not sulphur while proteins contain sulphur but not phosphorus. They cultured viruses that contained proteins with radioactive ( 3 5 S ) sulphur and they separately cultured viruses that contained D NA with radioactive ( 3 2 P)

344

phosphorus. They inected bacteria separately with the two types o viruses. They used a blender to separate the non- genetic component o the virus rom the cell and then centriuged the culture solution to concentrate the cells in a pellet. The cells were expected to have the radioactive genetic component o the virus in them. They measured the radioactivity in the pellet and the supernatant. Figure 3 represents the process and results o the experiment.

7. 1 D N A s t r u c t u r e A N D r e p l i c At i o N

radioactive protein ( 35 S) protein coat with 35 S bacteria

virus

radioactivity ( 35 S) in supernatant

bacterium

radioactive DNA ( 32 P) virus DNA with 32 P bacteria

bacterium

radioactivity ( 32 P) in pellet

Questions b) Explain why the genetic material should be ound in the pellet and not the supernatant. c) D etermine the percentage o the remains in the supernatant.

32

P that

d) D etermine the percentage o 35 S that remains in the supernatant. e) D iscuss the evidence that D NA is the chemical which transorms the bacteria into inected cells.

% of isotope in supernatant

a) Explain what a supernatant is. percentage of isotope in supernatant after 8 minutes agitation 100% 80% 60% 40% 20% 0% 35 S

32 P

 Figure 3

X-ay dfan an a vdn  ma  Making careul observations: Rosalind Franklins X-ray difraction provided crucial evidence that DNA was a helix. Two names are usually remembered in connection with the discovery o D NA, C rick and Watson. Flashes o insight led to their success, but they could not have achieved it without skilled experimental work and careul observations by other scientists. O ne o these was Erwin C harga. His research into the percentage base composition o D NA is described in the data- based question in sub-topic 2 .6 ( page 1 07) .

Another key fgure in the discovery o D NA was Rosalind Franklin. In 1 95 0, she became a research associate in the biophysics unit at Kings C ollege, London. The unit was already investigating the structure o D NA by X- ray diraction. Franklin had already become skilled in techniques o crystallography and X- ray diraction while researching other carbon compounds at an institute in Paris.

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At Kings C ollege she improved the resolution o a camera, so she could make more detailed measurements o the X-ray diraction patterns than had previously been possible. She also produced high quality samples o D NA with the molecules aligned in narrow bres. B y careul control o humidity two types o pure sample could be produced and as Franklin was unsure which represented the normal structure o D NA, she investigated both. Soon ater starting work at Kings C ollege, Franklin had obtained the sharpest X- ray diraction images o D NA in existence. They have been described as amongst the most beautiul X- ray photographs o any substance ever taken. Their implications are described in the next section. She was unwilling to publish her ndings until there was strong evidence. S he thereore embarked on a rigorous analysis o the

diraction patterns that allowed her to calculate the dimensions o the D NA helix. Without Franklins knowledge or permission, James Watson was shown the best diraction pattern and the calculations based on it. B eore Franklin could publish her results C rick and Watson had used them to build their model o D NA structure. It is widely accepted that Rosalind Franklin deserved a Nobel Prize or her research, but this never happened. C rick and Watson were awarded prizes in 1 962 , but she died o cancer in 1 95 8, aged thirty-seven. Nobel Prizes cannot be awarded posthumously, but Rosalind Franklin is remembered more than many prize winners. What we can remember rom her lie is that discoveries may sometimes be made through serendipity or fashes o insight, but the real oundations o science are rigorous experimental techniques and diligent observation.

Rosalind Franklins investigation of DNA structure Rosalind Franklin and Maurice Wilkins investigation o DNA structure by X-ray difraction. I a beam o X- rays is directed at a material, most o it passes through but some is scattered by the particles in the material. This scattering is called diraction. The wavelength o X- rays makes them particularly sensitive to diraction by the particles in biological molecules including D NA. In a crystal the particles are arranged in a regular repeating pattern, so the diraction occurs in a regular way. D NA cannot be crystallized but the molecules were arranged in an orderly enough array in Franklins samples or a diraction pattern to be obtained, rather than random scattering. An X-ray detector is placed close to the sample to collect the scattered rays. The sample can be rotated in three dierent dimensions to investigate the pattern o scattering. D iraction patterns can be recorded using X-ray lm. Franklin developed a high resolution camera containing X- ray lm to obtain very clear images o diraction patterns rom D NA. Figure 4 shows the most amous o these diraction patterns.

346

 Figure 4 Rosalind Franklins X-ray difraction photograph o DNA

From the diraction pattern in gure 4 Franklin was able to make a series o deductions about the structure o D NA: 

The cross in the centre o the pattern indicated that the molecule was helical in shape.



The angle o the cross shape showed the pitch ( steepness o angle) o the helix.

7. 1 D N A s t r u c t u r e A N D r e p l i c At i o N



The distance between the horizontal bars showed turns o the helix to be 3 .4 nm apart.



The distance between the middle o the diraction pattern and the top showed that there was a repeating structure within the molecule, with a distance o 0. 3 4 nm between

the repeats. This turned out to be the vertical distance between adj acent base pairs in the helix. These deductions that were made rom the X-ray diraction pattern o DNA were critically important in the discovery o the structure o DNA.

The Watson and Crick model suggested semiconservative replication DNA structure suggested a mechanism or DNA replication. S everal lines o experimental evidence came together to lead to the knowledge o the structure o D NA: molecular modelling pioneered by the Nobel prize winner Linus Pauling, X- ray diraction patterns discerned rom the careul photographs o Rosalind Franklin and the base composition studies o Erwin C harga. B ut insight and imagination played a role as well. O ne o Watson and C ricks frst models had the sugar- phosphate strands wrapped around one another with the nitrogen bases acing outwards. Rosalind Franklin countered this model with the knowledge that the nitrogen bases were relatively hydrophobic in comparison to the sugarphosphate backbone and would likely point in to the centre o the helix. Franklins X-ray diraction studies showed that the DNA helix was tightly packed so when Watson and C rick built their models, their choices required the bases to ft together such that the strands were not too ar apart. As they trialled various models, Watson and C rick ound the tight packing they were looking or would occur i a pyrimidine was paired with a purine and i the bases were upside down in relation to one another. In addition to being structurally similar, adenine has a surplus negative charge and thymine has a surplus positive charge so that pairing was electrically compatible. Pairing cytosine with guanine allows or the ormation o three hydrogen bonds which enhances stability. O nce the model was proposed, the complementary base pairing immediately suggested a mechanism by which D NA replication could occur  one o the key requirements that any structural model would have to address. The WatsonC rick model led to the hypothesis o semiconservative replication.

The role of nucleosomes in DNA packing Nucleosomes help to supercoil DNA. One dierence between eukaryotic DNA and bacterial DNA is that eukaryotic DNA is associated with proteins called histones. Most groups o prokaryotes have D NA that is not associated with histones, or proteins like histones. For this reason, prokaryotic DNA is reerred to as being naked.

toK Wha n d n hav whn h and dn dn fy mah xmna vdn? Chargaf wrote about his observations: the results serve to disprove the tetranucleotide hypothesis. It is, however, noteworthy - whether this is more than accidental, cannot yet be said - that in all deoxypentose nucleic acids examined thus ar the molar ratios o total purines to total pyrimidines and also o adenine to thymine and o guanine to cytosine were not ar rom 1 H. H. Bauer, author o the book Scientifc Literacy and the Myth o the Scientifc Method, argues that Chargaf needed to: stick his neck out beyond the actual results and say that they mean exact equality and hence some sort o pairing in the molecular structure . Watson and Crick, on the other hand were speculating and theorizing about the molecular nature and biological unctions o DNA and they postulated a structure in which the equalities are exactly one and the deviation orm this in the data could be regarded as experimental error. Ideas and theory turned out to be a better guide than raw data.

Histones are used by the cell to package the D NA into structures called nucleosomes. A nucleosome consists o a central core o eight histone

347

7

N U C L E I C AC I D S ( AH L ) H1 histone

proteins with D NA coiled around the proteins. The eight proteins, or octamer, consist o two copies o our dierent types o histones. A short section o linker D NA connects one nucleosome to the next. An additional histone protein molecule, called H1 , serves to bind the D NA to the core particle ( fgure 5 ) .

DNA nucleosome

30nm bre

 Figure 5

The association o histones with the D NA contributes to a pattern known as supercoiling. An analogy is i you twist an elastic band repeatedly eventually it orms an additional pattern o coils. S upercoiling allows a great length o D NA to be packed into a much smaller space within the nucleus. The nucleosome is an adaptation that acilitates the packing o the large genomes that eukaryotes possess. The H1 histone binds in such a way to orm a structure called the 3 0 nm fbre that acilitates urther packing.

Visualizing nucleosomes

Activity Determining packing ratio Packing ratio is defned as the length o DNA divided by the length into which it is packaged. Use the inormation below to estimate the packing ratio o:

Utilization o molecular visualization sotware to analyse the association between protein and DNA within a nucleosome. Visit the protein data bank at http://www.rcsb.org/pdb/home/home.do or download the image o a nucleosome rom the companion website or this textbook. 1

Rotate the molecule to see the two copies o each histone protein. In fgure 6, they are identifed by the tails that extend rom the core. Each protein has such a tail that extends out rom the core.

2

Note also the approximately 1 5 0 bp o D NA wrapped nearly twice around the octamer core.

3

Note the N- terminal tail that proj ects rom the histone core or each protein. C hemical modifcation o this tail is involved in regulating gene expression.

4

Visualize the positively charged amino acids on the nucleosome core. S uggest how they play a role in the association o the protein core with the negatively charged D NA.

(a) a nucleosome; and (b) chromosome 22 (one o the smallest human chromosomes) .

348



The distance between base pairs is 0.34 nm.



There is approximately 200 bp o DNA coiled around a nucleosome.



A nucleosome is approximately 10 nm long.



There is an estimated 5.0  10 7 total base pairs (bp) present in the shortest human autosome (chromosome 22).



Chromosome 22 in its most condensed orm is approximately 2 m long.

 Figure 6

7. 1 D N A s t r u c t u r e A N D r e p l i c At i o N

Daa-bad qn: Apoptosis and the length of DNA between nucleosomes Under natural conditions, programmed cell death sometimes occurs. This is known as apoptosis and it plays an important role in such processes as metamorphosis and embryological development. O ne mechanism involved in this auto-destruction is the digestion o D NA by enzymes called D NAases. The D NA associated with the nucleosome is normally not as accessible to the D NAase as the linking sections. D NA gets digested into ragments o lengths equal to multiples o the distance between nucleosomes.

Origin

 2000 bp  1500 bp  1000 bp  750 bp  500 bp

The let hand column o fgure 7 shows the results o separation by gel electrophoresis o the D NA released by the action o D NAase on rat liver cells. The right column represents ragments used as a reerence called a ladder.

 250 bp

 Figure 7

O nce the D NA had been cut, nucleosomes were digested by protease. 1

( iii) the length o D NA between two linker D NA regions with three nucleosomes between them.

Identiy on the diagram the ragment that represents: ( i) the length o D NA between the two sections o linker D NA on either side o one nucleosome; ( ii) the length o D NA between two linker D NA regions with two nucleosomes between them;

2

D educe the length o D NA associated with a nucleosome.

3

S uggest how the pattern in the lethand column would change i very high concentrations o D NAase were applied to the cells.

The leading strand and the lagging strand DNA replication is continuous on the leading strand and discontinuous on the lagging strand. B ecause the two strands o the D NA double helix are arranged in an anti-parallel ashion, synthesis on the two strands occurs in very dierent ways. One strand, the leading strand, is made continuously ollowing the ork as it opens. The other strand, known as the lagging strand, is made in ragments moving away rom the replication ork. New ragments are created on the lagging strand as the replication ork exposes more o the template strand. These ragments are called Okazaki ragments.

Proteins involved in replication DNA replication is carried out by a complex system of enzymes. Replication involves the ormation and movement o the replication ork and synthesis o the leading and lagging strands. Proteins are involved as enzymes at each stage but also serve a number o other unctions.

349

7

N U C L E I C AC I D S ( AH L ) The enzyme helicase unwinds the D NA at the replication ork and the enzyme topoisomerase releases the strain that develops ahead o the helicase. Single-stranded binding proteins keep the strands apart long enough to allow the template strand to be copied. S tarting replication requires an RNA primer. Note that on the lagging strand there are a number o primers but there is j ust one on the leading strand. The enzyme D NA primase creates one RNA primer on the leading strand and many RNA primers on the lagging strand. The RNA primer is necessary to initiate the activity o D NA polymerase. D NA polymerase is responsible or covalently linking the deoxyribonucleotide monophosphate to the 3  end o the growing strand. D ierent organisms have dierent kinds o D NA polymerases, each with dierent unctions such as proo- reading, polymerization and removal o RNA primers once they are no longer needed. D NA ligase connects the gaps between ragments. DNA topoisomerase

leading strand

DNA polymerase

5 3

parental DNA

primase RNA primer

DNA helicase

DNA ligase

DNA polymerase

3  lagging 5  strand

 Figure 8

The direction of replication DNA polymerases can only add nucleotides to the 3 end of a primer Within D NA molecules, D NA replication begins at sites called origins o replication. In prokaryotes there is one site and in eukaryotes there are many. Replication occurs in both directions away rom the origin. The result appears as a replication bubble in electron micrographs. The fve carbons o the deoxyribose sugar have a number ( see fgure 9) .

phosphate nitrogen base O

5 CH 2 4

H

1

H H 2

3 OH

350

OH T

H

 Figure 9

DNA growing strand

deoxyribose sugar

C A

template strand DNA

H  Figure 10

C

A G

T

G

G

C 5  end

OH base

3  end

sugar

phosphate

7. 1 D N A s t r u c t u r e A N D r e p l i c At i o N The phosphate group o new D NA nucleotides is added to the the 3  carbon o the deoxyribose o the nucleotide at the end o the chain. Replication thereore occurs in the 5  to 3  direction.

Non-coding regions o DNA have important unctions Some regions o DNA do not code or proteins but have other important unctions. The cellular machinery operates according to a genetic code. D NA is used as a guide or the production o polypeptides using the genetic code. However, only some D NA sequences code or the production o polypeptides. These are called coding sequences. There are a number o non- coding sequences ound in genomes. S ome o them have unctions, such as those sequences that are used as a guide to produce tRNA and rRNA. S ome non- coding regions play a role in the regulation o gene expression such as enhancers and silencers. In sub-topic 7.2 we will explore non-coding sequences called introns.

toK t wha xn d n hav a nq nby whn a dmay? Molecular biologist Elizabeth Blackburn is one o the most renowned original researchers in the feld o telomeres. She shared the Nobel Prize in Physiology or medicine or her co-discovery o telomerase. She made headlines in 2004 when she was dismissed rom the Presidents Council on Bioethics ater objecting to the councils call or a ban on stem cell research and or criticizing the suppression o relevant scientifc evidence in its fnal report.

Most o the eukaryotic genome is non- coding. Within the genome, especially in eukaryotes, repetitive sequences can be common. There are two types o repetitive sequences: moderately repetitive sequences and highly repetitive sequences ( satellite D NA) . Together they can orm between 5 and 60 per cent o the genome. In humans, nearly 60% o the D NA consists o repetitive sequences. O ne such area o repetitive sequences occurs on the ends o eukaryotic chromosomes called telomeres. The telomere serves a protective unction. D uring interphase, the enzymes that replicate D NA cannot continue replication all the way to the end o the chromosome. I cells went through the cell cycle without telomeres, they would lose the genes at the end o the chromosomes. S acrifcing the repetitive sequences ound in telomeres serves a protective unction.

 Figure 11 False colour scanning electron

micrograph with telomeres coloured pink. The grey region in the centre is the centromere which also consists of non-coding repetitive sequences

DNA profling Tandem repeats are used in DNA profling. A variable number tandem repeat ( VNTR) is a short nucleotide sequence that shows variations between individuals in terms o the number o times the sequence is repeated. E ach variety can be inherited as an allele. Analysis o VNTR allele combinations in individuals is the basis behind D NA profling or use in such applications as genealogical investigations. A locus is the physical location o a heritable element on the chromosome. In the hypothetical example shown in fgure 1 2, locus A has a VNTR

o the sequence AT and locus B has a VNTR o the sequence TC G. In the two individuals shown, there are two dierent alleles (varieties) o locus A, two repeats (allele A2) and our repeats (allele A4) . In the same individuals, there are three alleles or locus B , three repeats (allele B 3) , our repeats (allele B 4) and fve repeats (allele B 5 ) . The asterisk mark indicates where the restriction enzyme would cut. The D NA profle that would result is shown in the lower part o fgure 1 2 . Note that the two individuals have some bands in common and some unique bands.

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N U C L E I C AC I D S ( AH L )

Genealogists deduce paternal lineage by analysing short tandem repeats rom the Y-chromosome, and deduce maternal lineage by analysing

mitochondrial D NA variations in single nucleotides at specifc locations called hypervariable regions.

individual # 1

individual # 2

locus A allele A2 (2 repeats)

AT

AT

locus A allele A4 (4 repeats)

allele A2 (2 repeats)

AT

AT

allele A2 (2 repeats)

AT AT AT AT AT

AT

locus B allele B3 (3 repeats)

TCG TCG TCG

locus B allele B3 (3 repeats)

TCG TCG TCG

allele B4 (4 repeats)

TCG TCG TCG TCG

allele B5 (5 repeats)

TCG TCG TCG TCG TCG

DNA prole origin B5 B4

B3

B3 A4

A2 individual #1

A2 individual #2

 Figure 12

Activity Analysis o a DNA profle involving alleles o short tandem repeats o DNA

A logarithm is an alternative way to express an exponent. For example, log 1,000 = log 10 3 =3

log 100 = log 10 2 =2

In biology, very large changes in a variable are easier to represent graphically i logarithms are used.

 Figure 13

Gel electrophoresis. The outside columns represent ladders of known length. The two inside columns represent samples of unknown length

352

In the example (fgure 13), DNA ragments were separated using gel electrophoresis. The ragments vary in size rom 100 bp (base pairs) up to 5,000 bp. The two outside columns o the gel represent ladders, i.e. mixtures o DNA ragments o known size. These were used to obtain the data in table 1 and create the plot shown in fgure 14. The other inner columns shown in fgure 13 are unknowns.

Knwn add fagmn z (b) 5,000 2,000 850 400 100

base pairs

7. 1 D N A s t r u c t u r e A N D r e p l i c At i o N

Dan mvd (mm) 58 96 150 200 250

10 3

10 2

 Table 1

1

10 4

Using fgure 1 4 determine the size o D NA ragments in the two centre digests:

Fagmn Dan Fagmn Dan z (b) mvd (mm) z (b) mvd (mm) (mn 2) (mn 2) (mn 3) (mn 3) 60 70 70

160

130

200

10 1 50

100

150

200

250 distance / mm

 Figure 14

Distance moved as a function of fragment size in gel electrophoresis. Notice that the y-axis scale on this graph goes up in powers of 10. This is a logarithmic scale

Daa-bad qn: Analysis o DNA profles using D1S80 O ne commonly studied D NA locus is a VNTR named D 1 S 80. D 1 S 80 is located on human chromosome 1 . This locus is composed o repeating units o 1 6- nucleotide- long segments o D NA. The number o repeats varies rom one individual to the next with 2 9 known alleles ranging rom 1 5 repeats to 41 . In the image o a D NA profle ( fgure 1 5 ) the outside and inside lanes represent ladders representing multiples o one hundred and twenty- three bp. a) Identiy the lengths o the ragments represented by each o the bands in the ladder. b) Using a ruler measure the distance between the origin and the band. Use the length and distance data, to create a standard curve using a logarithmic graph. c) Measure the distance travelled by each band rom the origin.

 Figure 15

d) Using the standard curve, estimate the lengths o the bands in each individual. e) Estimate the number o repeats represented by each band. f) It is unclear whether the individual in lane 7 has two dierent copies o the same allele or dierent alleles. S uggest what could be done to urther resolve the genotype o the fnal individual.

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DNA sequencing Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing. The determination o the sequence o bases in a genome is carried out most commonly using a method that employs fuoresence. Many copies o the unknown D NA that is to be sequenced are placed into test tubes with all o the raw materials including deoxyribonucleotides and the enzymes necessary to carry out replication. In addition very small quantities o dideoxyribonucleotides that have been labelled with dierent fuorescent

markers are added. The dideoxyribonucleotides will be incorporated into some o the new D NA, but when they are incorporated, they will stop the replication at precisely the point where they were added. The ragments are separated by length using electrophoresis. The sequence o bases can be automatically analysed by comparing the colour o the fuorescence with the length o the ragment.

DNA to be sequenced A

T

A G A

C

T

A

G

C

C

primer extension reactions: ddA reaction: ddC reaction: TACTATGCC AG A TACTATGCCAG A ATG A ATGATAC primer ddG reaction: ddT reaction: TACTATGCC AG A TACTATGCCAG A ATG ATACG ATGAT

C

T A

to computer

T

mixture of nucleotides containing rare dideoxyribonucleotides (ddn) replication stops when a ddn is incorporated

column electrophoresis

electropherogram

T A

A

G

A G A C C G T A T C A T

T G

ddn that is on the end of the fragment

detector

laser  Figure 16

354

C

????????? 123456789

7. 2 t r A N s c r i p t i o N A N D g e N e e X p r e s s i o N

7.2 tann and n xn Understanding  Gene expression is regulated by proteins that     

bind to specifc base sequences in DNA. The environment o a cell and o an organism has an impact on gene expression. Nucleosomes help to regulate transcription in eukaryotes. Transcription occurs in a 5' to 3' direction. Eukaryotic cells modiy mRNA ater transcription. Splicing o mRNA increases the number o dierent proteins an organism can produce.

Applications  The promoter as an example o non-coding DNA

with a unction.

Skills  Analysis o changes in DNA methylation patterns.

Nature of science  Looking or patterns, trends and discrepancies:

there is mounting evidence that the environment can trigger heritable changes in epigenetic actors.

The function of the promoter The promoter as an example o non-coding DNA with a unction. Only some DNA sequences code or the production o polypeptides. These are called coding sequences. There are a number o non-coding sequences ound in genomes. Some o them have unctions, such as those sequences that produce tRNA and rRNA. Some non-coding regions play a role in the regulation o gene expression such as enhancers and silencers.

The promoter is a sequence that is located near a gene. It is the binding site o RNA polymerase, the enzyme that catalyses the ormation o the covalent bond between nucleotides during the synthesis o RNA. The promoter is not transcribed but plays a role in transcription.

Regulation of gene expression by proteins Gene expression is regulated by proteins that bind to specifc base sequences in DNA. S ome proteins are always necessary or the survival o the organism and are thereore expressed in an unregulated ashion. O ther proteins need to be produced at certain times and in certain amounts; i.e., their expression must be regulated. Gene expression is regulated in prokaryotes as a consequence o variations in environmental actors. For example, the genes responsible or the absorption and metabolism o lactose by E.coli are expressed in the presence o lactose and are not expressed in the absence o lactose. In this case, the breakdown o lactose results in regulation o gene expression by negative eedback. In the presence o lactose a repressor protein is deactivated ( fgure 1 ) . O nce the lactose has been broken

355

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N U C L E I C AC I D S ( AH L ) lactose not in the environment; repressor blocks transcription

down, the repressor protein is no longer deactivated and proceeds to block the expression o lactose metabolism genes. As in prokaryotes, eukaryotic genes are regulated in response to variations in environmental conditions. E ach cell o a multicellular eukaryotic organism expresses only a raction o its genes.

p ro m o te r

lactose present in the environment; repressor deactivated; genes involved in lactose use are transcribed promoter

There are a number o proteins whose binding to D NA regulates transcription. These include enhancers, silencers and promoter-proximal elements. Unlike the promoter sequence, the sequences linked to regulatory transcription actors are unique to the gene.

RNA polymerase

-galactosidase -

The regulation o eukaryotic gene expression is also a critical part o cellular dierentiation as well as the process o development. This is seen in the passage o an insect through its lie cycle stages or in human embryological development.

+

transacetylase permease -

-

 Figure 1

lactose

Regulatory sequences on the D NA which increase the rate o transcription when proteins bind to them are called enhancers. Those sequences on the D NA which decrease the rate o transcription when proteins bind to them are called silencers. While enhancers and silencers can be distant rom the promoter, another series o sequences called promoter-proximal elements are nearer to the promoter and binding o proteins to them is also necessary to initiate transcription.

The impact of the environment on gene expression The environment of a cell and of an organism has an impact on gene expression. In the history o Western thought, much debate has gone in to the naturenurture debate. This is a debate centred on the extent to which a particular human behaviour or phenotype should be attributed to the environment or to heredity. Much eort has gone into twin studies especially or twins raised apart.

Data-based questions: Identical twin studies Twin studies have been used to identiy the relative infuence o genetic actors and environmental actors in the onset o disease ( gure 2 ) . Identical twins have 1 00% o the same D NA while raternal twins have approximately 5 0% o the same D NA.

Questions 1

2

3

356

D etermine the percentage o identical twins where both have diabetes. [2 ] Explain why a higher percentage o identical twins sharing a trait suggests that a genetic component contributes to the onset o the trait. [3 ] With reerence to any our conditions, discuss the relative role o the environment and genetics in the onset o the condition. [3 ]

percent of twin pairs who share the trait 0% 100% greater height genetic inuence reading disability autism Alzheimers schizophrenia alcoholism bipolar disorder hypertension diabetes multiple sclerosis breast cancer Crohns disease stroke rheumatoid arthritis  Figure 2

identical twins greater fraternal twins environmental inuence

7. 2 t r A N s c r i p t i o N A N D g e N e e X p r e s s i o N The infuence o the environment on gene expression or some traits is unequivocal. Environmental actors can aect gene expression such as the production o skin pigmentation during exposure to sunlight in humans.

Avy exlan h a lu an f sam a

In embryonic development, the embryo contains an uneven distribution o chemicals called morphogens. C oncentrations o the morphogens aect gene expression contributing to dierent patterns o gene expression and thus dierent ates o the embryonic cells depending on their position in the embryo. In coat colour in cats, the C  gene codes or the production o the enzyme tyrosinase, the rst step in the production o pigment. A mutant allele o the gene, cs allows normal pigment production only at temperatures below body temperature. This mutant allele has been selected or in the selective breeding o S iamese cats. At higher temperatures, the protein product is inactive or less active, resulting in less pigment.

Nucleosomes regulate transcription Nucleosomes help to regulate transcription in eukaryotes. E ukaryotic D NA is associated with proteins called histones. C hemical modication o the tails o histones is an important actor in determining whether a gene will be expressed or not. A number o dierent types o modication can occur to the tails o histones including the addition o an acetyl group, the addition o a methyl group or the addition o a phosphate group. O  C H3 C -

Acetyl group

C H 3 - Methyl group

For example, residues o the amino acid lysine on histone tails can have acetyl groups either removed or added. Normally the lysine residues on histone tails bear a positive charge that can bind to the negatively charged D NA to orm a condensed structure that inhibits transcription. Histone acetylation neutralizes these positive charges allowing a less condensed structure with higher levels o transcription. C hemical modication o histone tails can either activate or deactivate genes by decreasing or increasing the accessibility o the gene to transcription actors.

G C M

M C

T

G

A

M C

T

G

G

A

C M

T

G

A

C M

NH 2 CH 3

C

Analysing methylation patterns Analysis of changes in DNA methylation patterns The addition o methyl groups directly to D NA is thought to play a role in gene expression. Whereas methylation o histones can promote or inhibit transcription, direct methylation o D NA tends to decrease gene expression. The amount o D NA methylation varies during a lietime and is aected by environmental actors.

N

C

C

C

O

N

 Figure 3

DNA methylation is the addition of a methyl group (green M) to the DNA base cytosine

357

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Data-basd qustions: Changes in methylation pattern with age in identical twins One study compared the methylation patterns o 3-year-old identical twins with 50-year-old identical twins. Methylation patterns were dyed red on one chromosome or one twin and dyed green or the other twin on the same chromosome. Chromosome pairs in each set o twins were digitally superimposed. The result would be a yellow colour i the patterns were the same. Dierences in patterns on the two chromosomes results in mixed patterns o green and red patches. This was done or our o the twenty-three chromosome pairs in the genome. 1

2

3

Explain the reason or yellow coloration i the methylation pattern is the same in the two twins.

[3 ]

Identiy the chromosome with the least changes as twins age.

[1 ]

Identiy the chromosomes with the most changes as twins age.

[1 ]

4

Explain how these dierences could arise. [3 ]

5

Predict with a reason whether identical twins will become more or less similar to

each other in their characteristics as they grow older.

[2 ]

 Figure 4

epigntics Looking for patterns, trends and discrepancies: there is mounting evidence that the environment can trigger heritable changes in epigenetic factors. The chemical modifcations o chromatin that impact gene expression, including acetylation, methylation and phosphorylation o amino acid tails o histones (fgure 5 ) as well as methylation o D NA(fgure 6) , all have an impact on gene expression and thus impact the visible characteristics o an individual (fgure 7) . These chemical modifcations are called epigenetic tags. There is mounting evidence that the chemical modifcations that occur to the hereditary material in one generation might, in certain circumstances, be passed on to the next generation both at the cellular as well as whole organism level. The sum o all the epigenetic tags constitutes the epigenome. D ierent cells have their own methylation pattern so that a unique set o proteins will be produced in order or that cell to perorm its unction. D uring cell division, the methylation pattern will be passed over to the daughter cell. In other words, the environment is aecting inheritance.

358

Sperm and eggs develop rom cells with epigenetic tags. When two reproductive cells meet, the epigenome is erased through a process called reprogramming. Ac M P

acetylation methylation phosphorylation

 Figure 5 Histone modifcations

NH 2

NH 2

C N C O

C

H N

C N

C

CH

H  Figure 6 DNA methylation

O

Me C

N H

CH

7. 2 t r A N s c r i p t i o N A N D g e N e e X p r e s s i o N

About 1 % o the epigenome is not erased and survives yielding a result called imprinting. For example, when a mammalian mother has gestational diabetes, the high levels o glucose in

the etal circulation trigger epigenetic changes in the daughters D NA such that she is predisposed to develop gestational diabetes hersel.

transcription possible gene switched on  active (open) chromatin  unmethylated cytosines (white circles)  acetylated histones

gene switched o  silent (condensed) chromatin  methylated cytosines (red circles)  deacetylated histones transcription prevented  Figure 7

The diagram compares the chemical modifcations that prevent transcription with the chemical modifcations that allow transcription

The direction o transcription Transcription occurs in a 5' to 3' direction. The synthesis o mRNA occurs in three stages: initiation, elongation and termination. Transcription begins near a site in the D NA called the promoter. O nce binding o the RNA polymerase occurs, the D NA is unwound by the RNA polymerase orming an open complex. The RNA polymerase slides along the D NA, synthesizing a single strand o RNA. base

RNA growing strand

OH C

U A template strand OH DNA

OH G

OH OH

OH A C T

G

G

3 end

C 5 end

sugar

phosphate

 Figure 8

Post-transcriptional modifcation Eukaryotic cells modify mRNA after transcription. The regulation o gene expression can occur at several points. Transcription, translation and post-translational regulation occur in both eukaryotes and prokaryotes. However, most regulation o prokaryotic gene expression occurs at transcription. In addition, post-transcriptional modifcation o RNA is a method o gene expression that does not occur in prokaryotes.

 Figure 9

Coloured transmission electron micrograph o DNA transcription coupled with translation in the bacterium Escherichia coli. During transcription, complementary messenger ribonucleic acid (mRNA) strands ( green) are synthesized using DNA (pink) as a template and immediately translated by ribosomes (blue)

359

7

N U C L E I C AC I D S ( AH L ) a)

O H N

H H

H

OH

HO

O

N

H2N

CH 2

O O

H H

P O

O O

P O

O O

P

O O

base

CH 2

O

H

H

O

OH

H

N N

O

O

CH 3

H P

O

O

CH 2 5

7-methylguanosine cap exon

b)

intron

exon

5

3 pre-mRNA

spliceosome sn RNPs exon

toK Hw d he crieria fr judgmen used change he cnclusins drawn frm he same daa? Estimates o the number o genes ound in the human genome fuctuated wildly in the time between 2000 and 2007. Reported as high as 120,000 in 2000, the current consensus view is that there are approximately 20,500. The reason or the uncertainty was due to the dierent criteria used or searching used by dierent gene-nding programs. Dening the criteria was problematic because:  small genes are diicult to detect; 



360

because o mRNA splicing, one gene can code or several protein products; some genes are nonprotein coding and two genes can overlap.

exon

5

3

excised intron 5

3 mature mRNA

c)

5

A

A poly A tail consisting of 100200 adenine nucleotides is added after transcription. A A A A 3 poly A tail

 Figure 10

O ne o the most signifcant dierences between eukaryotes and prokaryotes is the absence o a nuclear membrane surrounding the genetic material in prokaryotes. The absence o a compartment in prokaryotes means that transcription and translation can be coupled. The separation o the location o transcription and translation into separate compartments in eukaryotes allows or signifcant post-transcriptional modifcation to occur beore the mature transcript exits the nucleus. An example would be the removal o intervening sequences, or introns, rom the RNA transcript. Prokaryotic DNA does not contain introns. In eukaryotes, the immediate product o mRNA transcription is reerred to as pre- mRNA, as it must go through several stages o posttranscriptional modifcation to become mature mRNA. O ne o these stages is called RNA splicing, shown in fgure 1 1 b. Interspersed throughout the mRNA are sequences that will not

3

7. 2 t r A N s c r i p t i o N A N D g e N e e X p r e s s i o N

contribute to the ormation o the polypeptide. They are reerred to as intervening sequences, or introns. These introns must be removed. The remaining coding portions o the mRNA are called exons. These will be spliced together to orm the mature mRNA. Post-transcriptional modication also includes the addition o a 5  cap that usually occurs beore transcription has been completed (see gure 1 1 a) . A poly-A tail is added ater the transcript has been made (see gure 1 1 c) .

mRNA splicing Splicing o mRNA increases the number o diferent proteins an organism can produce. Alternative splicing is a process during gene expression whereby a single gene codes or multiple proteins. This occurs in genes with multiple exons. A particular exon may or may not be included in the nal messenger RNA. As a result, the proteins translated rom alternatively spliced mRNAs will dier in their amino acid sequence and possibly in their biological unctions. In mammals, the protein tropomyosin is encoded by a gene that has eleven exons. Tropomyosin pre- mRNA is spliced dierently in dierent tissues resulting in ve dierent orms o the protein. For example, in skeletal muscle, exon 2  is missing rom the mRNA and in smooth muscle, exons 3  and 1 0 are not present. In ruit fies, the Dscam protein is involved in guiding growing nerve cells to their targets. Research has shown that there are potentially 3 8, 000 dierent mRNAs possible based on the number o dierent introns in the gene that could be spliced alternatively.

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7.3 translaion Understanding  Initiation o translation involves assembly o     

 





the components that carry out the process. Synthesis o the polypeptide involves a repeated cycle o events. Disassembly o the components ollows termination o translation. Free ribosomes synthesize proteins or use primarily within the cell. Bound ribosomes synthesize proteins primarily or secretion or or use in lysosomes. Translation can occur immediately ater transcription in prokaryotes due to the absence o a nuclear membrane. The sequence and number o amino acids in the polypeptide is the primary structure. The secondary structure is the ormation o alpha helices and beta pleated sheets stabilized by hydrogen bonding. The tertiary structure is the urther olding o the polypeptide stabilized by interactions between R groups. The quaternary structure exists in proteins with more than one polypeptide chain.

Applications  tRNA-activating enzymes illustrate enzyme-

substrate specifcity and the role o phosphorylation.

Skills  The use o molecular visualization sotware to

analyse the structure o eukaryotic ribosomes and a tRNA molecule.  Identifcation o polysomes in an electron micrograph.

Nature of science  Developments in scientifc research ollow

improvements in computing: the use o computers has enabled scientists to make advances in bioinormatics applications such as locating genes within genomes and identiying conserved sequences.

The structure of the ribosome The use o molecular visualization sotware to analyse the structure o eukaryotic ribosomes and a tRNA molecule. Ribosome structure includes:

362



Proteins and ribosomal RNA molecules (rRNA) .



Two sub-units, one large and one small.



Three binding sites or tRNA on the surace o the ribosome. Two tRNA molecules can bind at the same time to the ribosome.



There is a binding site or mRNA on the surace o the ribosome.

E ach ribosome has three tRNA binding sites  the E or exit site, the P or peptidyl site and the A or aminoacyl site ( see fgure 1 ) . The protein data bank ( PD B ) is a public database containing data regarding the threedimensional structure or a large number o biological molecules. In 2 000, structural biologists Venkatraman Ramakrishnan, Thomas A. S teitz and Ada E . Yonath made the frst data about

7. 3 t r A N s l At i o N

position of growing polypeptide

tRNA structure A C

large sub-unit

5

binding sites for tRNA

3

C

double stranded sections linked by base pairing

site for attaching an amino acid loop of seven nucleotides

small sub-unit 5

extra loop

3 position of mRNA

loop of eight nucleotides

 Figure 1

ribosome subunits available through the PD B . In 2 009, they received a Nobel Prize or their work on the structure o ribosomes. Visit the protein databank to obtain images o the Thermus thermophilus ribosome ( images 1 j go and 1 giy) , or download these images rom the companion website to the textbook. Using Jmol, rotate the image to visualize the small sub-unit and the large sub- unit. In the image in fgure 2 , an mRNA molecule is coloured yellow. The pink,  purple and blue areas in the image represent the three tRNA binding sites with tRNA molecules bound.

anticodon loop

anticodon  Figure 3



a triplet o bases called the anticodon which is part o a loop o seven unpaired bases



two other loops



the base sequence C C A at the 3 ' end which orms a site or attaching an amino acid.

Visit the PD B to obtain an image o a tRNA molecule or download the image rom the companion site to this book to explore the structure in a programme such as Jmol. Figure 4 shows such an image. The parts marked green represent the amino acid binding site and the anticodon. The part in purple shows a region o the molecule where a triplet o bases are hydrogen bonded. This is shown in the second image.

 Figure 2

The generalized structure o a tRNA molecule is shown in fgure 3 . All tRNA molecules have: 

sections that become double-stranded by base pairing, creating loops

 Figure 4 Whole view of a

tRNA molecule with a close-up of a triplet of bases connected by hydrogen bonds

363

7

N U C L E I C AC I D S ( AH L )

tRNA-activating enzymes tRNA-activating enzymes illustrate enzyme-substrate specifcity and the role o phosphorylation. Each tRNA molecule is recognized by a tRNAactivating enzyme that attaches a specifc amino acid to the tRNA, using ATP or energy. The base sequence o tRNA molecules varies and this causes some variability in structure. Activation o a tRNA molecule involves the attachment o an amino acid to the 3' terminal o the tRNA by an enzyme called a tRNA-activating enzyme. There are twenty dierent tRNA-activating enzymes that are each specifc to one o the 2 0 amino acids and the correct tRNA molecule. The active site o the

activating enzyme is specifc to both the correct amino acid and the correct tRNA. Energy rom ATP is needed or the attachment o amino acids. Once ATP and an amino acid are attached to the active site o the enzyme, the amino acid is activated by the ormation o a bond between the enzyme and adenosine monophosphate (AMP) . Then the activated amino acid is covalently attached to the tRNA. Energy rom this bond is later used to link the amino acid to the growing polypeptide chain during translation. tRNA

ATP

charged tRNA

P P P

amino acid

P

aminoacyl-tRNA synthetase

P Pi pyrophosphate

A specic amino acid and ATP bind to the enzyme

The amino acid is a activated by the hydrolysis of ATP and covalent bonding of AMP

P AMP The correct tRNA binds to the active site. The amino acid binds to the attachment site on the tRNA and AMP is released

The activated tRNA is released

 Figure 5

Initiation of translation Met

3 U 5 A

A C U G

5 3

To begin the process o translation, an mRNA molecule binds to the small ribosomal subunit at an mRNA binding site. An initiator tRNA molecule carrying methionine then binds at the start codon  AUG .

initiator tRNA

3

5

start codon mRNA binding site  Figure 6

364

Initiation o translation involves assembly o the components that carry out the process.

The large ribosomal subunit then binds to the small one. small ribosomal subunit

The initiator tRNA is in the P site. The next codon signals another tRNA to bind. It occupies the A site. A peptide bond is ormed between the amino acids in the P and A site.

7. 3 t r A N s l At i o N

P site

Met

E

peptide bond forming

large ribosomal subunit E

A

E

3 3

5

P A site site

5

P A

 Figure 8

 Figure 7

Elongation of the polypeptide Synthesis of the polypeptide involves a repeated cycle of events. Following initiation, elongation occurs through a series of repeated steps. The ribosome translocates three bases along the mRNA, moving the tRNA in the P site to the E site, freeing it and allowing a tRNA with the appropriate anticodon to bind to the next codon and occupy the vacant A site.

E

E

3

P A 5

P A site site

 Figure 9

Termination of translation Disassembly of the components follows termination of translation. The process continues until a stop codon is reached when the free polypeptide is released. Note the direction of movement along the mRNA is from the 5 end to the 3  end. free polypeptide

3 5

3 5

stop codon (UAG, UAA, or UGA)  Figure 10

365

7

N U C L E I C AC I D S ( AH L )

Free ribosomes

toK Hw d wrds acquire heir meaning? Is a ribosome an organelle? Karl August Mbius is credited as the frst to establish the analogy between cellular substructures with defned unctions and the organs o the body. Early usage varied rom reerring only to the reproductive structures o protists, later ocusing on propulsion structures and later even including extracellular structures such as cell walls. The original defnition o an organelle as a subcellular unctional unit in general has emerged as the dominant defnition, and this would include ribosomes. A criterion in this case or defning an organelle is whether it can be isolated by a process known as cellular ractionation. Others limit the term to membrane-bound cell compartments and some cell biologists choose to limit the term even urther to those structures that originated rom endosymbiotic bacteria.

Free ribosomes synthesize proteins or use primarily within the cell. In eukaryotes, proteins unction in a particular cellular compartment. Proteins are synthesized either in the cytoplasm or at the endoplasmic reticulum depending on the fnal destination o the protein. Translation occurs more commonly in the cytosol. Proteins destined or use in the cytoplasm, mitochondria and chloroplasts are synthesized by ribosomes ree in the cytoplasm.

Bound ribosomes Bound ribosomes synthesize proteins primarily or secretion or or use in lysosomes. In eukaryotic cells, thousands o proteins are made. In many cases, proteins perorm a unction within a specifc compartment o the cell or they are secreted. Proteins must thereore be sorted so that they end up in their correct location. Proteins that are destined or use in the ER, the Golgi apparatus, lysosomes, the plasma membrane or outside the cell are synthesized by ribosomes bound to the ER. Whether the ribosome is ree in the cytosol or bound to the E R depends on the presence o a signal sequence on the polypeptide vesicle containing polypeptide

ribosome

mRNA signal sequence signal recognition protein (SRP) polypeptide

SRP receptor lumen of ER

 Figure 11

366

ER membrane

7. 3 t r A N s l At i o N

being translated. It is the frst part o the polypeptide translated. As the signal sequence is created it becomes bound to a signal recognition protein that stops the translation until it can bind to a receptor on the surace o the E R. O nce this happens, translation begins again with the polypeptide moving into the lumen o the E R as it is created.

The coupling o transcription and translation in prokaryotes Translation can occur immediately ater transcription in prokaryotes due to the absence o a nuclear membrane. In eukaryotes, cellular unctions are compartmentalized whereas in prokaryotes they are not. O nce transcription is complete in eukaryotes, the transcript is modifed in several ways beore exiting the nucleus. Thus there is a delay between transcription and translation due to compartmentalization. In prokaryotes, as soon as the mRNA is transcribed, translation begins.

Identifcation o polysomes Identifcation o polysomes in an electron micrograph. Polysomes are structures visible in an electron microscope. They appear as beads on a string. They represent multiple ribosomes attached to a single mRNA molecule. B ecause translation and transcription occur in the same

compartment in prokaryotes, as soon as the mRNA is transcribed, translation begins. Thus, multiple polysomes are visible associated with one gene. In eukaryotes, polysomes occur in both the cytoplasm and next to the E R.

 Figure 12

Strings of polysomes attached to a DNA molecule in a prokaryote. The arrow designates where investigators believe RNA polymerase is sitting at, or near, the initiation site for a gene

367

7

N U C L E I C AC I D S ( AH L )

polypeptide

ribosome mRNA  Figure 13

The image shows multiple ribosomes translating a single mRNA molecule within the cytoplasm at the same time. The beginning of the mRNA is to the right (at the arrow) . The polypeptides being synthesized get longer and longer, the closer the end of the mRNA the ribosomes get

Bioinformatics Developments in scientifc research ollow improvements in computing: the use o computers has enabled scientists to make advances in bioinormatics applications such as locating genes within genomes and identiying conserved sequences. B ioinormatics involves the use o computers to store and analyse the huge amounts o data being generated by the sequencing o genomes and the identication o gene and protein sequences. Such inormation is oten amassed in databases, or example, GenB ank ( a US -based database) , the D D B J ( D NA databank o Japan) or the nucleotide sequence database maintained by the E MB L ( the European Molecular B iology Laboratory) , which then become accessible to the global community including scientists and the general public.

The unctions o conserved sequences are oten investigated in model organisms such as E. coli, yeast ( S. cerevisiae) , ruit fies ( D. melanogaster) , a soil roundworm C. elegans, thale cress A. thalania and mice M. musculus. These particular organisms are oten used because, along with humans, their entire genomes have been sequenced. Functions are oten discovered by knockout studies where the conserved gene is disrupted or altered and the impact on the organisms phenotype is observed.

A scientist studying a particular genetic disorder in humans might identiy sequence similarities that exist in people with the disorder. They might then search or homologous sequences in other organisms. These sequences might have a common ancestral origin but have accumulated dierences over time due to random mutation. To carry out the search or a homologous nucleotide or amino acid sequence, the scientist would conduct a B LAS T search. The acronym stands or basic local alignment search tool. Sometimes the homologous sequences are identical or nearly identical across species. These are called conserved sequences. The act that they are conserved across species suggests they play a unctional role.

368

 Figure 14 Examples of model

organisms

In addition to the B LAS T program, there are other sotware programs available. C lustalW can be used to align homologous sequences to search or changes. PhyloWin can be used to construct evolutionary trees based on sequence similarities.

7. 3 t r A N s l At i o N

Primary structure The sequence and number of amino acids in the polypeptide is the primary structure. A chain of amino acids is called a polypeptide. Given that the 20 commonly occurring amino acids can be combined in any sequence, it should not be surprising that there is a huge diversity of proteins. The sequence of amino acids in a polypeptide is termed its primary structure.

Daa-baed quen The hemoglobin molecule transports oxygen in the blood. It consists of 4 polypeptide chains. In human adults the molecule has two kinds of chains, alpha chains and beta chains, and there are two each. The alpha chain has 1 41 amino acid residues and the beta chain has 1 46 amino acid residues. The primary sequence of both polypeptides is shown below. The single residue in the beta chain marked in blue is the site of a mutation in sickle cell anemia. In the mutation, the glutamic acid is replaced by valine. alpha chain: 1 val * leu ser pro ala asp lys thr asn val lys ala ala trp gly lys val gly ala his ala gly glu tyr gly ala glu ala leu glu arg met phe leu ser phe pro thr thr lys thr tyr phe pro his phe * asp leu ser his gly ser ala * * * * * gln val lys gly his gly lys lys val ala asp ala leu thr asn ala val ala his val asp asp met pro asn ala leu ser ala leu ser asp leu his ala his lys leu arg val asp pro val asp phe lys leu leu ser his cys leu leu val thr leu ala ala his leu pro ala

glu phe thr pro ala val his ala ser leu asp lys phe leu ala ser val ser thr val leu thr ser lys tyr arg 1 41 beta chain: 1 val his leu thr pro glu glu lys ser ala val thr ala leu trp gly lys val asn * * val asp glu val gly gly glu ala leu gly arg leu leu val val tyr pro trp thr gln arg phe phe glu ser phe gly asp leu ser thr pro asp ala val met gly asn pro lys val lys ala his gly lys lys val leu gly ala phe ser asp gly leu ala his leu asp asn leu lys gly thr phe ala thr leu ser glu leu his cys asp lys leu his val asp pro glu asn phe arg leu leu gly asn val leu val cys val leu ala his his phe gly lys glu phe thr pro pro val gln ala ala tyr gln lys val val ala gly val ala asp ala leu ala his lys tyr his 1 46 C ompare the primary structure of the two polypeptides. The asterix ( *) symbols indicates locations where sections of the amino acid sequence are missing to facilitate comparison.

[4]

Secondary structure The secondary structure is the formation of alpha helices and beta pleated sheets stabilized by hydrogen bonding. B ecause the chain of amino acids in a polypeptide has polar covalent bonds within its backbone, it tends to fold in such a way that hydrogen bonds form between the carboxyl ( C = O ) group of one residue and the amino group ( NH) group of an amino acid in another part of the chain. This results in the formation of patterns within the polypeptide called secondary structures. The - helix and the - pleated sheet are examples of secondary structures.

369

7

N U C L E I C AC I D S ( AH L ) (a) alpha helix H N

C

H N C

HO N C

O

(b) beta pleated sheet O

N

O H

C

N

O

C

C

C

H

C

C

O

C

C

H

O

N

C N

H C C N

H hydrogen bond

O C

C N

 Figure 15 The structure of insulin

showing three areas where the -helix can be seen. It also shows the quaternary structure of insulin, i.e. the relative positions of the two polypeptides

N

C

C

H

C

N

O

H O O H C CN C N C C N C C C C N N C C H O H H O O

C

H

O

CN O

H

C H

O

O

C N

C

H

H C N O

O C N C H

C

H CN O

O C

C

O  Figure 16 Two examples of protein

secondary structure

Tertiary structure The tertiary structure is the further folding of the polypeptide stabilized by interactions between R groups. Tertiary structure reers to the overall three- dimensional shape o the protein ( fgure 1 8) . This shape is a consequence o the interaction o R- groups with one another and with the surrounding water medium. There are several dierent types o interaction. 

Positively charged R-groups will interact with negatively charged R-groups.



Hydrophobic amino acids will orientate themselves toward the centre o the polypeptide to avoid contact with water, while hydrophilic amino acids will orientate themselves outward.



Polar R- groups will orm hydrogen bonds with other polar R-groups.



The R-group o the amino acid cysteine can orm a covalent bond with the R- group o another cysteine orming what is called a disulphide bridge.

H3C H3C

CH 2 hydrogen OH bond O OH

C CH 2

hydrophobic interaction

CH CH 3 CH 3 CH

CH 2 S

polypeptide backbone

S

CH 2

disulphide bridge

O  Figure 17

Collagenthe quaternary structure consists of three polypeptides wound together to fom a tough, rope-like protein

370

CH 2 CH 2 CH 2 CH 2

O

NH 3 ionic bond

C

CH 2

 Figure 18 R-group interactions contribute to tertiary

structure

7. 3 t r A N s l At i o N

Quartenary structure

beta chain

beta chain

The quaternary structure exists in proteins with more than one polypeptide chain. Proteins can be ormed rom a single polypeptide chain or rom more than one polypeptide chain. Lysozyme is composed o a single chain, so lysozyme is both a polypeptide and a protein. Insulin is ormed rom two polypeptides, and hemoglobin is made up o our chains. Quaternary structure reers to the way polypeptides ft together when there is more than one chain. It also reers to the addition o non- polypeptide components. The quaternary structure o the hemoglobin molecule consists o our polypeptide chains and our heme groups.

alpha chain

heme

alpha chain

 Figure 19

The biological activity o a protein is related to its primary, secondary, tertiary and quaternary structure. C ertain treatments such as exposure to high temperatures, or changes in pH can cause alterations in the structure o a protein and thereore disrupt its biological activity. When a protein has permanently lost its structure it is said to be denatured.

The quaternary structure of hemoglobin in adults consists of four chains: two -chains and two -chains. Each subunit contains a molecule called a heme group

Daa-baed quen or the changes in hemoglobin type during development and ater birth. [3 ]

Hemoglobin is a protein composed o two pairs o globin subunits. During the process o development rom conception through to 6 months ater birth, human hemoglobin changes in composition. Adult hemoglobin consists o two alpha- and two betaglobin subunits. Four other polypeptides are ound during development: zeta, delta, epsilon and gamma.

b) C ompare changes in the amount o the gamma- globin gene with beta- globin.

[3 ]

c) D etermine the composition o the hemoglobin at 1 0 weeks o gestation and at 6 months o age.

[2 ]

d) S tate the source o oxygen or the etus.

[1 ]

e) The dierent types o hemoglobin have dierent afnities or oxygen. Suggest reasons

alpha-globin gamma-globin beta-globin delta-globin epsilon-globin zeta-globin % hemoglobin

Figure 2 0 illustrates the changes in hemoglobin composition during gestation and ater birth in a human. a) S tate which two subunits are present in highest amounts early in gestation. [1 ]

Key

50 40 30 20 10 0

10 20 30 Weeks of gestation

40 Birth

2

4 6 Month of age

 Figure 20

371

37

N U C L E I C AC I D S

Questions 1

D ierent samples o bacteria were supplied with radioactive nucleoside triphosphates or a series o times ( 1 0, 3 0 or 60 seconds) . This was the pulse period. This was ollowed by adding a large excess o non-radioactive nucleoside triphosphates or a longer period o time. This is called the chase period. The appearance o radioactive nucleotides ( incorporated during the pulse) in parts o the product D NA give an indication o the process o converting intermediates to fnal products.

2

B A

C

D NA was isolated rom the bacterial cells, denatured ( separated into two strands by heat) and centriuged to separate molecules by size. The closer to the top o the centriuge tube, the smaller the molecule.

D

a) C ompare the sample that was pulsed or 1 0 seconds with the sample that was pulsed or 3 0 seconds. [2 ]

E  Figure 22

b) Explain why the sample that was pulsed or 3 0 seconds provides evidence or the presence o both a leading strand and many lagging strands.

a) What part o the nucleotide is labelled A? [1 ]

c) Explain why the sample that was pulsed or 60 seconds provides evidence or the activity o D NA ligase. Radioactivity cpm / 0.1 m 1

With reerence to Figure 2 2 , answer the ollowing questions.

6,000

b) What kind o bond orms between the structures labelled B ?

[1 ]

c) What kind o bond is indicated by label C ?

[1 ]

d) What sub-unit is indicated by label D ?

[1 ]

e) What sub-unit is indicated by label E?

[1 ]

60 sec

3

5,000 4,000

Reer to fgure 2 3 when answering the ollowing questions. V

CH 2 OH

30 sec

I

O

H

3,000 IV 2,000

H H

H OH II

1,000

10 sec

OH

H

III 0

 Figure 21

372

0

1 2 3 Distance from top

 Figure 23

a) S tate what molecule is represented.

[1 ]

b) S tate whether the molecule would be ound in D NA or RNA.

[1 ]

c) S tate the part o the molecule to which phosphates bind.

[1 ]

d) Identiy the part o the molecule that reers to the 3  end.

[1 ]

8

M ETAB O LI SM , CE LL RE SPI RATI O N AN D PH O TO SYN TH E SI S ( AH L) CE LL B I O LO GY Introduction Life is sustained by a complex web of chemical reactions inside cells. These metabolic reactions are regulated in response to the needs of the cell and the organism. Energy is converted to a

usable form in cell respiration. In photosynthesis light energy is converted into chemical energy and a huge diversity of carbon compounds is produced.

8.1 Metabolism Understanding  Metabolic pathways consist o chains and

cycles o enzyme-catalysed reactions.  Enzymes lower the activation energy o the chemical reactions that they catalyse.  Enzyme inhibitors can be competitive or non-competitive.  Metabolic pathways can be controlled by end-product inhibition.

Applications  End-product inhibition o the pathway that

converts threonine to isoleucine.  Use o databases to identiy potential new anti-malarial drugs.

Skills  Distinguishing diferent types o inhibition rom

graphs at specied substrate concentration.  Calculating and plotting rates o reaction rom raw experimental results.

Nature of science  Developments in scientic research ollow improvements in computing: developments in bioinormatics,

such as the interrogation o databases, have acilitated research into metabolic pathways.

373

8

M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) initial TREAD substrate BREAD BREED BLEED intermediates BLEND BLIND BLINK end product

Figure 1 Word game analogy for metabolic pathways

Metabolic pathways Metabolic pathways consist of chains and cycles of enzyme-catalysed reactions. The word metabolism was introduced in the 1 9th century by the German cytologist and physiologist Theodor S chwann, to reer to the chemical changes that take place in living cells. It is now known that a huge range o chemical reactions occur in cells, catalysed by over 5 , 000 dierent types o enzyme. Although metabolism is very complex, there are some common patterns. 1

Most chemical changes happen not in one large j ump, but in a sequence o small steps, together orming what is called a metabolic pathway. The word game in fgure 1 is an analogy.

2

Most metabolic pathways involve a chain o reactions. Figure 2 shows a reaction chain that is used by cells to convert phenylalanine into umarate and acetoacetate, which can be used as energy sources in respiration. Phenylalanine causes severe health problems i there is an excess o it in the blood.

3

S ome metabolic pathways orm a cycle rather than a chain. In this type o pathway, the end product o one reaction is the reactant that starts the rest o the pathway.

phenylalanine I tyrosine II hydroxyphenylpyruvate III homogentisate

input: 3 CO 2

IV

RuBP

4-maleylacetoacetate V

3 ADP

3-PGA Calvin cycle

3 ATP

NADH + H + NAD + FADH 2

4-fumarylacetoacetate 5 G3P

6 G3P

Figure 2 Example of a metabolic pathway

output: 1 G3P

Krebs cycle

6 ATP

FAD

6 ADP + P 6 NADPH

C 4 compound C 6 compound NADH + H + + NAD NAD + + NADH + H CO 2 C5 compound

6 NADP+

VI fumarate + acetoacetate

acetyl group C 2

glucose and other compounds

CO 2

Figure 3

Enzymes and activation energy Enzymes lower the activation energy of the chemical reactions that they catalyse. C hemical reactions are not single- step processes. S ubstrates have to pass through a transition state beore they are converted into products. E nergy is required to reach the transition state, and although energy is released in going rom the transition state to the product, some energy must be put in to reach the transition state. This is called the activation energy. The activation energy is used to break or weaken bonds in the substrates. Figure 4 shows these energy

374

8 . 1 M e Tab O li s M changes for an exergonic ( energy releasing) reaction that is and is not catalysed by an enzyme. (b)

transition state

energy

activation energy

transition state

energy

(a)

substrate

activation energy substrate

product

product

progress of reaction

progress of reaction

Figure 4 Graphs showing activation energy (a) without an enzyme and (b) with an enzyme When an enzyme catalyses a reaction, the substrate binds to the active site and is altered to reach the transition state. It is then converted into the products, which separate from the active site. This binding lowers the overall energy level of the transition state. The activation energy of the reaction is therefore reduced. The net amount of energy released by the reaction is unchanged by the involvement of the enzyme. However as the activation energy is reduced, the rate of the reaction is greatly increased, typically by a factor of a million or more.

Types of enzyme inhibitors Enzyme inhibitors can be competitive or non-competitive. Some chemical substances bind to enzymes and reduce the activity of the enzyme. They are therefore known as inhibitors. The two main types are competitive and non- competitive inhibitors. C ompetitive inhibitors interfere with the active site so that the substrate cannot bind. Non- competitive inhibitors bind at a location other than the active site. This results in a change of shape in the enzyme so that the enzyme cannot bind to the substrate. Table 1 shows examples of each type. substrate competitive inhibitor

active site is blocked by competitor

Figure 6

non-competitive inhibitor

binding of inhibitor changes shape of active site

no inhibition

Figure 5 A molecular model o the restriction enzyme EcoRV (purple and pink) bound to a DNA molecule (deoxyribonucleic acid, yellow and orange) . Restriction enzymes, also known as restriction endonucleases, recognize specifc nucleotide sequences and cut the DNA at these sites. They are ound in bacteria and archaea and are thought to have evolved as a deence against viral inection

TOK To wht xtnt houd thc contrn th dvopmnt of knowdg n cnc? Sarin was a chemical developed as an insectide beore being applied is a chemical weapon. It is a competitive inhibitor o the neurotransmitter acetylcholinesterase. Chemical weapons would not exist without the activities o scientists. In act, the name Sarin is an acronym o the surnames o the scientists who frst synthesized it. Fritz Haber received the 1918 Nobel Prize or Chemistry or his work in developing the chemistry behind the industrial production o ammonia ertilizer. Some scientists boycotted the award ceremony because Haber had been instrumental in encouraging and developing the use o chlorine gas in the First World War. Haber is quoted as saying: "During peace time a scientist belongs to the World, but during war time he belongs to his country."

375

8

M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L )

enzym dihydropteroate synthetase

sutrat

inhtor

para-aminobenzoate

bndng

suladiazine

OC

N SO 2

O

N

N H H 2N

H 2N

phosphoructokinase

The inhibitor binds reversibly to the enzymes active site. While it remains bound, substrates cannot bind. This is competitive inhibition.

ructose-6-phosphate

xylitol-5-phosphate

P

OH

OH

CH 2

CH 2

C

H

C

H

C

H

O

H2C

C

The inhibitor binds reversibly to a site away rom the active site. While it remains bound, the active site is distorted and substrate cannot bind. This is non-competitive inhibition.

OH P CH 2

C H

OH

HO

H

OH

OH C

C

OH

H

Table 1 Examples of each type of inhibitor

Efects o enzyme inhibitors Distinguishing diferent types o inhibition rom graphs at specied substrate concentration.

The orange line represents the effect of substrate concentration on enzyme activity in the absence of an inhibitor. The red line shows the effect of substrate concentration on the rate of reaction when a competitive inhibitor is present. When the concentration of substrate begins to exceed the amount of inhibitor, the maximum rate of the uninhibited enzyme can be achieved; however, it takes a much higher concentration of substrate to achieve this maximum rate. The blue line shows the effect of substrate concentration on the rate of reaction when a non- competitive inhibitor is present. In the presence of a non- competitive inhibitor, the enzyme does not reach the same maximum rate because the binding of the non- competitive

376

inhibitor prevents some of the enzymes from being able to react regardless of substrate concentration. Those enzymes that do not bind inhibitors follow the same pattern as the normal enzyme. It takes approximately the same concentration of enzyme to reach the maximum rate, but the maximum rate is lower than the uninhibited enzyme. maximum rate of reaction rate of reaction

Figure 7 represents the effect of substrate concentration on the rate of an enzyme controlled reaction.

normal enzyme competitive inhibitor non-competitive inhibitor

substrate concentration

Figure 7

8 . 1 M e Tab O li s M

End-product inhibition Metabolic pathways can be controlled by end-product inhibition. Many enzymes are regulated by chemical substances that bind to special sites on the enzyme away rom the active site. These are called allosteric interactions and the binding site is called an allosteric site. In many cases, the enzyme that is regulated catalyses one o the rst reactions in a metabolic pathway and the substance that binds to the allosteric site is the end product o the pathway. The end product acts as an inhibitor. The pathway works rapidly in cells with a shortage o end product but can be switched o completely in cells where there is an excess.

An example of end-product inhibition

initial substrate (threonine) threonine in active site

active site no longer binds to threonine

enzyme 1 (threonine deaminase) intermediate A enzyme 2

isoleucine in allosteric site

End-product inhibition o the pathway that converts threonine to isoleucine. Through a series o ve reactions, the amino acid threonine is converted to isoleucine. As the concentration o isoleucine builds up, it binds to the allosteric site o the rst enzyme in the chain, threonine deaminase, thus acting as a non- competitive inhibitor ( gure 8) .

feedback inhibition

To see why this is such an economical way to control metabolic pathways, we need to understand how the concentration o the product o a reaction can infuence the rate o reaction. Reactions oten do not go to completion  instead an equilibrium position is reached with a characteristic ratio o substrates and products. So, i the concentration o products increases, a reaction will eventually slow down and stop. This eect reverberates back through a metabolic pathway when the end product accumulates, with all the intermediates accumulating. Endproduct inhibition prevents this build-up o intermediate products.

intermediate B enzyme 3 intermediate C enzyme 4 intermediate D enzyme 5 end product (isoleucine)

Figure 8

Investigating metabolism through bioinformatics Developments in scientifc research ollow improvements in computing: developments in bioinormatics, such as the interrogation o databases, have acilitated research into metabolic pathways. Computers have increased the capacity o scientists to organize, store, retrieve and analyse biological data. Bioinormatics is an approach whereby multiple research groups can add inormation to a database enabling other groups to query the database. O ne promising bioinormatics technique that has acilitated research into metabolic pathways is reerred to as chemogenomics. S ometimes when a chemical binds to a target site, it can

signicantly alter metabolic activity. S cientists looking to develop new drugs test massive libraries o chemicals individually on a range o related organisms. For each organism a range o target sites are identied and a range o chemicals which are known to work on those sites are tested. O ne researcher called chemogenomics  the chemical universe tested against the target universe .

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Chemogenomics applied to malaria drugs Use of databases to identify potential new anti-malarial drugs. Malaria is a disease caused by the pathogen Plasmodium falciparum. The increasing resistance o P. falciparum to anti-malarial drugs such as chloroquine, the dependence o all new drug combinations on a narrow range o medicines and increasing global eorts to eradicate malaria all drive the need to develop new anti-malarial drugs. Plasmodium falciparum strain 3 D 7 is a variety o the malarial parasite or which the genome has been sequenced. In one study, approximately 3 1 0, 000 chemicals were screened against

a chloroquine- sensitive 3 D 7 strain and the chloroquine-resistant K1 strain to see i these chemicals inhibited metabolism. O ther related and unrelated organisms, including human cell lines, were also screened. O ne promising outcome was the identifcation o 1 9 new chemicals that inhibit the enzymes normally targeted by anti-malarial drugs and 1 5 chemicals that bind to a total o 61 dierent malarial proteins. This provides other scientists with possible lines o investigation in the search or new anti-malarials.

Calculating rates of reaction Calculating and plotting rates of reaction from raw experimental results. A large number o dierent protocols are available or investigating enzyme activity. D etermining the rate o an enzyme-controlled reaction involves measuring either the rate o disappearance o a

substrate or the rate o appearance o a product. Sometimes this will require conversion o units to yield a rate unit which should include s - 1 .

Data-basd qustions: The efectiveness o enzymes The degree to which enzymes increase the rate o reactions varies greatly. B y calculating the ratio between the rate o reactions with and without an enzyme catalyst, the afnity between an enzyme and its substrate can be estimated. Table 2 shows the rates o our reactions with and without an enzyme. The ratio between these rates has been calculated or one o the reactions. 1

S tate which enzyme catalyses the reaction with the slowest rate in the absence o an enzyme. [1 ]

enzym

State which enzyme catalyses its reaction at the most rapid rate. [1 ]

3

C alculate the ratios between the rate o reaction with and without an enzyme or ketosteroid isomerase, nuclease and O MP decarboxylase. [3 ]

4

D iscuss which o the enzymes is the more eective catalyst. [3 ]

5

Explain how the enzymes increase the rate o the reactions that they catalyse.

Rat without nzym/s 1

Rat with nzym/s 1

Ratio btwn rat with and without nzym

Carbonic anhydrase

1.3  10 1

1.0  10 6

7.7  10 6

Ketosteroid isomerase

1.7  10 7

6.4  10 4

Nuclease

1.7  10 13

9.5  10 6

OMP decarboxylase

2.8  10 16

3.9  10 8

Table 2

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2

[2 ]

8 . 1 M e Tab O li s M

oxygen/%

Dt-d quton: Calculating rates of reaction 22.0 21.5 21.0 20.5 20.0 19.5 19.0 18.5 18.0

actvty For each o the ollowing enzyme experiments, describe how the rate o reaction can be determined:

0

10 51C

20 4C

30

40 time/s 21C

50

60

70

34C

Figure 9 Percentage of oxygen concentration over time at various temperatures after adding catalase to a 1.5% hydrogen peroxide solution Ten drops o a commercial catalase solution were added to our reaction vessels containing a 1 .5 % hydrogen peroxide solution. Each o the solutions had been kept at a dierent temperature. The % oxygen in the reaction vessel was determined using a data logger in a set-up similar to fgure 1 0.

Figure 10 1

E xplain the variation in the % oxygen at time zero.

2

Determine the rate o reaction at each temperature using the graph.

3

C onstruct a scatter plot o reaction rate versus temperature.

) Paper discs soaked in the enzyme catalase are added to diferent concentrations o hydrogen peroxide. The reaction produces oxygen bubbles. ) Lipase catalyses the breakdown o triglycerides to atty acids and water. The pH o the reaction solution will lower as the reaction proceeds. c) Papain is a protease that can be extracted rom pineapple ruits. Gelatin cubes will be digested by papain. d) Catechol oxidase converts catechol to a yellow pigment in cut ruit. It can be extracted rom bananas. The yellow pigment reacts with oxygen in the air to turn brown.

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8.2 Cell respiration Understanding

Applications

 Cell respiration involves the oxidation and      





  

reduction o compounds. Phosphorylation o molecules makes them less stable. In glycolysis, glucose is converted to pyruvate. Glycolysis gives a small net gain o ATP without the use o oxygen. In aerobic cell respiration pyruvate is decarboxylated and oxidized. In the link reaction pyruvate is converted into acetyl coenzyme A. In the Krebs cycle, the oxidation o acetyl groups is coupled to the reduction o hydrogen carriers, liberating carbon dioxide. Energy released by oxidation reactions is carried to the cristae o the inner mitochondrial membrane by reduced NAD and FAD. Transer o electrons between carriers in the electron transport chain is coupled to proton pumping. In chemiosmosis protons difuse through ATP synthase to generate ATP. Oxygen is needed to bind with the ree protons to orm water to maintain the hydrogen gradient. The structure o the mitochondrion is adapted to the unction it perorms.

 Electron tomography used to produce images

o active mitochondria.

Skills  Analysis o diagrams o the pathways o aerobic

respiration to deduce where decarboxylation and oxidation reactions occur.  Annotation o a diagram to indicate the adaptations o a mitochondrion to its unction.

Nature of science  Paradigm shits: the chemiosmotic theory led to

a paradigm shit in the eld o bioenergetics.

Oxidation and reduction Cell respiration involves the oxidation and reduction o compounds. O xidation and reduction are chemical processes that always occur together. This happens because they involve transfer of electrons from one substance to another. O xidation is the loss of electrons from a substance and reduction is the gain of electrons. A useful example to help visualize this in the laboratory is in the B enedicts test, a test for certain types of sugar. The test involves the

380

8 . 2 C e l l R e s p i R aT i O n

use o copper sulphate solution, containing copper ions with a charge o two positive ( C u 2 + ) . C u 2+ oten imparts a blue or green colour to solutions. These copper ions are reduced and become atoms o copper by being given electrons. C opper atoms are insoluble and orm a red or orange precipitate. The electrons come rom sugar molecules, which are thereore oxidized. Electron carriers are substances that can accept and give up electrons as required. They oten link oxidations and reductions in cells. The main electron carrier in respiration is NAD ( nicotinamide adenine dinucleotide) . In photosynthesis a phosphorylated version o NAD is used, NAD P ( nicotinamide adenine dinucleotide phosphate) . The structure o the NAD molecule is shown in fgure 1 .

adenine base ribose sugar

phosphates

ribose sugar

The equation below shows the basic reaction. NAD + 2 electrons  reduced NAD The chemical details are a little more complicated. NAD initially has one positive charge and exists as NAD + . It accepts two electrons in the ollowing way: two hydrogen atoms are removed rom the substance that is being reduced. O ne o the hydrogen atoms is split into a proton and an electron. The NAD + accepts the electron, and the proton ( H + ) is released. The NAD accepts both the electron and proton o the other hydrogen atom. The reaction can be shown in two ways:

nicotinamide base

Figure 1 Structure of NAD

NAD + + 2 H + + 2 electrons ( 2e )  NAD H + H + NAD + + 2 H  NAD H + H + This reaction demonstrates that reduction can be achieved by accepting atoms o hydrogen, because they have an electron. O xidation can thereore be achieved by losing hydrogen atoms. O xidation and reduction can also occur through loss or gain o atoms o oxygen. There are ewer examples o this in biochemical processes, perhaps because in the early evolution o lie oxygen was absent rom the atmosphere. A ew types o bacteria can oxidize hydrocarbons using oxygen: 1 O  C H C H O H C 7 H 1 5 C H 3 + _ 7 15 2 2 2 n- octane n- octanol Nitriying bacteria oxidize nitrite ions to nitrate. 1 O  NO NO -2 + _ 3 2 2 Adding oxygen atoms to a molecule or ion is oxidation, because the oxygen atoms have a high afnity or electrons and so tend to draw them away rom other parts o the molecule or ion. In a similar way, losing oxygen atoms is reduction.

Phosphorylation Phosphorylation of molecules makes them less stable. Phosphorylation is the addition o a phosphate molecule ( PO 3) to 4 an organic molecule. B iochemists indicate that certain amino acid sequences tend to act as binding sites or the phosphate molecule on proteins. For many reactions, the purpose o phosphorylation is to make

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M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) the phosphorylated molecule more unstable; i.e., more likely to react. Phosphorylation can be said to activate the molecule. The hydrolysis o ATP releases energy to the environment and is thereore termed an exergonic reaction. Many chemical reactions in the body are endergonic ( energy absorbing) and thereore do not proceed spontaneously unless coupled with an exergonic reaction that releases more energy. For example, depicted below is the frst reaction in the series o reactions known as glycolysis. Glucose-6- phosphate

Glucose ATP AD P

The conversion o glucose to glucose-6- phosphate is endergonic and the hydrolysis o ATP is exergonic. B ecause the reactions are coupled, the combined reaction proceeds spontaneously. Many metabolic reactions are coupled to the hydrolysis o ATP.

Glycolysis and ATP Glycolysis gives a small net gain of ATP without the use of oxygen. The most signifcant consequence o glycolysis is the production o a small yield o ATP without the use o any oxygen, by converting sugar into pyruvate. This cannot be done as a single- step process and instead is an example o a metabolic pathway, composed o many small steps. The frst o these may seem rather perverse: ATP is used up in phosphorylating sugar. Glucose ATP AD P

Glucose- 6- phosphate  Fructose6phosphate

Fructose- 1 , 6- bisphosphate

ATP AD P However, these phosphorylation reactions reduce the activation energy required or the reactions that ollow and so make them much more likely to occur.

Pyruvate is a product of glycolysis In glycolysis, glucose is converted to pyruvate. In the next step, the ructose bisphosphate is split to orm two molecules o triose phosphate. E ach o these triose phosphates is then oxidized to glycerate- 3 - phosphate in a reaction that yields enough energy to make ATP. This oxidation is carried out by removing hydrogen. Note that it is hydrogen atoms that are removed. I only hydrogen ions were removed ( H + ) , no electrons would be removed and it would not be an oxidation. The hydrogen is accepted by NAD + , which becomes NAD H + H + . In the fnal stages o glycolysis, the phosphate group is transerred to AD P to produce more ATP and also pyruvate. These stages are summarized in the equation below, which occurs twice per glucose.

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8 . 2 C e l l R e s p i R aT i O n

NAD + NAD H + H + triose phosphate

glycerate- 3 - phosphate

The fate of pyruvate

Glucose

In aerobic cell respiration pyruvate is decarboxylated and oxidized.

reduced NAD

pyruvate

ATP

Pyruvate

Two molecules o pyruvate are produced in glycolysis per molecule o glucose. I oxygen is available, this pyruvate is absorbed into the mitochondrion, where it is ully oxidized. 2 C H 3 C O C O O H + 5 O 2  6C O 2 + 4H 2 O

Glycolysis

reduced NAD

Link reaction Acetyl CoA

reduced FAD  Electron transport  Oxidative reduced phosphorylation NAD  Chemiosmosis

Krebs cycle

ATP

As with glycolysis, this is not a single-step process. C arbon and oxygen are removed in the orm o carbon dioxide, in reactions called decarboxylations. The oxidation o pyruvate is achieved by the removal o pairs ATP o hydrogen atoms. The hydrogen carrier NAD + , and a Figure 2 A summary of aerobic respiration related compound called FAD , accept these hydrogen atoms and pass them on to the electron transport chain where oxidative phosphorylation will occur. These reactions are summarized in fgure 2 . O

The link reaction In the link reaction pyruvate is converted into acetyl coenzyme A.

In the Krebs cycle, the oxidation of acetyl groups is coupled to the reduction of hydrogen carriers. This cycle has several names but is oten called the Krebs cycle, in honour o the biochemist who was awarded the Nobel Prize or its discovery. The link reaction involves one decarboxylation and one oxidation. There are two more decarboxylations and our more oxidations in the Krebs cycle. I glucose as oxidized by burning in air, energy would be released as heat. Most o the energy released in the oxidations o the link reaction and the Krebs cycle is used to reduce hydrogen carriers ( NAD + and FAD ) .

O

S

CoA

C

O

C

O

CH 3

CO 2 NAD + reduced NAD CH 3

Figure 3 The link reaction

The frst step, represented by fgure 3 , occurs ater the pyruvate, which has been produced in the cytoplasm, is shuttled into the mitochondrial matrix. O nce there, the pyruvate is decarboxylated and oxidized to orm an acetyl group. Two high energy electrons are removed rom pyruvate. These react with NAD + to produce reduced NAD . This is called the link reaction, because it links glycolysis with the cycle o reactions that ollow.

The Krebs cycle

CoA-SH C

CO 2

pyruvic acid NAD + reduced NAD acetyl-CoA citric acid (6C)

CoA OAA (4C)

reduced NAD

NAD + reduced NAD CO 2

NAD +

CO 2

NAD + reduced NAD

FADH 2 FAD

ATP

ADP+ i P

Figure 4 Summary of the Krebs cycle

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TOK What kinds o explanations do scientists ofer, and how do these explanations compare with those ofered in other areas o knowledge? Hans Krebs was awarded the Nobel Prize in 1953. The two nal paragraphs o the lecture that he gave on this occasion are reproduced here. The reactions o the cycle have been ound to occur in representatives o all orms o lie, rom unicellular bacteria and protozoa to the highest mammals. The study o intermediary metabolism shows that the basic metabolic processes, in particular those providing energy and those leading to the synthesis o cell constituents, are also shared by all orms o lie. The existence o common eatures in dierent orms o lie indicates some relationship between the dierent organisms, and according to the concept o evolution these relations stem rom the circumstance that the higher organisms, in the course o millions o years, have gradually evolved rom simpler ones. The concept o evolution postulates that living organisms have common roots, and in turn the existence o common eatures is powerul support or the concept o evolution. The presence o the same mechanism o energy production in all orms o lie suggests two other inerences: frstly that the mechanism o energy production has arisen very early in the evolutionary process; and secondly that lie, in its present orms, has arisen only once. 1 Outline the argument or similarities o metabolism as evidence or evolution. 2 Are there any alternative explanations or the similarities?

384

The energy thereore remains in chemical orm and can be passed on to the nal part o aerobic cell respiration: oxidative phosphorylation. For every turn o the cycle, the production o reduced NAD occurs three times, decarboxylation occurs twice and the reduction o FAD occurs once. O ne molecule o ATP is also generated.

Oxidative phosphorylation Energy released by oxidation reactions is carried to the cristae o the mitochondria by reduced NAD and FAD. In aerobic respiration, there are several points where energy released by oxidation reactions is coupled to the reduction o mainly NAD but also FAD . Reduced NAD is produced during glycolysis, the link reaction and the Krebs cycle. FAD H 2 is produced during the Krebs cycle. The nal part o aerobic respiration is called oxidative phosphorylation, because AD P is phosphorylated to produce ATP, using energy released by oxidation. The substances oxidized include the FAD H 2 generated in the Krebs cycle and the reduced NAD generated in glycolysis, the link reaction and the Krebs cycle. Thus these molecules are used to carry the energy released in these stages to the mitochondrial cristae.

The electron transport chain Transer o electrons between carriers in the electron transport chain is coupled to proton pumping. The nal part o aerobic respiration is called oxidative phosphorylation, because AD P is phosphorylated to produce ATP, using energy released by oxidation. The main substance oxidized is reduced NAD . The energy is not released in a single large step, but in a series o small steps, carried out by a chain o electron carriers. Reduced NAD and FAD H 2 donate their electrons to electron carriers. As the electrons are passed rom carrier to carrier, energy is utilized to transer protons across the inner membrane rom the matrix into the intermembrane space. The protons then fow through ATP synthase down their concentration gradient providing the energy needed to make ATP.

Chemiosmosis In chemiosmosis protons difuse through ATP synthase to generate ATP. The mechanism used to couple the release o energy by oxidation to ATP production remained a mystery or many years, but is now known to be chemiosmosis. This happens in the inner mitochondrion membrane. It is called chemiosmosis because a chemical substance ( H + ) moves across a membrane, down the concentration gradient. This releases the energy needed or the enzyme ATP synthase to make ATP. The main steps in the process are as ollows ( also see gure 5 ) .

8 . 2 C e l l R e s p i R aT i O n



NAD H + H + supplies pairs o hydrogen atoms to the rst carrier in the chain, with the NAD + returning to the matrix.



The hydrogen atoms are split, to release two electrons, which pass rom carrier to carrier in the chain.









Energy is released as the electrons pass rom carrier to carrier, and three o these use this energy to transer protons (H + ) across the inner mitochondrial membrane, rom the matrix to the intermembrane space. As electrons continue to fow along the chain and more and more protons are pumped across the inner mitochondrial membrane, a concentration gradient o protons builds up. This proton gradient is a store o potential energy. To allow electrons to continue to fow, they must be transerred to a terminal electron acceptor at the end o the chain. In aerobic respiration this is oxygen, which briefy becomes  O 2 , but then combines with two H + ions rom the matrix to become water. Protons pass back rom the intermembrane space to the matrix through ATP synthase. As they are moving down the concentration gradient, energy is released and this is used by ATP synthase to phosphorylate AD P.

inter inner mitochondrial membrane space membrane

matrix

NADH + H + H+ NAD +

2e -

FADH 2

H+

FAD H 2O

H+ H+

2H +  O2O2

H+ ATP ADP +Pi low H + concentration

The role of oxygen Oxygen is needed to bind with the free protons to form water to maintain the hydrogen gradient.

H+

high H + concentration

Figure 5 Summary of oxidative phosphorylation

O xygen is the nal electron acceptor in the mitochondrial electron transport chain. The reduction o the oxygen molecule involves both accepting electrons and orming a covalent bond with hydrogen. B y using up hydrogen, the proton gradient across the inner mitochondrial membrane is maintained so that chemiosmosis can continue.

Dt-bd quto: Oxygen consumption by mitochondria Figure 6 shows the results o an experiment in which mitochondria were extracted rom liver cells and were kept in a fuid medium, in which oxygen levels were monitored. Pyruvate was added at point I on the graph, and AD P was added at points II, III and IV.

1

Explain why oxygen consumption by the mitochondria could not begin unless pyruvate had been added. [3 ]

2

D educe what prevented oxygen consumption between points I and II.

[2 ]

Predict, with reasons, what would have happened i AD P had not been added at point III.

[2 ]

D iscuss the possible reasons or oxygen consumption not being resumed ater AD P was added at point IV.

[3 ]

oxygen saturation / %

3 I

II

100 III

4 50

IV

0 time

Figure 6 Results of oxygen consumption experiment

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The chemiosmotic theory Paradigm shits: the chemiosmotic theory produced a paradigm shit in the feld o bioenergetics. In 1 961 Peter Mitchell proposed the chemiosmotic hypothesis to explain the coupling o electron transport in the inner mitochondrial membrane to ATP synthesis. His hypothesis was a radical departure rom previous hypotheses and only ater many years was it generally accepted. He was awarded the Nobel Prize or Chemistry in 1 978 and part o the Banquet Speech that he gave is reproduced here: Emile Zola described a work o art as a corner o nature seen through a temperament. The philosopher Karl Popper, the economist F.A. Hayek and the art historian K.H. Gombrich have shown that the creative process in science and art consists o two main activities: an imaginative jumping orward to a new abstraction or simplifed representation, ollowed by a critical looking back to see how nature appears in the light o the new vision. The imaginative leap orward is a hazardous, unreasonable activity. Reason can be used only when looking critically back. Moreover, in the experimental sciences, the scientifc raternity must test a new theory

The fnal outcome cannot be known, either to the originator o a new theory, or to his colleagues and critics, who are bent on alsiying it. Thus, the scientifc innovator may eel all the more lonely and uncertain. On the other hand, aced with a new theory, the members o the scientifc establishment are oten more vulnerable than the lonely innovator. For, i the innovator should happen to be right, the ensuing upheaval o the established order may be very painul and uncongenial to those who have long committed themselves to develop and serve it. Such, I believe, has been the case in the feld o knowledge with which my work has been involved. Naturally I have been deeply moved, and not a little astonished, by the accidents o ortune that have brought me to this point.

Structure and function in the mitochondrion

Examine fgure 7 showing an electron micrograph o a mitochondrion and a drawing representing that mitochondrion.

The structure o the mitochondrion is adapted to the unction it perorms.

The mitochondrion is a semi- autonomous organelle in that it can grow and reproduce itsel but it still depends on the rest o the cell or resources and is otherwise part o the cellular system. 70S ribosomes and a naked loop o D NA are ound within the mitochondrial matrix.

There is oten a clear relationship between the structures o the parts o living organisms and the unctions they perorm. This can be explained in terms o natural selection and evolution. The mitochondrion can be used as an example. I mitochondrial structure varied, those organisms with the mitochondria that produced ATP most efciently would have an advantage. They would have an increased chance o survival and would tend to produce more ospring. These ospring would inherit the type o mitochondria that produce ATP more efciently. I this trend continued, the structure o mitochondria would gradually evolve to become more and more efcient. This is called adaptation  a change in structure so that something carries out its unction more efciently.

386

to destruction, i possible. Meanwhile, the creator o a theory may have a very lonely time, especially i his colleagues fnd his views o nature unamiliar and difcult to appreciate.

The mitochondrion is the site o aerobic respiration. The outer mitochondrial membrane separates the contents o the mitochondrion rom the rest o the cell creating a compartment specialized or the biochemical reactions o aerobic respiration. The inner mitochondrial membrane is the site o oxidative phosphorylation. It contains electron transport chains and ATP synthase, which carry out oxidative phosphorylation. Cristae are tubular projections o the inner membrane which increase the surace area available or oxidative phosphorylation. The intermembrane space is the location where protons build up as a consequence o the electron

8 . 2 C e l l R e s p i R aT i O n transport chain. The proton build-up is used to produce ATP via the ATP synthase. The volume o the space is small, so a concentration gradient across the inner membrane can be built up rapidly.

The matrix is the site o the Krebs cycle and the link reaction. The matrix fuid contains the enzymes necessary to support these reaction systems.

Annotating a diagram of a mitochondrion Annotation o a diagram to indicate the adaptations o a mitochondrion to its unction. Outer mitochondrial membrane separates the contents of the mitochondrion Matrix from the rest of the cell, creating a cellular contains enzymes for the Krebs cycle and the link reaction compartment with ideal conditions for Intermembrane space aerobic respiration Proteins are pumped Inner mitochondrial into this space by the membrane contains electron transport chain. electron transport The space is small so the chanins and ATP synthase concentration builds up quickly Cristae are projections of the inner membrane Ribosome DNA which increase the surface area available for for expression of oxidative phosphorylation mitochondrial genes

Figure 7

actvty

0.1m

a)

b)

d)

c)

Figure 8 Electron micrographs of mitochondria: (a) from a bean plant (b) from mouse liver (c) from axolotl sperm (d) from bat pancreas

Study the electron micrographs in gure 8 and then answer the multiple-choice questions. 1 The fuid-lled centre o the mitochondrion is called the matrix. What separates the matrix rom the cytoplasm around the mitochondrion?

80S ribosomes. Which o these hypotheses is consistent with this observation? (i) Protein is synthesized in the mitochondrion. (ii) Ribosomes in mitochondria have evolved rom ribosomes in bacteria.

) One wall.

c) Two membranes.

(iii) Ribosomes are produced by aerobic cell respiration.

b) One membrane.

d) One wall and one membrane.

) (i) only

c) (i) and (ii)

b) (ii) only

d) (i) , (ii) and (iii)

2 The mitochondrion matrix contains 70S ribosomes, whereas the cytoplasm o eukaryotic cells contains

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Mitochondrial membranes are dynamic Electron tomography used to produce images o active mitochondria. Ideas in science sometimes change gradually. B ut sometimes they remain stable or years or even decades and then undergo a sudden change. This can be due to the insight or enthusiasm o a particular scientist, or team. The development o new techniques can sometimes be the stimulus. The technique o electron tomography has recently allowed three-dimensional images o the interior o mitochondria to be made. O ne o the leaders in this feld is D r. C armen Mannella, ormer D irector, D ivision o Molecular Medicine, Wadsworth C enter, Albany NY: Resource or Visualization o B iological C omplexity. He recently gave this brie comment on developments in our understanding o mitochondrial structure and unction. The new take-home message about the mitochondrial inner membrane is that the cristae

are not simple inoldings but are invaginations, dening micro-compartments in the organelle. The cristae originate at narrow openings (crista junctions) that likely restrict diusion o proteins and metabolites between the compartments. The membranes are not only very fexible but also dynamic, undergoing usion and ssion in response to changes in metabolism and physiological stimuli. The working hypothesis is that the observed changes in membrane shape (topology) are not random and passive but rather a specic mechanism by which mitochondrial unction is regulated by changes in internal diusion pathways, e.g., allowing more ecient utilization o ADP. It appears that there are specic proteins and lipids that actively regulate the topology o the inner membrane. This is a bit speculative at the time but it gives a sense o where things are headed in the eld.

Figure 9 Three images of the inner mitochondrial membrane of mitochondria from liver cells show the dynamic nature of this membrane

TOK There are some scientic elds that depend entirely upon technology or their existence, or example, spectroscopy, radio or X-ray astronomy. What are the knowledge implications o this? Could there be problems o knowledge that are unknown now, because the technology needed to reveal them does not exist yet?

388

activity Answer the ollowing questions with respect to the three images in gure 9 . ) The diameter o the mitochondrion was 700 nm. Calculate the magnication o the image.

[3]

b) Electron tomography has shown that cristae are dynamic structures and that the volume o the intracristal compartment increases when the mitochondrion is active in electron transport. Suggest how electron transport could cause an increase in the volume o fuid inside the cristae. [2] c) Junctions between the cristae and boundary region o the inner mitochondrial membrane can have the shape o slots or tubes and can be narrow or wide. Suggest how narrow tubular connections could help in ATP synthesis by one o the cristae in a mitochondrion. [2]

8 . 3 ph O TO s yn Th e s i s

8.3 potot Understanding  Light-dependent reactions take place in the         

   

intermembrane space o the thylakoids. Reduced NADP and ATP are produced in the light-dependent reactions. Light-independent reactions take place in the stroma. Absorption o light by photosystems generates excited electrons. Photolysis o water generates electrons or use in the light-dependent reactions. Transer o excited electrons occurs between carriers in thylakoid membranes. Excited electrons rom Photosystem II are used to generate a proton gradient. ATP synthase in thylakoids generates ATP using the proton gradient. Excited electrons rom Photosystem I are used to reduce NADP. In the light-independent reactions a carboxylase catalyses the carboxylation o ribulose bisphosphate. Glycerate 3-phosphate is reduced to triose phosphate using reduced NADP and ATP. Triose phosphate is used to regenerate RuBP and produce carbohydrates. Ribulose bisphosphate is reormed using ATP. The structure o the chloroplast is adapted to its unction in photosynthesis.

Applications  Calvins experiment to elucidate the

carboxylation o RuBP.

Skills  Annotation o a diagram to indicate the

adaptations o a chloroplast to its unction.

Nature of science  Developments in scientifc research ollow

improvements in apparatus: sources o 1 4 C and autoradiography enabled Calvin to elucidate the pathways o carbon fxation.

Location of light-dependent reactions Light-dependent reactions take place in the intermembrane space o the thylakoids. Research into photosynthesis has shown that it consists of two very different parts, one of which uses light directly ( light- dependent reactions) and the other does not use light directly ( light-independent

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M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) reactions) . The light- independent reactions can only carry on in darkness or a ew seconds because they depend on substances produced by the light-dependent reactions which rapidly run out. The chloroplast has an outer membrane and an inner membrane. The inner membrane encloses a third system o interconnected membranes called the thylakoid membranes. Within the thylakoid is a compartment called the thylakoid space. The light-dependent reactions take place in the thylakoid space and across the thylakoid membranes.

Data-based questions: Freeze-fracture images of chloroplasts I chloroplasts are rozen rapidly in liquid nitrogen and then split, they racture across planes o weakness. These planes o weakness are usually the centres o membranes, between the two layers o phospholipid, where there are no hydrogen bonds attracting water molecules to each other. Structures within the membrane such as the photosystems are then visible in electron micrographs ( see fgure 1 ) . 1

2

3

D escribe the evidence, visible in the electron micrograph, or chloroplasts having many layers o membrane.

[2 ]

Explain how photosystems become visible as lumps in reeze- racture electron micrographs o chloroplasts.

[2 ]

S ome membranes contain large particles arranged in rectangular arrays. These are Photosystem II. They have a diameter o 1 8 nm. C alculate the magnifcation o the electron micrograph. [3 ]

4

Other membranes visible in the electron micrograph contain a variety o other structures. Use the inormation on the ollowing pages to deduce what these are.

[3]

Figure 1 Freeze-fracture electron micrograph of spinach chloroplast

The products of the light-dependent reactions Reduced NADP and ATP are produced in the light-dependent reactions. Light energy is converted into chemical energy in the orm o ATP and reduced NAD P in the light reacations. The ATP and reduced NAD P serve as energy sources or the light- independent reactions.

The location of the light-independent reactions Light-independent reactions take place in the stroma. The inner membrane o the chloroplast encloses a compartment called the stroma. This is a thick protein-rich medium containing enzymes or use in the light-independent reactions, also known as the C alvin

390

8 . 3 ph O TO s yn Th e s i s

cycle. In the light-independent reactions the C alvin cycle is an anabolic pathway that requires endergonic reactions to be coupled to the hydrolysis o ATP and the oxidation o reduced NAD P. Figure 2 summarizes the processes o both the light- dependent and lightindependent reactions. outer membrane of chloroplast

inner membrane of chloroplast CO 2

thylakoid membrane light energy

thylakoid space

P1 + ADP ATP

Calvin cycle

NADP NADPH + H + sugars

2e H 2O

light-independent reactions - photolysis - photoactivation - electron transport - chemiosmosis - ATP synthesis - reduction of NADP

2H +

+

1 2

O2

light-independent reactions - carbon xation - carboxylation of RuBP - production of triose phosphate - ATP and NADPH as energy sources - ATP used to regenerate RuBP - ATP used to produce carbohydrates

Figure 2

Photoactivation Absorption of light by photosystems generates excited electrons. C hlorophyll and the accessory pigments are grouped together in large light- harvesting arrays called photosystems. These photosystems are located in the thylakoids, an arrangement o membranes inside the chloroplast. There are two types o light- harvesting arrays, called Photosystems I and II. In addition to light- harvesting arrays, the photosystems have reaction centres ( fgure 3 ) . B oth types o photosystem contain many chlorophyll molecules, which absorb light energy and pass it to two special chlorophyll molecules in the reaction centre o the photosystem. Like other chlorophylls, when these special chlorophyll molecules absorb the energy rom a photon o light an electron within the molecule becomes excited. The chlorophyll is then p hotoactivated. The chlorophylls at the reaction centre have the special property o being able to donate excited electrons to an electron acceptor.

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M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) Photosystem II

light

light harvesting array

Rather conusingly, Photosystem II, rather than Photosystem I, is where the light- dependent reactions o photosynthesis begin. The electron acceptor or this photosystem is called plastoquinone. It collects two excited electrons rom Photosystem II and then moves away to another position in the membrane. Plastoquinone is hydrophobic, so although it is not in a xed position, it remains within the membrane.

reaction centre

primary acceptor e-

plastoquinone Absorption o two photons o light causes the production

transfer of electrons pigment molecules

chlorophyll molecules that transfer electrons

Figure 3 Diagram showing the relationship between the light-harvesting array, the reaction centre and plastoquinone

o one reduced plastoquinone, with one o the chlorophylls at the reaction centre having lost two electrons to a plastoquinone molecule. Photosystem II can repeat this process, to produce a second reduced plastoquinone, so the chlorophyll at the reaction centre has lost our electrons and two plastoquinone molecules have been reduced.

Photolysis Photolysis of water generates electrons for use in the light-dependent reactions. O nce the plastoquinone becomes reduced, the chlorophyll in the reaction centre is then a powerul oxidizing agent and causes the water molecules nearest to it to split and give up electrons, to replace those that it has lost: 2 H 2 O  O 2 + 4H + + 4e The splitting o water, called photolysis, is how oxygen is generated in photosynthesis. O xygen is a waste product and diuses away. The useul product o Photosystem II is the reduced plastoquinone, which not only carries a pair o electrons, but also much o the energy absorbed rom light. This energy drives all the subsequent reactions o photosynthesis.

The electron transport chain Transfer of excited electrons occurs between carriers in thylakoid membranes. The production o ATP, using energy derived rom light is called photophosphorylation. It is carried out by the thylakoids. These are regular stacks o membranes, with very small fuid-lled spaces inside ( see gure 4) . The thylakoid membranes contain the ollowing structures:

Figure 4 Electron micrograph of thylakoids  75,000

392



Photosystem II



ATP synthase



a chain o electron carriers



Photosystem I.

Reduced plastoquinone is needed, carrying the pair o excited electrons rom the reaction centre o Photosystem II. Plastoquinone carries the electrons to the start o the chain o electron carriers.

8 . 3 ph O TO s yn Th e s i s

The proton gradient Excited electrons from Photosystem II are used to generate a proton gradient. O nce plastoquinone transers its electrons, the electrons are then passed rom carrier to carrier in this chain. As the electrons pass, energy is released, which is used to pump protons across the thylakoid membrane, into the space inside the thylakoids. A concentration gradient o protons develops across the thylakoid membrane, which is a store o potential energy. Photolysis, which takes place in the fuid inside the thylakoids, also contributes to the proton gradient.

stroma (low H + concentration)

Photosystem II

light

2 H+

cytochrome complex light

NADP+ reductase

Photosystem I Fd

NADP+ + H + NADPH

Pq H2O thylakoid space (high H + concentration)

1 2

O2 +2 H +

Pc

2 H+ to Calvin cycle

stroma (low H + concentration)

thylakoid membrane

ATP synthase ADP + P1

ATP H+

Figure 5

Chemiosmosis ATP synthase in thylakoids generates ATP using the proton gradient. The protons can travel back across the membrane, down the concentration gradient, by passing through the enzyme ATP synthase. The energy released by the passage o protons down their concentration gradient is used to make ATP rom AD P and inorganic phosphate. This method o producing ATP is strikingly similar to the process that occurs inside the mitochondrion and is given the same name: chemiosmosis. When the electrons reach the end o the chain o carriers they are passed to plastocyanin, a water-soluble electron acceptor in the fuid inside the thylakoids. Reduced plastocyanin is needed in the next stage o photosynthesis.

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M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L )

Data-based questions: Evidence for chemiosmosis One o the rst experiments to give evidence or ATP production by chemiosmosis was perormed in the summer o 1 966 by Andr Jagendor. Thylakoids were incubated or several hours in darkness, in acids with a pH ranging rom 3.8 to 5.2. The lower the pH o an acid, the higher its concentration o protons. During the incubation,

protons diused into the space inside the thylakoids, until the concentrations inside and outside were equal. The thylakoids were then transerred, still in darkness, into a solution o ADP and phosphate that was more alkaline. There was a brie burst o ATP production by the thylakoids. The graph shows the yield o ATP at three acid incubation pHs and a range o pHs o the ADP solution. 1

ATP production / mol

3.8

a) D escribe the relationship between pH o ADP solution and ATP yield, when acid incubation was at pH 3 .8. [2 ] b) Explain why the pH o the AD P solution aects the ATP yield. [2 ]

2

4.8

Explain the eect o changing the pH o acid incubation on the yield o ATP.

[2 ]

Explain why there was only a short burst o ATP production.

[2 ]

Explain the reason or perorming the experiment in darkness.

[2 ]

5.2

3 6.5

7.0 7.5 8.0 pH of ADP solution

8.5

Figure 6 Results of Jagendorf experiment

4

Reduction of NADP Excited electrons from Photosystem I are used to reduce NADP. The remaining parts o the light-dependent reactions involve Photosystem I. The useul product o these reactions is reduced NAD P, which is needed in the light- independent reactions o photosynthesis. Reduced NAD P has a similar role to reduced NAD in cell respiration: it carries a pair o electrons that can be used to carry out reduction reactions. uid in thylakoid H 2O 2H +

thylakoid membrane Photosystem II 2e -

1 2 O2

plastocyanin

uid outside thylakoid

plastoquinone

electron transport chain

Photosystem II

ferredoxin

NADP

Figure 7 Summary of the lightdependent reactions of photosynthesis

394

C hlorophyll molecules within Photosystem I absorb light energy and pass it to the special two chlorophyll molecules in the reaction centre. This raises an electron in one o the chlorophylls to a high energy level. As with Photosystem II, this is called photoactivation. The excited electron passes along a chain o carriers in Photosystem I, at the end o which it is passed to erredoxin, a protein in the fuid outside the thylakoid. Two molecules o reduced erredoxin are then used to reduce NAD P, to orm reduced NAD P. The electron that Photosystem I donated to the chain o electron carriers is replaced by an electron carried by plastocyanin. Photosystems I and II are thereore linked: electrons excited in Photosystem II are passed along the chain o carriers to plastocyanin, which transers them to Photosystem I. The electrons are re-excited with light energy and are eventually used to reduce NAD P. The supply o NAD P sometimes runs out. When this happens the electrons return to the electron transport chain that links the two photosystems, rather than being passed to NAD P. As the electrons fow

8 . 3 ph O TO s yn Th e s i s

back along the electron transport chain to Photosystem I, they cause pumping o protons, which allows ATP production. This process is cyclic photophosphorylation.

ribulose bisphosphate

Carbon fxation In the light-independent reactions a carboxylase catalyses the carboxylation of ribulose bisphosphate. C arbon dioxide is the carbon source or all organisms that carry out photosynthesis. The carbon xation reaction in which it is converted into another carbon compound is arguably the most important in all living organisms. In plants and algae it occurs in the stroma  the fuid that surrounds the thylakoids in the chloroplast. The product o this carbon xation reaction is a three- carbon compound: glycerate 3 - phosphate. As so oten occurs in biological research, the details o the reaction were a surprise when they were discovered. C arbon dioxide does not react with a two- carbon compound to produce glycerate 3 -phosphate. Instead, it reacts with a ve-carbon compound called ribulose bisphosphate ( RuB P) , to produce two molecules o glycerate 3 - phosphate. The enzyme that catalyses this reaction is called ribulose bisphosphate carboxylase, usually abbreviated to rubisco. The stroma contains large amounts o rubisco to maximize carbon xation.

The role o reduced NADP and ATP in the Calvin cycle

CO 2 rubisco 2 glycerate 3-phosphate 2ATP 2ADP + 2 phosphates

2(NADPH + H + ) 2NADP+ 2 triose phosphate

Figure 8 Summary o carbon fxation reactions

Glycerate 3-phosphate is reduced to triose phosphate using reduced NADP and ATP. RuB P is a 5 -carbon sugar derivative, but when it is converted to glycerate 3-phosphate by adding carbon and oxygen, the amount o hydrogen in relation to oxygen is reduced. In sugars and other carbohydrates, the ratio o hydrogen to oxygen is 2 :1 . Hydrogen has to be added to glycerate 3-phosphate by a reduction reaction to produce carbohydrate. This involves both ATP and reduced NADP, produced by the light-dependent reactions o photosynthesis. ATP provides the energy needed to perorm the reduction and reduced NAD P provides the hydrogen atoms. The product is a three-carbon sugar derivative, triose phosphate.

The ate o triose phosphate Triose phosphate is used to regenerate RuBP and produce carbohydrates. The rst carbohydrate produced by the light-independent reactions o photosynthesis is triose phosphate. Two triose phosphate molecules can be combined to orm hexose phosphate and hexose phosphate can be combined by condensation reactions to orm starch. However, i all o the triose phosphate produced by photosynthesis was converted to hexose or starch, the supplies o RuB P in the chloroplast would soon be used up. Some triose phosphate in the chloroplast thereore has to be

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To what extent is it acceptable to adjust empirical evidence to conform to theoretical prediction? One o the most amous experiments in the history o biology is that o the Flemish scientist Johannes Baptista van Helmont, published in 1648. It is regarded as the rst quantitative biology experiment and also changed our understanding o the growth o plants. At this time, plants were thought to be soil-eaters. To test this idea, van Helmont put 200 pounds (90 kg) o dry soil in large pot and in it planted a willow tree, which had a mass o 5 pounds (2.2 kg). He attempted to keep dust out o the pot by covering it with a perorated metal plate. He watered the tree with rainwater or distilled water over a period o ve years. When the willow was reweighed at the end o this time it had increased to 169 pounds (76 kg). Ater drying the soil rom the pot he ound that it had remained almost unchanged in mass, having lost only one eighth o a pound (about 50g). Removal o soil rom willow roots is very difcult as soil particles inevitably get stuck to the roots. van Helmont's masses or the soil beore and ater the ve-year period are thereore surprisingly close. Some have questioned whether van Helmont made his data t pre-decided conclusions.

used to regenerate RuB P. This process is a conversion o 3 - carbon sugars into 5 - carbon sugars and it cannot be done in a single step. Instead a series o reactions take place. As RuB P is both consumed and produced in the light- independent reactions o photosynthesis, these reactions orm a cycle. It is called the C alvin cycle to honour Melvin C alvin, who was given the Nobel Prize or C hemistry in 1 9 6 1 or his work in elucidating this process. For the C alvin cycle to continue indefnitely, as much RuB P must be produced as consumed. I three RUB P molecules are used, six triose phosphates are produced. Five o these are needed to regenerate the three RuB P molecules. This leaves j ust one triose phosphate or conversion to hexose, starch or other products o photosynthesis. To produce one molecule o glucose or example, six turns o the C alvin cycle are needed, each o which contributes one o the fxed carbon atoms in the glucose.

Data-based questions: The eect o light and dark on carbon dioxide fxation One o the pioneers o photosynthesis research was James B assham. The results o one o his experiments are shown in fgure 9. C oncentrations o ribulose bisphosphate and glycerate 3 -phosphate were monitored in a culture o cells o the alga, Scenedesmus. The algae were kept in bright light and then in the dark. light relative concentration

TOK

396

glycerate 3 - phosphate ribulose bisphosphate

1 What evidence against the hypothesis that plants are soil eaters does van Helmont's experiment provide? 2 van Helmont concluded rom his results that, 164 pounds o Wood, Barks, and Roots, arose out o water only. (164 pounds is 73 kg.) This was not a new idea - 2000 years earlier the Greek philosopher Thales had stated that all matter arose rom water. To what extent was van Helmont's conclusion correct?

dark

0

100

200

300

400

500

600

700

Figure 9 Results of Bassham experiment 1 2

3

C ompare the eects o the dark period on the concentrations o ribulose bisphosphate and glycerate 3 - phosphate.

[2 ]

Explain the change that took place in the 2 5 seconds ater the start o darkness, to the concentration o: a) glycerate 3 - phosphate [3 ] b) ribulose bisphosphate.

[1 ]

Predict what the eect would be o turning the light back on ater the period o darkness.

[2 ]

8 . 3 ph O TO s yn Th e s i s

4

Predict the eect o reducing the carbon dioxide concentration rom 1 . 0% to 0. 003 % , instead o changing rom light to darkness: a) on glycerate 3 - phosphate concentration [2 ] b) on ribulose bisphosphate concentration.

5 triose phosphate

[2 ]

3ATP 3(ADP + phosphate)

RuBP regeneration

3 ribulose bisphosphate

Ribulose bisphosphate is reormed using ATP. In the last phase o the C alvin cycle, a series o enzyme- catalysed reactions convert triose phosphate molecules into RuB P. Ater the RuB P is regenerated, it can serve to fx C O 2 and begin the cycle again. Figure 1 0 summarizes the regeneration process.

Figure 10 Summary of RuBP regeneration

Calvins lollipop apparatus Developments in scientifc research ollow improvements in apparatus: sources o 14 C and autoradiography enabled Calvin to elucidate the pathways o carbon fxation.

1

E xplain the evidence rom the graph that convinced C alvin that glycerate 3 - phosphate is the frst product o carbon dioxide fxation. [4]

2

E xplain the evidence rom the graph or the conversion o glycerate 3 - phosphate to triose phosphate and other sugar phosphates. [4]

3

Using the data in the graph, estimate how rapidly carbon dioxide can diuse into cells and be converted with RuB P to glycerate 3 - phosphate.

to pump circulating air and CO 2

funnel for adding algae

syringe for injecting H 14CO 3 -

light

algal suspension in nutrient medium

solenoid control valve for rapid sampling

hot methanol to kill samples rapidly

Figure 11 Calvins lollipop apparatus

% radioactivity

Sometimes progress in biological research suddenly becomes possible because o other discoveries. Martin Kamen and Samuel Ruben discovered 1 4C in 1 945 . The hal-lie o this radioactive isotope o carbon makes it ideal or use in tracing the pathways o photosynthesis. Figure 1 1 shows apparatus used by Melvin C alvin and his team. At the start o their experiment, they replaced the 1 2 C O 2 supplied to algae with 1 4C O 2 . They took samples o the algae at very short time intervals and ound what carbon compounds in the algae contained radioactive 1 4C . The results are shown in fgure 1 2 . The amount o radioactivity o each carbon compound is shown as a percentage o the total amount o radioactivity.

70 60 50 40 30 20 10 0 4 8 12 16 0 seconds

1

2 3 minutes

4

time after introducing 14C glycerate-3-phosphate

[2 ]

malate and aspartate

triose phosphate and other sugar phosphates alanine

Figure 12 Graph showing Calvin's results

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M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L )

Chloroplast structure and function The structure of the chloroplast is adapted to its function in photosynthesis. C hloroplasts are quite variable in structure but share certain eatures: 

a double membrane orming the outer chlorop last envelop e



an extensive system o internal membranes called thylakoids, which are an intense green colour



small fuid- lled spaces inside the thylakoids



a colourless fuid around the thylakoids called stroma that contains many dierent enzymes.



In most chloroplasts there are stacks o thylakoids, called grana. I a chloroplast has been photosynthesizing rapidly then there may be starch grains or lip id drop lets in the stroma.

thylakoid one thylakoid

granum  a stack of thylakoids granum  a stack of thylakoids

Figure 13 Electron micrograph of pea chloroplast

Figure 14 Drawing of part of the pea chloroplast to show the arrangement of the thylakoid membranes

Data-based questions: Photosynthesis in Zea mays Zea mays uses a modied version o photosynthesis, reerred to as C 4 physiology. The processes o photolysis and the C alvin cycle are separated by being carried out in dierent types o chloroplast. O ne o the advantages is that carbon dioxide can be xed even when it is at very low concentrations, so the stomata do not need to be opened as widely as in plants that do not have C 4 physiology. This helps to conserve water in the plant, so is useul in dry habitats. The electron micrograph ( gure 1 5 ) shows the two types o chloroplast in the leaves o Zea mays. O ne type ( C hloroplast X) is rom mesophyll tissue and the other ( C hloroplast Y) is rom the sheath o cells around the vascular tissue that transports materials to and rom the lea.

398

8 . 3 ph O TO s yn Th e s i s

Chloroplast X

Chloroplast Y

Figure 15 Two types of chloroplast in Zea mays leaf 1

D raw a small portion of each chloroplast to show its structure.

[5 ]

2

C ompare the structure of the two types of chloroplast.

[4]

3

D educe, with a reason: a) which type of chloroplast has the greater light absorption capacity

[2 ]

b) which is the only type of chloroplast to carry out the reactions of the Calvin cycle [2] c) which is the only type of chloroplast to produce oxygen.

[2 ]

Diagram showing chloroplast structure function relationship Annotation of a diagram to indicate the adaptations of a chloroplast to its function. There is a clear relationship between the structure of the chloroplast and its function. 1

C hlorop lasts absorb light. Pigment molecules, arranged in photosystems in the thylakoid membranes, carry out light absorption. The large area of thylakoid membranes ensures that the chloroplast has a large light- absorbing capacity. The thylakoids are often arranged in stacks called grana. Leaves that are brightly illuminated typically have chloroplasts with deep grana, which allow more light to be absorbed.

2

C hlorop lasts p roduce ATP by p hotop hosp horylation. A proton gradient is needed. This develops between the inside and

399

8

M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L )

outside o the thylakoids. The volume o fuid inside the thylakoids is very small, so when protons are pumped in, a proton gradient develops ater relatively ew photons o light have been absorbed, allowing ATP synthesis to begin. 3

Chloroplasts carry out the many chemical reactions of the Calvin cycle. The stroma is a compartment o the plant cell in which the enzymes needed or the Calvin cycle are kept together with their substrates and products. This concentration o enzymes and substrates speeds up the whole Calvin cycle. ATP and reduced NADP, needed or the Calvin cycle, are easily available because the thylakoids, where they are produced, are distributed throughout the stroma. thylakoid membranes stroma containing 70S ribsomes and

granum

naked DNA

inner

outer

membrane

membrane

chloroplast envelope

400

starch grain lipid droplet

QuesTiOn s

Questions 1

a) S tate the meaning o the term metabolic pathway. [2 ] Glucose phosphate ( G6P) is converted to pyruvate in one o the metabolic pathways o cell respiration. This process happens whether oxygen is available or not.

percentage

Figure 1 6 shows the concentrations o the intermediates o this pathway in rat heart tissue. The concentrations are shown as a percentage o the concentrations in the heart when it has been starved o oxygen.

intensity. The lamp was controlled by an electronic timer, which switched it o at night. A light meter was placed against the side o the ermenter, near the base, to measure the intensity o light passing through the liquid in the ermenter. The maximum reading it could give was 1 , 2 00 lux. At the start o the experiment, a small quantity o Chlorella, a type o algae, was added to the fuid in the ermenter. Figure 1 7 shows the light intensity measured over the 45 days o the experiment. a) The light intensity ollowed a similar pattern, every day rom D ay 1 2 onwards.

400 350 300 250 200 150 100 50

(i)

Outline the daily changes in light intensity over a typical day ater Day 1 2. [2]

( ii) Explain these daily changes in light intensity. [2 ]

G6P F6P

FDP DHAP G3P 3PGA 2PGA PEP metabolic intermediate

PYR

b) Each day there is a maximum light intensity. O utline the trends in maximum light intensity. ( i)

 Figure 16

rom D ay 1 to D ay 1 2 [1 ]

( ii) rom D ay 1 3 to D ay 3 8 [2 ]

( i)

increased in concentration most [1 ]

( ii) decreased in concentration most [1 ] ( iii) did not change in concentration. [1 ] c) ( i)

The concentrations shown in Figure 1 6 suggest that the rate o this metabolic pathway has been greater than is needed by the heart cells. E xplain how the data in the bar chart shows this. [2 ]

( ii) B ecause rate o the pathway has been greater than necessary, the enzyme catalysing one o the reactions in the pathway has been inhibited. D educe which reaction this enzyme catalyses, giving reasons or your answer. [3 ]

( iii) rom D ay 3 9 to D ay 45 . [2 ] c) Explain why the light intensity when the light was switched on was lower at the end o the experiment than at the start. [3 ] d) S uggest reasons or the trend in maximum daily light intensity between D ay 3 9 and D ay 45 . [3 ]

1000

light intensity/lux

b) C ompared with concentrations during oxygen starvation, state which metabolic intermediate:

800 600 400 200

2

Water with mineral nutrients dissolved in it was sterilized and then placed in a 2 dm 3 ermenter. The temperature was kept at 2 5 C . The ermenter was kept in natural sunlight, but a lamp was also used to increase the light

0 0

10

20 time/days

30

40

 Figure 17

401

8

M e Tab O li s M , C e ll R e s p i R aT i O n an D p h O TO s yn T h e s i s ( ah l ) 3

At the start o glycolysis, glucose is phosphorylated to produce glucose 6- phosphate, which is converted into ructose 6- phosphate. A second phosphorylation reaction is then carried out, in which ructose 6- phosphate is converted into ructose 1 , 6- bisphosphate. This reaction is catalysed by the enzyme phosphoructokinase. B iochemists measured the enzyme activity o phosphoructokinase ( the rate at which it catalysed the reaction) at dierent concentrations o ructose 6-phosphate. The enzyme activity was measured with a low concentration o ATP and a high concentration o ATP in the reaction mixture. The graph below shows the results. low ATP concentration

Dt

RQ

Lipid

0.71

Carbohydrate

1.00

Protein

0.74

Source: Walsberg and Wolf, Journal of Experimental Biology, (1995) , 198, pages 213219. Reproduced by permission of The Company of Biologists Ltd. In an experiment to assess RQ values or house sparrows, the birds were ed a diet o pure mealworms ( beetle larvae) or millet ( a type o grain) . The graph below shows the RQ values o a house sparrow ed on a high carbohydrate diet ( millet) and a high lipid diet ( mealworms) .

enzyme activity

1.0 high ATP concentration respiratory quotient

0.9

fructose 6-phosphate concentration

millet mealworms

0.8

0.7

a) ( i)

Using only the data in the above graph, outline the eect o increasing ructose 6- phosphate concentration on the activity o phosphoructokinase, at a low ATP concentration. [2 ]

( ii) E xplain how increases in ructose 6- phosphate concentration aect the activity o the enzyme. [2 ] b) ( i)

O utline the eect o increasing the ATP concentration on the activity o phosphoructokinase. [2 ]

( ii) S uggest an advantage to living organisms o the eect o ATP on phosphoructokinase. 4

402

[1 ]

The respiratory quotient ( RQ) is a measure o the metabolic activity o an animal. It is the ratio o C O 2 produced to O 2 consumed. In general, the lower the RQ value the higher the energy yield. The RQ is dependent on the diet consumed by the animal. The ollowing table lists the typical RQ values or specifed diets.

0.6 0

1

2

3 4 5 time after feeding/h

6

Source: Walsberg and Wolf, Journal of Experimental Biology, (1995) , 198, pages 213219. Reproduced by permission of The Company of Biologists Ltd. a) C ompare the RQ values or millet and mealworms between 1 hour and 6 hours ater eeding. [2 ] The expected RQ value or house sparrows metabolizing millet is 0.93 . The expected value when metabolizing mealworms is 0. 75 . b) E xplain why the expected RQ values or millet and mealworms are dierent. [2 ] c) S uggest reasons or ( i)

the high initial RQ values or house sparrows ed on millet; [1 ]

( ii) the rapid all in RQ values or house sparrows ed on millet. [1 ]

7

9C E LPLL BA NI OTLBOIGOYL O G Y ( A H L ) Introduction

Plants are highly diverse in structure and physiology. They act as the producers in almost all terrestrial ecosystems. S tructure and unction are correlated in the xylem and phloem o plants.

Plants have sophisticated methods o adapting their growth to environmental conditions. Reproduction in fowering plants is infuenced by both the biotic and abiotic environment.

9.1 Transport in the xylem of plants Understanding  Transpiration is the inevitable consequence o  

 

gas exchange in the lea. Plants transport water rom the roots to the leaves to replace losses rom transpiration. The cohesive property o water and the structure o the xylem vessels allow transport under tension. The adhesive property o water and evaporation generate tension orces in lea cell walls. Active uptake o mineral ions in the roots causes absorption o water by osmosis.

Applications  Adaptations o plants in deserts and in saline

soils or water conservation.  Models o water transport in xylem using simple apparatus including blotting or lter paper, porous pots and capillary tubing.

Skills  Drawing the structure o primary xylem vessels

in sections o stems based on microscope images.  Measurement o transpiration rates using potometers. (Practical 7)  Design o an experiment to test hypotheses about the efect o temperature or humidity on transpiration rates.

Nature of science  Use models as representations o the real world: mechanisms involved in water transport in the xylem

can be investigated using apparatus and materials that show similarities in structure to plant tissues.

403

9

PL AN T B I O LO G Y ( AH L )

Transpiration Transpiration is the inevitable consequence o gas exchange in the lea. Plant leaves are the primary organ of photosynthesis. Photosynthesis involves the synthesis of carbohydrates using light energy. C arbon dioxide is used as a raw material. O xygen is produced as a waste product. Exchange of these two gases must take place to sustain photosynthesis. Absorption of carbon dioxide is essential for photosynthesis and the waxy cuticle has very low permeability to it, so pores through the epidermis are needed. These pores are called stomata. Figure 1 shows that the problem for plants is that if stomata allow carbon dioxide to be absorbed, they will usually also allow water vapour to escape. This is an intractable problem for plants and other organisms: having gas exchange without water loss. The loss of water vapour from the leaves and stems of plants is called transpiration. water CO 2 O2  Figure 1

Plants minimize water losses through stomata using guard cells. These are the cells that are found in pairs, one on either side of a stoma. Guard cells control the aperture of the stoma and can adj ust from wide open to fully closed. S tomata are found in nearly all groups of land plants for at least part of the plants life cycle. The exception is a group called the liverworts.

Modelling water transport Models o water transport in xylem using simple apparatus including blotting or flter paper, porous pots and capillary tubing. porous pot

plant

water

 Figure 2

Porous pots can be used to model evaporation rom leaves. Water flls pores within the pot demonstrating adhesion to the clay molecules within the pot. As the water is drawn into the pot, cohesion causes water molecules to be drawn up the glass tubing

404

 Figure 3

Capillary tubes dipped into water with dye and mercury. Unlike water, there is no adhesion o mercury to the glass nor is there cohesion between mercury atoms, so the mercury does not climb the glass

9 .1 Tr an s po r T i n Th e x yle m o f pl an Ts

 Figure 4 The ability

of adhesive forces to result in the movement of water is demonstrated in this image. A folded paper towel with one end immersed in water will transport water into an empty container by capillary action

Using a potometer Measurement o transpiration rates using potometers. (Practical 7) Mechanisms involved in water transport in the xylem can be investigated using apparatus and materials that show similarities in structure to plant tissues. Figure 5 shows a potometer. This is a device used to measure water uptake in plants. The apparatus consists o a leay shoot in a tube ( right) , a reservoir ( let o shoot) , and a graduated capillary tube ( horizontal) . A bubble in the capillary tube marks the zero point. As the plant takes up water through its roots, the bubble will move along the capillary tube. The progress o the bubble is being timed here, along with noting the distance travelled. The tap below the reservoir allows the bubble to be reset to carry out new measurements.

 Figure 5

Efect o humidity on transpiration Design o an experiment to test hypotheses about the efect o temperature or humidity on transpiration rates. The rate o transpiration is difcult to measure directly. Instead, the rate o water uptake is usually measured, using a potometer. Figure 6 shows one type o potometer. To design an investigation you will need to discuss the ollowing questions. 1

How will you measure the rate o transpiration in your investigation?

405

9

PL AN T B I O LO G Y ( AH L )

2

What biotic or abiotic actor will you investigate?

3

How will you vary the level o this actor?

4

How many results do you need, at each level o the actor that you are varying?

5

How will you keep other actors constant, so that they do not aect the rate o transpiration?

fresh shoot, cut under water and transferred to apparatus under water to avoid introducing air bubbles reservoir from which water can be let into thecapillary tube, pushing the air bubble back to the start of the tube tap

air tight seal

capillary tube  Figure 7

Longitudinal section through a rhubarb stem, Rheum rhaponticum. Cut xylem vessels are coloured brown. Xylem vessels are reinorced and strengthened with spiral bands o lignin. Spiral bands allow xylem vessels to elongate and grow lengthwise

scale calibrated in mm 3 air bubble moves along tube as water is absorbed by shoot  Figure 6 Diagram

o a potometer

Xylem structure helps withstand low pressure The cohesive property of water and the structure of the xylem vessels allow transport under tension. The structure o xylem vessels allows them to transport water inside plants very efciently. Xylem vessels are long continuous tubes. Their walls are thickened, and the thickenings are impregnated with a polymer called lignin. This strengthens the walls, so that they can withstand very low pressures without collapsing.

 Figure 8 Light micrograph

o a vertical section o the primary wood or xylem o a tree showing wood vessels with lignifed supporting thickenings

406

Xylem vessels are ormed rom fles o cells, arranged end-to- end. In owering plants, the cell wall material in some areas between adj acent cells in the fle is largely removed and the plasma membranes and contents o the cells break down ( see fgures 7 and 8) . When mature, these xylem cells are nonliving, so the ow o water along them must be a passive process. The pressure inside xylem vessels is usually much lower than atmospheric pressure but the rigid structure prevents the xylem vessels rom collapsing.

9 .1 Tr an s po r T i n Th e x yle m o f pl an Ts Water molecules are polar and the partial negative charge on the oxygen atom in one water molecule attracts the hydrogen atom in a neighbouring water molecule. This is termed cohesion. Water is also attracted to hydrophilic parts o the cell walls o xylem. This is termed adhesion. As a result o the connections between the molecules, water can be pulled up rom the xylem in a continuous stream.

Dt-bd qut: The Renner experiment Figure 9 shows the results o an experiment by the German plant physiologist O tto Renner in 1 91 2 . A transpiring woody shoot was placed in a potometer and the rate o water uptake was measured. A clamp was attached to the stem to restrict the fow o water up to the leaves. Later on, the top o the shoot, with all o its leaves, was removed. A vacuum pump was then attached to the top o the shoot.

and the rate caused by the leaves immediately beore the shoot top was cut o. [2 ] 4

The water in the potometer was at atmospheric pressure. The vacuum pump generated a pressure o zero. D iscuss what the results o the experiment showed about the pressures generated in the xylem by the leaves o the shoot. [2 ]

1

D escribe the eect o clamping the stem on the rate o water uptake. [3 ]

2

E xplain the eect o cutting o the top o the shoot on the rate o water uptake. [3 ]

3

C alculate the dierence between the rate o water uptake caused by the vacuum pump

water uptake / cm 3 h 1

stem clamped

Questions

20

shoot removed

10 9 8 7 6 5 4 3 2 1

vacuum pump

1

2

3

4

time (hrs)  Figure 9

Results of the Renner experiment

Tension in leaf cell walls maintains the transpiration stream The adhesive property of water and evaporation generate tension forces in leaf cell walls. When water evaporates rom the surace o the wall in a lea, adhesion causes water to be drawn through the cell wall rom the nearest available supply to replace the water lost by evaporation. The nearest available supply is the xylem vessels in the veins o the lea. Even i the pressure in the xylem is already low, the orce o adhesion between water and cell walls in the lea is strong enough to suck water out o the xylem, urther reducing its pressure. The low pressure generates a pulling orce that is transmitted though the water in the xylem vessels down the stem and to the ends o the xylem in the roots. This is called transpiration- pull and is strong enough to move water upwards, against the orce o gravity, to the top o the tallest tree. For the plant, it is a passive process, with all the energy needed or it coming rom the thermal energy ( heat) that causes transpiration. The pulling o water upwards in xylem vessels depends on the cohesion that exists between water molecules. Many liquids

407

9

PL AN T B I O LO G Y ( AH L ) would be unable to resist the very low pressures in xylem vessels and the column o liquid would break. This is called cavitation and it does occasionally happen even with water, but it is unusual. E ven though water is a liquid, it can transmit pulling orces in the same way as a solid length o rope does.

Active transport of minerals in the roots Active uptake of mineral ions in the roots causes absorption of water by osmosis. Water is absorbed into root cells by osmosis. This happens because the solute concentration inside the root cells is greater than that in the water in the soil. Most o the solutes in both the root cells and the soil are mineral ions. The concentrations o mineral ions in the root can be 1 00 or more times higher than those in the soil. These concentration gradients are established by active transport, using protein pumps in the plasma membranes o root cells. There are separate pumps or each type o ion that the plant requires. Mineral ions can only be absorbed by active transport i they make contact with an appropriate pump protein. This can occur by diusion, or by mass ow when water carrying the ions drains through the soil. Some ions move through the soil very slowly because the ions bind to the surace o soil particles. To overcome this problem, certain plants have developed a relationship with a ungus. The ungus grows on the surace o the roots and sometimes even into the cells o the root. The thread-like hyphae o the ungus grow out into the soil and absorb mineral ions such as phosphate rom the surace o soil particles. These ions are supplied to the roots, allowing the plant to grow successully in mineral-defcient soils. This relationship is ound in many trees, in members o the heather

Data-based questions: Fungal hyphae and mineral ion absorption Figure 1 0 shows the results o an experiment in which seedlings o Sitka spruce, Picea sitchensis, were grown or 6 months in sterilized soil either with or without ungi added: C was the control with no ungi added. The species o ungi added were:

b) E xplain the eects o the ungi on the growth o tree seedlings. [2 ] 2

408

a)

S tate the relationship between root growth and shoot growth in the tree seedlings.

[1 ]

shoot dry mass (g)

D iscuss the eects o the fve species o ungi on the growth o the roots and shoots o the tree seedlings. [4]

root dry mass (g)

a)

[1 ]

c) Using the data in Figure 1 0, deduce whether the eects o closely related ungi on tree growth are the same. [2 ] 0.5

I = Laccaria laccata; II = Laccaria ameythestea; III = Thelophora terrestris rom a tree nursery; IV = Thelophora terrestris rom a orest; V = Paxillus involutus; VI = Pisolithus tinctorius. 1

b) Suggest a reason or the relationship.

0.4 0.3 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5

 Figure 10

C

I

II

III

V IV VI

Results of Sitka spruce experiment

9 .1 Tr an s po r T i n Th e x yle m o f pl an Ts

amily and in orchids. Most, but not all, o these plants supply sugars and other nutrients to the ungus, so both the ungus and the plant beneft. This is an example o a mutualistic relationship.

Replacing losses from transpiration Plants transport water from roots to leaves to replace losses from transpiration. The movement o water rom roots to leaves is summarized in fgure 1 1 . Water leaving through stomata by transpiration is replaced by water rom xylem. Water in the xylem climbs the stem through the pull o transpiration combined with the orces o adhesion and cohesion. Water moves rom soil into roots by osmosis due to the active transport o minerals into the roots. Once the water is in the root it travels to the xylem through cell walls (the apoplast pathway) and through cytoplasm (the symplast pathway) .

water from xylem

1 cohesion 2 adhesion 1 2 water leaving through stomata

xylem cell

water molecule

root hair

epidermal cell cytoplasm

soil particle

apoplastic movement Casparian strip

water moves from soil into roots

root hair absorbs water from the soil

symplastic movement

xylem vessel and tracheids

 Figure 11

Adaptations for water conservation Adaptation of plants in deserts and in saline soils for water conservation. Xerophytes are plants adapted to growing in deserts and other dry habitats. There are various strategies that plants can use to survive in these habitats, including increasing the rate o water

uptake rom the soil and reducing the rate o water loss by transpiration. Some xerophytes are ephemeral, with a very short lie cycle that is completed in the brie period when water is

409

9

PL AN T B I O LO G Y ( AH L )

available ater rainall. They then remain dormant as embryos inside seeds until the next rains, sometimes years later. Other plants are perennial and rely on storage o water in specialized leaves, stems or roots. Most cacti are xerophytes, with leaves that are so reduced in size that they usually only consist o spines. The stems contain water storage tissue and become swollen ater rainall. Pleats allow the stem to expand and contract in volume rapidly. The epidermis o cactus stems has a thick waxy cuticle and unlike most plant stems there are stomata, though they are spaced more widely than in leaves. The stomata usually open at night rather than in the day, when it is much cooler and transpiration occurs more slowly. C arbon dioxide is absorbed at night and stored in the orm o a our-carbon compound, malic acid. C arbon dioxide is released rom the malic acid during the day, allowing photosynthesis even with the stomata closed. This is called C rassulacean acid metabolism. Plants such as cacti that use this system are called C AM plants. C 4 physiology also helps to reduce transpiration.

in these xerophytes are oten very similar to those o cacti. Some Arican species o Euphorbia or example, are difcult to distinguish rom cacti until they produce owers. Marram Grass (Ammophila arenaria) is a xerophyte, i.e. it is a plant adapted or dry conditions. It has a rolled lea. This creates a localized environment o water vapour which helps to prevent losses o water. The stomata sit in small pits within the curls o the structure, which make them less likely to open and to lose water. The olded leaves have hairs on the inside to slow or stop air movement, much like many other xerophytes. This slowing o air movement once again reduces the amount o water vapour being lost.

reduced leaf spine swollen stem

Gymnocalycium baldianum (cactus) viewed from above

 Figure 13

Saline soils are those that contain high concentrations o salts. Plants that live in saline soils are called halophytes. Halophytes have several adaptations or water conservation: 

the leaves are reduced to small scaly structures or spines



the leaves are shed when water is scarce and the stem becomes green and takes over the unction o photosynthesis when the leaves are absent



water storage structures develop in the leaves



they have a thick cuticle and a multiple layered epidermis



they have sunken stomata



they have long roots, which go in search o water



they have structures or removing salt build-up.

10 mm

E uphorbia obesa viewed from above swollen stem

5 mm  Figure 12

Xerophytes

C acti are native plants o North and South America. Xerophytes in other parts o the world belong to dierent plant amilies. The adaptations

410

9 .1 Tr an s po r T i n Th e x yle m o f pl an Ts

Drawing xylem vessels Drawing the structure of primary xylem vessels in sections of stems based on microscope images. Primary xylem vessels are visible in cross sections o young stems such as in young Helianthus. Figure 1 6 shows a longitudinal section through a stem illustrating the structure o xylem. Primary xylem has a thin primary wall that is unlignifed and reely permeable, plus lignifed secondary thickening o the epidermis wall that is usually annular or helical. The thickening cortex allows the xylem vessel to continue growing in length because the rings o annular thickening can move pith urther apart or helical thickening can be stretched so the pitch o the helix is greater.

xylem vascular cambium bundle phloem

O nce extension growth o a root or stem is complete the plant produces secondary xylem which is much more extensively lignifed. S econdary thickening o its cell wall provides more strength but does not allow growth in length.  Figure 14

thickenings of xylem vessel wall impregnated with lignin

 Figure 15 Light micrograph o a

section through a young stem rom a sunfower (Helianthus annuus) , showing one o the many vascular bundles. The vascular bundles have an outer layer o sclerenchyma tissue (crimson) . Next is the phloem (dark blue) with phloem tubes, parenchyma and companion cells. Then the xylem (red) and at the end o the xylem are patches o bres (red) . In between the phloem and xylem is the cambium (light blue)

continuous tubular structure  Figure 16 Structure o xylem

vessels

411

9

PL AN T B I O LO G Y ( AH L )

9.2 Transport in the phloem of plants Understanding  Plants transport organic compounds rom  

 

sources to sinks. Incompressibility o water allows transport by hydrostatic pressure gradients. Active transport is used to load organic compounds into phloem sieve tubes at the source. High concentrations o solutes in the phloem at the source lead to water uptake by osmosis. Raised hydrostatic pressure causes the contents o the phloem to fow toward sinks.

Applications  Structureunction relationships o phloem

sieve tubes.

Skills  Analysis o data rom experiments measuring

phloem transport rates using aphid stylets and radioactively-labelled carbon dioxide.  Identication o xylem and phloem in microscope images o stem and root.

Nature of science  Developments in scientic research ollow improvements in apparatus: experimental methods or

measuring phloem transport rates using aphid stylets and radioactively-labelled carbon dioxide were only possible when radioisotopes became available.

xylem

Translocation occurs from source to sink

phloem

water

source (leaf cell)

Plants transport organic compounds rom sources to sinks. sucrose

transpiration stream

companion cell sieve plate

sink (root cell)

412

Phloem transports organic compounds throughout the plant. The transport o organic solutes in a plant is called translocation. Phloem links parts o the plant that need a supply o sugars and other solutes such as amino acids to other parts that have a surplus. Table 1 classifes parts o the plant into sources (areas where sugars and amino acids are loaded into the phloem) and sinks (where the sugars and amino acids are unloaded and used) . Figure 2 shows the results o a simple experiment in which two rings o bark were removed rom an apple tree. The bark contains the phloem tissue. The eects on apple growth are clearly visible.

water

 Figure 1

Phloem tissue is ound throughout plants, including the stems, roots and leaves. Phloem is composed o sieve tubes. S ieve tubes are composed o columns o specialized cells called sieve tube cells. Individual sieve tube cells are separated by perorated walls called sieve plates. S ieve tube cells are closely associated with companion cells ( fgure 1 ) .

companion cell

S ometimes sinks turn into sources, or vice versa. For this reason the tubes in phloem must be able to transport biochemicals in either direction and, unlike the blood system o animals, there are no valves or central pump in phloem. However there are similarities between transport in phloem and blood vessels: in both systems a uid ows inside tubes because o pressure gradients. E nergy is needed to generate

9 . 2 Tr an s po r T i n Th e ph lo e m o f pl an Ts the pressures, so the ow o blood and the movement o phloem sap are both active processes.

suc

sk

Photosynthetic tissues: mature green leaves  green stems. Storage organs that are unloading their stores:  storage tissues in germinating seeds  tap roots or tubers at the start of the growth season. 

Roots that are growing or absorbing mineral ions using energy from cell respiration. Parts of the plant that are growing or developing food stores:  developing fruits  developing seeds  growing leaves  developing tap roots or tubers.

actvty 1

State which the sources and which the sinks are in this part of the apple tree. [2]

2

) Compare the sizes of the apples.

[2]

b) Explain the conclusions that can be drawn from the sizes of the apples. [4]

 Table 1

Phloem loading Active transport is used to load organic compounds into phloem sieve tubes at the source. The data in table 2 indicates that sucrose is transported in the phloem. S ucrose is the most prevalent solute in phloem sap. Sucrose is not as readily available or plant tissues to metabolize directly in respiration and thereore makes a good transport orm o carbohydrate as it will not be metabolized during transport. Plants dier in the mechanism by which they bring sugars into the phloem, a process called phloem loading. In some species, a signifcant amount travels through cell walls rom mesophyll cells to the cell walls o companion cells, and sometimes sieve cells, where a sucrose transport protein then actively transports the sugar in. This is reerred to as the apoplast route.

 Figure 2

Results of apple tree ringing experiment

In this case, a concentration gradient o sucrose is established by active transport. Figure 3 shows that this is achieved by a mechanism whereby H + ions are actively transported out o the companion cell rom surrounding tissues using ATP as an energy source. The build-up o H+ then ows down its concentration gradient through a co-transport protein. The energy released is used to carry sucrose into the companion cell-sieve tube complex. [outside cell] - high H + concentration H+ co-transporter

S sucrose gradient

proton gradient

proton pump

low H +

ATP

ADP+P H+

H+

S

[inside cell] - low H + concentration  Figure 3

Movement of sucrose (S) across a sieve tube membrane

413

9

PL AN T B I O LO G Y ( AH L ) In other species, much o the sucrose travels between cells through connections between cells called plasmodesmata ( singular plasmodesma) . This is reerred to as the symplast route. O nce the sucrose reaches the companion cell it is converted to an oligosaccharide to maintain the sucrose concentration gradient. mesophyll cell cell wall plasma membrane plasmodesmata

mesophyll cell

sieve-tube member companion (transfer) cell

phloem parenchyma cell

symplast route apoplast route

 Figure 4

Data-based questions: Carbohydrates in cyclamen 1

C hoose a suitable presentation ormat to display the data in table 2 , including the standard error values. You can use graphing sotware or you can draw graphs, tables, charts or diagrams by hand.

plant art

2

D escribe the trends in the data and suggest reasons or them based on your knowledge o photosynthesis, the structure o disaccharides and polysaccharides and the transport and storage o carbohydrates in plants.

mean carbohydrate content (g g1 fresh ass  standard error of ean) sucrose glucose fructose starch

Leaf blade Vascular bundle in the leaf stalk, consisting of xylem and phloem Tissue surrounding the vascular bundle in the leaf stalk Buds, roots and tubers (underground storage organs)

1,312  212 5,757 1,190

210 88 479 280

494 653 1,303 879

62 25 0.5 ) so we rej ect the alternate hypothesis and accept the null hypothesis.

Data-based questions: Using the chi-squared test Warren and Hutt ( 1 9 3 6 ) test- crossed a double heterozygote or two pairs o alleles in hens: one or the presence ( C r) or absence ( cr) o a crest and one or white ( I) or non- white ( i) plumage.

1

2

3 For their F 2 cross, there was a total o 75 4 ospring.

[4]

C alculate the expected values, assuming independent assortment.

[4]

D etermine the number o degrees o reedom.

[2 ]

4

Find the critical region or chi- squared at a signifcance level o 5 % . [2 ]

3 3 7 were non- white, non-crested;

5

C alculate chi-squared.

3 4 were non- white crested; and

6

S tate the two alternative hypotheses, H 0 and H 1 and evaluate them using the calculated value or chi-squared. [4]

3 3 7 were white, crested;

46 were white, non- crested.

454

C onstruct a contingency table o observed values.

[4]

1 0 . 3 G e n e P o o l s a n D s P e c i at i o n

10.3 G p d p Understnding  A gene pool consists o all the genes and their

   

diferent alleles, present in an interbreeding population. Evolution requires that allele requencies change with time in populations. Reproductive isolation o populations can be temporal, behavioural or geographic. Speciation due to divergence o isolated populations can be gradual. Speciation can occur abruptly.

applictions  Identiying examples o directional, stabilizing

and disruptive.  Speciation in the genus Allium by polyploidy.

Skills  Comparison o allele requencies o

geographically isolated populations.

Nture of science  Looking or patterns, trends and discrepancies:

patterns o chromosome number in some genera can be explained by speciation due to polyploidy.

Gene pools A gene pool consists o all the genes and their diferent alleles, present in an interbreeding population. The most commonly accepted denition o a species is the biological species concept. This denes a species as a group o potentially interbreeding populations, with a common gene pool that is reproductively isolated rom other species. S ome populations o the same species are geographically isolated so it is possible or multiple gene pools to exist or the same species. Individuals that reproduce contribute to the gene pool o the next generation. Genetic equilibrium exists when all members o a population have an equal chance o contributing to the uture gene pool.

allele frequency nd evolution Evolution requires that allele requencies change with time in populations. Evolution is dened as the cumulative change in the heritable characteristics o a population over time. Evolution can occur due to a number o reasons such as mutations introducing new alleles, selection pressures avouring the reproduction o some varieties over others and barriers to gene fow emerging between dierent populations. I a population is small, random events can also have a signicant eect on allele requency.

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Patterns of natural selection

activity In the cross depicted in gure 1, the requency o fower colour phenotypes in Japanese our oclocks is shown over three generations. The genotype CRCR yields red fowers, the genotype CW CW yields white fowers and because the alleles are co-dominant, the genotype CRCW yields pink fowers: 

in the rst generation, 50% o the population is red and 50% is white



in the second generation, 100% o the fowers are pink



in the third generation, there are 50% pink, 25% white and 25% red.

Show that the allele requency is 50% CR and 50% CW in each o the three generations. While phenotype requencies can change between generations, it is possible that allele requency is not changing. This population is not evolving because allele requencies are not changing. eggs

Identiying examples o directional, stabilizing and disruptive selection. Fitness o a genotype or phenotype is the likelihood that it will be ound in the next generation. S election pressures are environmental actors that act selectively on certain phenotypes resulting in natural selection. There are three patterns o natural selection: stabilizing selection, disruptive selection, and directional selection. In stabilizing selection, selection pressures act to remove extreme varieties. For example, average birth weights o human babies are avoured over low birth weight or high birth weight. A clutch is the number o eggs a emale lays in a particular reproductive event. S mall clutch sizes may mean that none o the ospring survive into the next generation. Very large clutch sizes may mean higher mortality as the parent cannot provide adequate nutrition and resources and may impact their own survival to the next season. This means that a medium clutch size is avoured. In disruptive natural selection, selection pressures act to remove intermediate varieties, avouring the extremes. One example is in the red crossbill Loxia curvirostra. The asymmetric lower part o the bill o red crossbills is an adaptation to extract seeds rom conier cones. An ancestor with a straight bill could have experienced disruptive selection, given that a lower part o the bill crossed to either side enables a more efcient exploitation o conier cones. B oth let over right and right over let individuals exist within the same population allowing them to access seeds rom cones hanging in dierent positions. In directional selection, the population changes as one extreme o a range o variation is better adapted.

CRCR sperm

C WC W

F1 generation all C R C W

 Figure 1

CRCR

CRCW

C R C W C WC W F2 generation 1:2:1

A change in phenotypic frequency between generations does not necessarily indicate that evolution is occurring

Dt-bsed questions: Stabilizing selection A population o bighorn sheep ( Ovis canadensis) on Ram Mountain in Alberta, C anada, has been monitored since the 1 970s. Hunters can buy a licence to shoot male bighorn sheep on the mountain. The large horns o this species are very attractive to hunters, who display them as hunting trophies. Most horn growth takes place between the second and the ourth year o lie in male bighorn sheep. They use their horns or fghting other males during the breeding season to try to deend groups o emales and then mate with them. Figure 2 shows the mean horn length o our- year- old males on Ram Mountain, between 1 975 and 2 002 . a) O utline the trend in horn length over the study period. b) Explain the concept o directional selection reerring to this example.

456

1 0 . 3 G e n e P o o l s a n D s P e c i at i o n

c) D iscuss the trade- o between short and long horns as an adaptation in this case.

mean horn length /cm

80 70 60 50 40 0 1970

1975

1980

1985 1990 year

1995

2000

2005

 Figure 2

Source: Reprinted with permission from Macmillan Publishers Ltd: David W. Coltman, Undesirable evolutionary consequences of trophy hunting, Nature, vol. 426, issue 6967, pp. 655658

D-bd qu Researchers carried out a study on 3 , 760 children born in a London hospital over a period o 1 2 years. D ata was collected on the childrens mass at birth and their mortality rate. The purpose o the study was to determine how natural selection acts on mass at birth. The chart in fgure 3 shows the requency o babies o each mass at birth. The line superimposed on the bar chart indicates the percentage mortality rate ( the children that did not survive or more than 4 weeks) .

a) Identiy the mode value or mass at birth. b) Identiy the optimum mass at birth or survival. c) O utline the relationship between mass at birth and mortality. d) Explain how this example illustrates the pattern o natural selection called stabilizing selection.

100

600

400

10

200

mortality/% (log scale)

frequency of mass at birth

800

0 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 mass at birth/kg  Figure 3

Source: W H Dowderswell, (1984) Evolution, A Modern Synthesis, page 101

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G e n e t i cs an d e vo l u t i o n ( aH l )

Data-based questions

a) D etermine the mean proximity to emales achieved by 3 5 3 9 cm males by: a. sneaking b. ghting. b) D etermine the size range that gets nearest to the emales by:

a. ghting b. sneaking. c) Identiy a size o male sh that never gets within 1 00 cm ( 1 m) by ollowing either strategy. d) Explain how this example illustrates the pattern o natural selection known as disruptive selection. 10 10 proximity to female(cm)

In coho salmon ( Oncorhynchus kisutch), some males reach maturity as much as 5 0% earlier and as small as 3 0% o the body size o other males in the population. S uccess in spawning ( breeding) depends on the male releasing sperm in close proximity to the egg-laying emale. S mall and large males employ dierent strategies to gain access to emales. The small-sized males called j acks are specialized at sneaking. The large- sized males are specialized at ghting and coercing emales to spawn. In contrast, intermediate- sized males are at a competitive disadvantage to both j acks and large males as they are more targeted or ghts which they lose and are more likely to be prevented rom sneaking. The graph in gure 4 shows the average proximity to emales achieved by the two strategies.

40

120

2

8

3

40

sneaking

12

5

ghting

28 3

3 200 2529

8

6

3

3539 4549 5559 male body size(cm)

6569

Nature, Vol. 313, No. 5997, pp. 4748, 3 January 1985  Figure 4 Efect o body size and courting strategy on proximity to emales

there are diferen caegories o reproducive isolaion Reproductive isolation of populations can be temporal, behavioural or geographic. S peciation is the ormation o a new species by the splitting o an existing population. Various barriers can isolate the gene pool o one population rom that o another population. S peciation may occur when this happens. I the isolation occurs because o geographic separation o populations, then the speciation is termed allopatric speciation. The cichlids ( sh) are one o the largest amilies o vertebrates. Most species o cichlids occur in three East Arican lakes, Lake Victoria, Lake Tanganyika and Lake Malawi. Annual fuctuations in water levels lead to isolation o populations that are then subj ect to dierent selection pressures. When the rainy season comes, the populations are recombined but can then be reproductively isolated. This can result in the ormation o new species. S ometimes isolation o gene pools occurs within the same geographic area. I speciation occurs, then the process is termed sympatric speciation. For example, isolation can be behavioural. When closely

458

1 0 . 3 G e n e P o o l s a n D s P e c i at i o n

D-bd qu: Lacewing songs S ongs are part o the process o mate selection in members o dierent species within the genus Chrysoperla ( lacewings) . Males and emales o the same species have precisely the same song and during the pre-mating period take turns making the songs. The oscillograph or two species o lacewings are shown in gure 5 . 1

2

3

4 2 0 -2 -4 0

5

10

15

20

25

30

(b) 4

C ompare the songs o the two species o lacewings.

[3 ]

Explain why dierences in mating songs might lead to speciation.

[3 ]

2 0

The ranges o the two species currently overlap. S uggest how dierences in song could have developed:

-2 -4 1

2

3

4

5

6

7

8

9

10 11 12

 Figure 5 Pre-mating songs of lacewings: (a)

a) by allopatric speciation b) by sympatric speciation.

(a)

[4]

C. lucasina and (b) C. mediterranea. C. lucasina ranges across most of Europe and eastward into western Asia, as well as across the northern quarter of Africa. C. mediterranea ranges across southern to central Europe and across the north African Mediterranean

related individuals dier in their courtship behaviour, they are oten only successul in attracting members o their own population. There can be temporal isolation o gene pools in the same area. Populations may mate or fower at dierent seasons or dierent times o day. For example, three tropical orchid species o the genus Dendrobium each fower or a single day. Flowering occurs in response to sudden drops in temperature in all three species. However, the lapse between the stimulus and fowering is 8 days in one species, 9 in another, and 1 0 to 1 1 in the third. Isolation o the gene pools occurs because, at the time the fowers o one species are open, those o the other species have already withered or have not yet matured.

diferent populations have iferent allele requencies Comparison of allele frequencies of geographically isolated populations. O nline databases such as the Allele Frequency D atabase ( AlFreD ) hosted by Yale University contains the requencies o a variety o human populations. Most human populations are no longer in geographic isolation because o the ease o travel and the signicant culture to culture contact that exists due to globalization. Nonetheless, patterns o variation do exist, especially when comparing remote island populations with mainland populations.

PanI is a gene in cod sh that codes or an integral membrane protein called pantophysin. Two alleles o the gene, PanI A and PanI B , code or versions o pantophysin that dier by our amino acids in one region o the protein. S amples o cod sh were collected rom 2 3  locations in the north Atlantic and were tested to nd the proportions o PanI A and PanI B alleles in each population. The results are shown in pie charts, numbered 1 2 3 , on the

459

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G e n e t i cs an d e vo l u t i o n ( aH l )

map in fgure 6 . The proportions o alleles in a population are called the allele requencies. The requency o an allele can vary rom 0 . 0 to 1 . 0 . The light grey sectors o the pie charts show the allele requency o PanI A and the black sectors show the allele requency o PanI B . 1

S tate the two populations with the highest PanIB allele requencies. [2 ]

2

D educe the allele requencies o a population in which hal o the cod fsh had the genotype PanIA PanI A, and hal had the genotype PanI A PanIB . [2 ]

3

Suggest two populations which are likely geographically isolated.

Suggest two possible reasons why the PanI B allele is more common in population 1 4 than population 2 1 . [2 ]

The coherence test o truth flters knowledge claims through existing theories that are well established. I the new knowledge claim does not ft, it is more likely to be greeted with skepticism. While polyploidy does occur in fsh and amphibians, it has always been unexpected in mammals. The sex determination system in mammals is very sensitive to extra sex chromosomes. Since the existence o a tetraploid mammal was frst claimed, the response has been skepticism. Though there is still no reasonable answer to the question o the T. barrerae origin.

460

Gradualism in speciation Speciation due to divergence o isolated populations can be gradual. There are two theories about the pace o evolutionary change. Gradualism, as depicted in fgure 7, is the idea that species slowly change through a series o intermediate orms. The axis label structure might reer to such things as beak length in birds or cranial capacity in hominids. frequency

What role does expectation have in determining the response of scientists to unexpected discoveries?

Source: R A J Case, et al., (2005) , Marine Ecology Progress Series, 201, pages 267278

structure

e

TOK

 Figure 6

tim

4

[2 ]

 Figure 7 In the gradualist framework, new species emerge from a long sequence of

intermediate forms Gradualism was, or a long time, the dominant ramework in palaeontology. However, it was conronted by gaps in the ossil record, i. e. an absence o intermediate orms. Gradualism predicted that evolution occurred by a long sequence o continuous intermediate orms. The absence o these intermediate orms was explained as imperections in the ossil record.

1 0 . 3 G e n e P o o l s a n D s P e c i at i o n

Punctuated equilibrium

gradualism

Speciation can occur abruptly. Punctuated equilibrium holds that long periods of relative stability in a species are punctuated by periods of rapid evolution. According to the theory of punctuated equilibrium, gaps in the fossil record might not be gaps at all, as there was no long sequence of intermediate forms. Events such as geographic isolation ( allopatric speciation) and the opening of new niches within a shared geographic range can lead to rapid speciation.

morphology

Rapid change is much more common in organisms with short generation times like prokaryotes and insects. Figure 8 compares the two models. The top model shows the gradualist model with slow change over geological time. The punctuated equilibrium model on the bottom consists of relatively rapid changes over a short period of time followed by periods of stability.

time punctuated equilibrium  Figure 8

Polyploidy can lead to speciation Looking for patterns, trends and discrepancies: patterns of chromosome number in some genera can be explained by speciation due to polyploidy. A polyploid organism is one that has more than two sets of homologous chromosomes. Polyploidy can result from hybridization events between different species. There are also polyploids whose chromosomes originate from the same ancestral species. This can occur when chromosomes duplicate in preparation for meiosis but then meiosis doesnt occur. The result is a diploid gamete that when fused with a haploid gamete produces a fertile offspring. In other words, the polyploid has now become reproductively isolated from the original population. The polyploid plant can selfpollinate or it can mate with other polyploid plants. Polyploidy can lead to sympatric speciation. Polyploidy occurs most commonly in plants, though it does also occur in less complex animals. The red viscacha (Tympanoctomys barrerae), a rodent from Argentina, has the highest chromosome number of any mammal and it has been hypothesized that this is the result of polyploidy. Its chromosome number is 1 02 and its cells are roughly twice normal size. Its closest living relative is Octomys mimax, the Andean viscacha-rat of the same family, whose 2n = 56. Researchers propose that an Octomys-like ancestor produced tetraploid offspring (i.e. 4n = 1 1 2) that were reproductively isolated from their parent species, eventually shedding some of the additional chromosomes gained at this doubling. Recent scholarship has tested this hypothesis but results are

ambiguous: probes detected only two copies of each autosome pair but it has also been observed that there are several genes that exist in four copies.

 Figure 9

Tympanoctomys barrerae

 Figure 10

Octomys mimax

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G e n e t i cs an d e vo l u t i o n ( aH l )

Polyploidy has occurred frequently in Allium Speciation in the genus Allium by polyploidy. Estimates of the number of species of angiosperms that have experienced a polyploidy event range between 5 0 to 70% .

Many species of Allium reproduce asexually and polyploidy may confer an advantage over diploidy under certain selection pressures.

The Allium genus includes onions, leeks, garlic and chives, and as such has played an important role in the food of multiple cultures. D etermining the number of species in the genus presents a challenge to taxonomists as polyploidy events are common within the genus. These result in a number of reproductively isolated but otherwise similar populations.

Wild onion (Allium canadense) is a native of North America. The diploid number for the plant is 1 4. However, there are variants such as A. c. ecristatum ( 2 n = 2 8) and A. c. lavendulae ( 2 n = 2 8) .

 Figure 11

2n=16

462

Metaphase chromosomes of Allium angulosum,

Allium angulosum and Allium oleraceum are two species that occur in Lithuania. O ne is a diploid plant with 1 6 chromosomes and one is a tetraploid plant with 3 2 chromosomes.

 Figure 12

2n=32

Metaphase chromosomes of Allium oleraceum,

Question s

Questions 1

Identiy the stages o meiosis shown in fgures 1 3 and 1 4.

( i) D educe the chromosome number o nuclei in their lea cells. Give two reasons or your answer. [3 ] ( ii) S uggest a disadvantage to S. arcticum and S. olafi o having more D NA than other bog mosses. [1 ] d) It is unusual or plants and animals to have an odd number o chromosomes in their nuclei. Explain how mosses can have odd numbers o chromosomes in their lea cells. [2 ]

Sphagnum M f nmbr f pc Dna/pg chrmm S. aongstroemii 0.47 19 S. arcticum 0.95 S. balticum 0.45 19 S. fmbriatum 0.48 19 S. olafi 0.92 S. teres 0.42 19 S. tundrae 0.44 19 S. warnstorfi 0.48 19

 Figure 13

 Table 1

3  Figure 14

2

The D NA content o cells can be estimated using a stain that binds specifcally to D NA. A narrow beam o light is then passed through a stained nucleus and the amount o light absorbed by the stain is measured, to give an estimate o the quantity o D NA. The results in table 1 are or lea cells in a species o bog moss ( Sphagnum) rom the S valbard islands. a) C ompare the D NA content o the bog mosses.

[2 ]

b) S uggest a reason or six o the species o bog moss on the S valbard islands all having the same number o chromosomes in their nuclei. [2 ] c) S. arcticum and S. olafi probably arose as new species when meiosis ailed to occur in one their ancestors.

The mechanisms o speciation in erns have been studied in temperate and tropical habitats. O ne group o three species rom the genus Polypodium lives in rocky areas in temperate orests in North America. Members o this group have similar morphology ( orm and structure) . Another group o our species rom the genus Pleopeltis live at dierent altitudes in tropical mountains in Mexico and C entral America. Members o this group are morphologically distinct. D ata rom the dierent species within each group was compared in order to study the mechanisms o speciation. Genetic identity was determined by comparing the similarities o certain proteins and genes in each species. Values between 0 and 1 were assigned to pairs o species to indicate the degree o similarity in genetic identity. A value o 1 would mean that all the genetic actors studied were identical between the species being compared.

463

10

G e n e t i cs an D e vo l u t i o n a) C ompare the geographic distributions o the two groups. [1 ]

c) S uggest how the process o speciation could have occurred in Polypodium.

b) ( i) Identiy, giving a reason, which group, Polypodium or Pleopeltis, is most genetically diverse. [1 ]

d) E xplain which o the two groups has most probably been genetically isolated or the longest period o time. [2 ]

( ii) Identiy the two species that are most similar genetically. [1 ]

4

[1 ]

In Zea mays, the allele or coloured seed (C) is dominant over the allele or colourless seed (c) . The allele or starchy endosperm (W) is dominant over the allele or waxy endosperm (w) . Pure breeding plants with coloured seeds and starchy endosperm were crossed with pure breeding plants with colourless seeds and waxy endosperm. a) S tate the genotype and the phenotype o the F 1 individuals produced as a result o this cross.

Po. sibiricum

genotype . .. ... ... ............... ........ ... .............. 0.435

Po. amorphum

0.608 0.338 Po. appalachianum

Pl. polyepis

phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2 ] b) The F 1 plants were crossed with plants that had the genotype c c w w. C alculate the expected ratio o phenotypes in the F 2 generation, assuming that there is independent assortment. E xpected ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3 ] The observed percentages o phenotypes in the F 2 generation are shown below.

Pl. crassinervata

Pl. conzattii Pl. mexicana Pl. polyepis 0.925

0.836

Pl. conzattii

Pl. mexicana

0.792

0.870

Pl. crassinervata  Figure 15 The approximate distribution

in North America o the three species o Polypodium (Po.) and a summary o genetic identity

Source: C Haufer, E Hooper and J Therrien, (2000) , Plant Species Biology, 15, pages 223236

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coloured starchy colourless starchy

3 7% 1 4%

coloured waxy colourless waxy

1 6% 33%

The observed results dier signifcantly rom the results expected on the basis o independent assortment. c) S tate the name o a statistical test that could be used to show that the observed and the expected results are signifcantly dierent. [1 ] d) Explain the reasons or the observed results o the cross diering signifcantly rom the expected results. [2 ]

11 A n I m A L p h Ys I O L O G Y ( A h L ) CE LL B I O LO GY Itroductio

Immunity is based on recognition of self and destruction of foreign material. The roles of the musculoskeletal system are movement, support and protection. All animals excrete nitrogenous

waste products and some animals also balance water and solute concentrations. S exual reproduction involves the development and fusion of haploid gametes.

11.1 Antibody production and vaccination Udertadig  Every organism has unique molecules on the           

surace o their cells. B lymphocytes are activated by T lymphocytes in mammals. Plasma cells secrete antibodies. Activated B cells multiply to orm a clone o plasma cells and memory cells. Antibodies aid the destruction o pathogens. Immunity depends upon the persistence o memory cells. Vaccines contain antigens that trigger immunity but do not cause the disease. Pathogens can be species-specifc although others can cross species barriers. White cells release histamine in response to allergens. Histamines cause allergic symptoms. Fusion o a tumour cell with an antibodyproducing plasma cell creates a hybridoma cell. Monoclonal antibodies are produced by hybridoma cells.

Alicatio  Antigens on the surace o red blood cells

stimulate antibody production in a person with a dierent blood group.  Smallpox was the frst inectious disease o humans to have been eradicated by vaccination.  Monoclonal antibodies to hCG are used in pregnancy test kits.

skill  Analysis o epidemiological data related to

vaccination programmes.

nature of ciece  Consider ethical implications o research:

Jenner tested his vaccine or smallpox on a child.

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AN I M AL P H YS I O LO G Y ( AH L )

Antigens in blood transfusion Every organism has unique molecules on the surace o their cells. Any oreign molecule that can trigger an immune response is reerred to as an antigen. The most common antigens are proteins and very large polysaccharides. S uch molecules are ound on the surace o cancer cells, parasites and bacteria, on pollen grains and on the envelopes o viruses. As an example, gure 1 shows a representation o an infuenza virus. Hemagglutinin and neuraminidase are two antigens ound on the surace o the virus. Hemagglutinin allows the virus to stick to host cells. Neuraminidase helps with the release o newly- ormed virus particles. The surace o our own cells contains proteins and polypeptides. Immune systems unction based on recognizing the distinction between oreign antigens and sel. Figure 2 shows a mixture o pollen grains rom several species. The antigens on the surace o these grains are responsible or triggering immune responses that are called allergies or hay ever in common language. hemagglutinin

Figure 2 Pollen grains

lipid membrane other protein genetic material (RNA) neuraminidase

Figure 1 Infuenza virus

Antigens in blood transfusion Antigens on the surace o red blood cells stimulate antibody production in a person with a diferent blood group. B lood groups are based on the presence or absence o certain types o antigens on the surace o red blood cells. Knowledge o this is important in the medical procedure called transusion where a patient is given blood rom a donor. The AB O blood group and the Rhesus ( Rh) blood group are the two most important antigen systems in blood transusions as mismatches between donor and recipient can lead to an immune response. In gure 3 , the dierences between the three A, B and O phenotypes are displayed. All three alleles involve a basic antigen sequence called antigen H. In blood type A and B , this antigen H is modied by the addition o an additional molecule. I the additional molecule is galactose, antigen B results. I the additional molecule is N-acetylgalactosamine,

466

antigen A results. B lood type AB involves the presence o both types o antigens.

O

A

B

AB

Key red blood cell

Figure 3

N acetyl-galactosamine N acetyl-glucosamine

fucose galactose

1 1 . 1 A n t i b o d y p r o d u c t i o n A n d vA c c i n At i o n

I a recipient is given a transusion involving the wrong type o blood, the result is an immune response called agglutination ollowed by hemolysis where red blood cells are destroyed and blood may coagulate in the vessels ( fgure 4) .

red blood cells with antibodies from agglutination surface antigens from recipient (clumping) an incom atable donor

hemolysis

Figure 4 B lood typing involves mixing samples o blood with antibodies. Figure 5 shows the result o a blood group test showing reactions between blood types ( rows) and antibody serums ( columns) . The frst column shows the bloods appearance prior to the tests. There are our human blood types: A, B , AB and O . Type A blood has type A antigens ( surace proteins) on its blood cells. Type B blood has type B antigens. Mixing type A blood with anti- A+ B serum causes an agglutination reaction, producing dense red dots that are dierent rom the control in the frst column. Type B blood undergoes the same reaction with anti- B serum and anti A+ B serum. AB blood agglutinates in all three anti- serums. Type O blood has neither the A or B antigen, so it does not react to the serums.

Figure 5

The specifc immune response B lymphocytes are activated by T lymphocytes in mammals. The principle o  challenge and response has been used to explain how the immune system produces the large amounts o the specifc antibodies that are needed to fght an inection, and avoid producing any o the hundreds o thousands o other types o antibodies that could be produced. Antigens on the surace o pathogens that have invaded the body are the  challenge . The  response involves the ollowing stages. Pathogens are ingested by macrophages, and antigens rom them are displayed in the plasma membrane o the macrophages. Lymphocytes called helper T cells each have an antibody-like receptor protein in their plasma membranes, which can bind to antigens displayed by macrophages. O  the many types o helper T cell, only a ew have receptor proteins that ft the antigen. These helper T cells bind and are activated by the macrophage.

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AN I M AL P H YS I O LO G Y ( AH L ) 1 Macrophage ingests pathogen and displays antigens from it

2 Helper T cell specic to the antigen is activated by the macrophage

The activated helper T cells then bind to lymphocytes called B cells. Again, only B cells that have a receptor protein to which the antigen binds are selected and undergo the binding process. The helper T cell activates the selected B cells, both by means o the binding and by release o a signalling protein.

The role of plasma cells 3 B cell specic to the antigen is activated by proteins from the helper T cell

5 B cell also divides to produce memory cells

4 B cell divides repeatedly to produce antibodysecreting plasma cells

Plasma cells secrete antibodies. Plasma cells are mature B lymphocytes (white blood cells) that produce and secrete large number o antibodies during an immune response. Figure 7 shows a plasma cell. The cells cytoplasm (orange) contains an unusually extensive network o rough endoplasmic reticulum (rER) . rER manuactures, modifes and transports proteins, in this case, the antibodies. The cell produces a lot o the same type o protein meaning that the range o genes expressed is lower than a typical cell. This explains the staining pattern o the nucleus where dark staining indicates unexpressed genes.

Clonal selection and memory cell formation Activated B cells multiply to form a clone of plasma cells and memory cells. 6 Antibodies produced by the clone of plasma cells are specic to antigens on the pathogen and help to destroy it.

Figure 6 The stages in antibody production

The activated B cells divide many times by mitosis, generating a clone o plasma cells that all produce the same antibody type. The generation o large numbers o plasma cells that produce one specifc antibody type is known as clonal selection. The antibodies are secreted and help to destroy the pathogen in ways described below. These antibodies only persist in the body or a ew weeks or months and the plasma cells that produce them are also gradually lost ater the inection has been overcome and the antigens associated with it are no longer present. Although most o the clone o B cells become active plasma cells, a smaller number become memory cells, which remain long ater the inection. These memory cells remain inactive unless the same pathogen inects the body again, in which case they become active and respond very rapidly. Immunity to an inectious disease involves either having antibodies against the pathogen, or memory cells that allow rapid production o the antibody.

The role of antibodies Figure 7 A plasma cell

Antibodies aid the destruction of pathogens. Antibodies aid in the destruction o pathogens in a number o ways. 

468

O p sonization: They make a pathogen more recognizable to phagocytes so they are more readily enguled. O nce bound, they can link the pathogen to phagocytes.

1 1 . 1 A n t i b o d y p r o d u c t i o n A n d vA c c i n At i o n



Neutralization of viruses and bacteria: Antibodies can prevent viruses rom docking to host cells so that they cannot enter the cells.



Neutralization of toxins: Some antibodies can bind to the toxins produced by pathogens, preventing them rom aecting susceptible cells.



Activation of comp lement: The complement system is a collection o proteins which ultimately lead to the peroration o the membranes o pathogens. Antibodies bound to the surace o a pathogen activate a complement cascade which leads to the ormation o a membrane attack complex that orms a pore in the membrane o the pathogen allowing water and ions to enter into the cell ultimately causing the cell to lyse.



Agglutination: Antibodies can cause sticking together or agglutination o pathogens so they are prevented rom entering cells and are easier or phagocytes to ingest. The large agglutinated mass can be fltered by the lymphatic system and then phagocytized. The agglutination process can be dangerous i it occurs as a result o an incorrect blood transusion.

Figure 8 summarizes some o the modes o action o antibodies. function of antibodies activation of complement complement

agglutination reduces number of pathogenic units to be engulfed bacterium

lysis

bacteria opsonization phagocyte

neutralization blocks adhesion of bacteria and docking of viruses to cells blocks activity of toxins virus bacterium

coating antigen with antibody enhances phagocytosis

toxin

Figure 8

Immunity Immunity depends upon the persistence o memory cells. Immunity to a disease is due either to the presence o antibodies that recognize antigens associated with the disease, or to memory cells that allow production o these antibodies. Immunity develops when the immune system is challenged by a specifc antigen and produces antibodies and memory cells in response. Figure 9 distinguishes a primary immune response ( launched the frst time the pathogen inects

toK Wha a game he ells s a he essee f smallx skles? Once wild smallpox had been eradicated there remained the challenge o what to do with samples o smallpox still in the hands o researchers and the military. Despite calls or the remaining stockpiles to be eradicated by the WHO, both the US and Russia have delayed complying with this directive. Game theory is a branch o mathematics that makes predictions about human behaviour when negotiations are being undertaken. In terms o payo, i one side reneges and the other proceeds on the basis o trust, the gain to the deal breaker is maximized. In this case, they are no longer threatened by the adversary but retain the ability to threaten. I both parties renege, the risk remains that the virus will be used as a weapon in both the frst attack and in retaliation. Maximum net gain or all would involve both parties complying with the directive but this involves trust and risk taking.

469

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AN I M AL P H YS I O LO G Y ( AH L ) the body) and the secondary immune response which is launched the second time the pathogen inects the body. Memory cells ensure that the second time an antigen is encountered, the body is ready to respond rapidly by producing more antibodies at a aster rate.

concentration of antibody

secondary response

primary response

0

10

rst encounter with antigen

20

30 40 50 time/days second encounter with antigen

60

Figure 9 The secondary immune response

Vaccines lead to immunity Vaccines contain antigens that trigger immunity but do not cause the disease. A vaccine is introduced into the body, usually by inj ection. The vaccine may contain a live attenuated ( weakened) version o the pathogen, or some derivative o it that contains antigens rom the pathogen. This stimulates a primary immune response. I the actual microorganism enters the body as a result o inection, it will be destroyed by the antibodies in a secondary immune response.

Figure 10

Figure 1 0 shows a phagocyte engulfng a Mycobacterium bovis bacterium ( orange) . This is the strain o the bacterium used in the vaccination or tuberculosis ( TB ) . The bacteria are live but attenuated ( weakened) and not as pathogenic as their relative Mycobacterium tuberculosis. The vaccine primes the immune system to produce antibodies that act on both species o bacteria, without causing the disease, so that it responds more rapidly i inected with Mycobacterium tuberculosis ( TB ) bacteria.

Ethical considerations of Jenners vaccine experiments Consider ethical implications of research: Jenner tested his vaccine for smallpox on a child. E dward Jenner was an 1 8th century scientist who noted that a milkmaid claimed that because she had caught the disease cowpox

470

she would never develop smallpox. He inected an eight- year- old boy with cowpox. Ater a brie illness, the boy recovered. Jenner then

1 1 . 1 A n t i b o d y p r o d u c t i o n A n d vA c c i n At i o n

purposely inected the boy with smallpox to conrm that he had the ability to resist the disease. He was the rst person to use human beings as research subj ects in testing a vaccine. He did not do any preliminary laboratory research nor any preliminary animal studies beore experimenting with human beings, his subj ect was a small child well below the age o consent, and he deliberately inected him with an extremely virulent, oten atal, disease- causing agent.

Jenners experiments were perormed well beore the ormulation o any statements o ethical principles or the protection o human research subjects. The Nuremberg Trials condemned medical experiments on children. These trials that ollowed the Second World War resulted in the Nuremberg Code or the protection o research subjects, and later the World Health Organizations International Ethical Guidelines or Biomedical Research Involving Human Subjects (1 993) . Jenners experiments would not be approved by a modern ethical review committee.

The eradication of smallpox Smallpox was the frst inectious disease o humans to have been eradicated by vaccination. The eorts to eradicate smallpox are an example o the contributions that intergovernmental organizations can make to address issues o global concern. The rst such eort was launched in 1 950 by the Pan American Health Organization. The World Health Assembly passed a resolution in 1 959 to undertake a global initiative to eradicate smallpox. It met with mixed success until a well-unded Smallpox Eradication Unit was established in 1 967. The last known case o wild smallpox was in 1 977 in Somalia, though there were two accidental inections ater this. The campaign was successul or several reasons: 

O nly humans can catch and transmit smallpox. There is no animal reservoir where

the disease could be maintained and re- emerge. This is the reason a yellow ever eradication eort ailed in the early 1 900s. 

S ymptoms o inection emerge quite quickly and are readily visible allowing teams to  ring vaccinate all o the people who might have come in contact with the aficted person. In contrast, eorts to eradicate polio have been hampered because inected persons do not always present readily recognized symptoms.



Immunity to smallpox is long-lasting unlike such conditions as malaria where reinection is more common.

Vaccines and epidemiology Analysis o epidemiological data related to vaccination programmes. Epidemiology is the study o the distribution, patterns and causes o disease in a population. The spread o disease is monitored in order to predict and minimize the harm caused by outbreaks as well as to determine the actors contributing to the outbreak. Epidemiologists would be involved in planning and evaluating vaccination programmes. An eort to achieve the global eradication o polio was begun in 1 988, as a combined eort between

the World Health O rganization ( WHO ) , UNIC EF and the Rotary Foundation. S imilarly, UNIC EF is leading a worldwide initiative to prevent tetanus through vaccination. A small number o polio cases are the result o a ailure in vaccination programmes. Figure 1 1 shows the incidence o wild rather than vaccineinduced polio cases in India over a seven-year period. Epidemiologists would investigate to

471

AN I M AL P H YS I O LO G Y ( AH L )

determine the causes of the two peaks in numbers. Figure 1 2 shows the geographic distribution of polio cases over a 1 3-year period in India. Epidemiologists would use information about geographic distribution to determine origins of outbreaks so they could focus resources on those areas. They could track incidence to determine the effectiveness of reduction campaigns. It is heartening to know that by 2 01 2 , India had been declared polio-free. The concern is that polio-free countries can still see some polio cases if infected individuals cross borders. 1998

1999

1,934 cases

number of cases

11

1800 1600 1400 1200 1000 800 600 400 200 0

1600

873 676 265

2003

66 2000 2001 2002 2003 2004 2005 2006 2007 year

Figure 11 2000

2001

265 cases

268 cases

2004

255 cases

1,600 cases

2006

2007

134 cases

2008

874 cases

676 cases

559 cases

2010

472

255 134

1,126 cases

2002

Figure 12

268

2011

2005

66 cases

2009

1 1 . 1 A n t i b o d y p r o d u c t i o n A n d vA c c i n At i o n

daa-ase qess: Polio incidence in 2012 Figure 1 3 provides data about polio incidence in the three countries where wild polio was still endemic as o mid- 2 01 2 .

4

Identiy one country where the situation appears to have improved between 201 1 and 201 2. (2)

5

Given that in 1 988 there were an estimated 350,000 cases o polio globally, discuss the success o the polio eradication programme. (5)

1

D efne the term endemic

(1 )

2

Identiy the three countries where polio was still endemic as o mid- 2 01 2 .

(1 )

6

Identiy the strain o polio virus which is the most prevalent.

Suggest some o the challenges an epidemiologist might ace in gathering reliable data. (5)

(1 )

7

Research to fnd the status o polio eradication in these countries.

3

wild poliovirus (WPV) cases

Afghanistan year-to-date 2012 year-to-date 2011 total in 2011 date of most recent case WPV1 WPV2 W1W3 total WPV1 WPV2 W1W3 total 80 30 June 2012 13 0 0 13 11 0 0 11 Pakistan year-to-date 2012 year-to-date 2011 total in 2011 date of most recent case WPV1 WPV3 W1W3 total WPV1 WPV3 W1W3 total 198 22 June 2012 20 2 1 23 58 1 0 59

Nigeria year-to-date 2012 year-to-date 2011 total in 2011 date of most recent case WPV1 WPV3 W1W3 total WPV1 WPV3 W1W3 total 62 22 June 2012 55 42 13 0 14 6 0 20 Global total cases YTD 2012 YTD 2011 total in 2011 globally 96 274 650 in endemic countries 91 91 341 in non-endemic countries 5 183 309

Figure 13

Zoonosis are a growing global health concern Pathogens can be species-specifc although others can cross species barriers. Pathogens are oten highly specialized with a narrow range o hosts. There are viruses that are specifc to birds, pigs and bacteria or example. There are bacterial pathogens that only cause disease in humans. Humans are the only known organism susceptible to such pathogens as syphilis, polio and measles, but we are resistant to canine distemper virus, or example. The bacterium Mycobacterium tuberculosis does not cause disease in rogs because rogs rarely reach the 37 C temperature necessary to support the prolieration o the bacterium. Rats injected with the diphtheria toxin do not become ill because their cells lack the receptor that would bring the toxin into the cell.

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AN I M AL P H YS I O LO G Y ( AH L ) A zoonosis is a pathogen which can cross a species barrier. This is an emerging global health concern. Bubonic plague, Rocky Mountain spotted ever, Lyme disease, bird fu and West Nile virus are all zoonotic diseases. The major actor contributing to the increased appearance o zoonotic diseases is the growth o contact between animals and humans by such means as humans living in close contact with livestock or disruption o habitats.

Figure 14 A thermal scanning camera is being used to monitor the skin temperature o passengers arriving at Nizhny Novgorod airport, in Russia. Raised skin temperature can be an indicator o ever rom illnesses. Such cameras have been used widely to screen or possible carriers o various possible zoonotic epidemic infuenzas such as bird fu and swine fu

For example, in the late 1 990s in Malaysia, intensive pig arming in the habitat o bats inected with the Nipah virus eventually saw the virus move rom the bats to the pigs to the humans and resulted in over 1 00 human deaths.

The immune system produces histamines White cells release histamine in response to allergens. Mast cells are immune cells ound in connective tissue that secrete histamine in response to inection. Histamine is also released by basophils which circulate in the blood. Histamine causes the dilation o the small blood vessels in the inected area causing the vessels to become leaky. This increases the fow o fuid containing immune components to the inected area and it allows some o the immune components to leave the blood vessel resulting in both specic and non- specic responses.

Efects o histamines Histamines cause allergic symptoms. Histamine is a contributor to a number o symptoms o allergic reactions. C ells in a variety o tissues have membrane-bound histamine receptors. Histamine plays a role in bringing on the symptoms o allergy in the nose ( itching, fuid build-up, sneezing, mucus secretion and infammation) . Histamine also plays a role in the ormation o allergic rashes and in the dangerous swelling known as anaphylaxis. To lessen the eects o allergic responses, anti-histamines can be taken.

The process or creating hybridoma cells Figure 15 The rash across the body o this male patient is due to the release o excessive histamines in response to taking Amoxicillin (penicillin) antibiotic

Fusion of a tumour cell with an antibody-producing plasma cell creates a hybridoma cell. Monoclonal antibodies are highly specic, puried antibodies that are produced by a clone o cells, derived rom a single cell. They recognize only one antigen. plasma cells

immunize mouse

isolate spleen B cells antigen and dye used to screen to nd desired hybridoma hybridomas

cell culture

474

Figure 16

myeloma cells

1 1 . 1 A n t i b o d y p r o d u c t i o n A n d vA c c i n At i o n To produce the clone o cells that will manuacture a monoclonal antibody, the antigen recognized by the antibody is inj ected into a mouse, or other mammal. In response to this challenge, the mouses immune system makes plasma B cells that are capable o producing the desired antibody. Plasma cells are removed rom the spleen o the mouse. They will be o many dierent types with only some producing the desired antibody. The B cells are used with cancer cells called myeloma cells. The cells ormed by usion o plasma B cells and myeloma cells are called hybridoma cells.

production of monoclonal antibodies h CG hCG hCG

dye

B

dye

h CG

O nce identifed, the desired hybridoma cell is allowed to divide and orm a clone. These cells can be cultured in a ermenter where they will secrete huge amounts o monoclonal antibody. Figure 1 7 shows a 2 000- litre ermenter used in the commercial production o monoclonal antibodies. The hybridoma cell is multiplied in the ermenter to produce large numbers o genetically identical copies, each secreting the antibody produced by the original lymphocyte.

A

dye

dye

dye

dye

dye

dye

h CG

B ecause the ull diversity o B cells are used with the myeloma cells, many dierent hybridomas are produced and they are individually tested to fnd one that produces the required antibody.

Figure 17

C

dye

h CG

Monoclonal antibodies are produced by hybridoma cells.

dye dye

h CG

Monoclonal antibodies are used both or treatment and diagnosis o diseases. E xamples include the test or malaria that can be used to identiy whether either humans or mosquitoes are inected with the malarial parasite, the test or the HIV pathogen or the creation o antibodies or inj ection into rabies victims.

dye dye

D

pregnancy tests emloy monoclonal antibodies Monoclonal antibodies to hCG are used in pregnancy test kits. Monoclonal antibodies are used in a broad range o diagnostic tests, including tests or HIV antibodies and or an enzyme released during heart attacks. Pregnancy test kits are available that use monoclonal antibodies to detect hC G ( human chorionic gonadotrophin) . hC G is uniquely produced during pregnancy by the developing embryo and later the placenta. The urine o a pregnant woman contains detectable levels o hC G. Figure 1 8 shows how the pregnancy test strip works. At point C , there are antibodies to hC G immobilized in the strip. At point B there are ree antibodies to hC G attached to a dye. At point D there are immobilized antibodies that bind to the dye- bearing antibodies. Urine applied to the end o a test strip washes antibodies down the strip.

dye

dye dye

dye

dye

dye

dye

Figure 18

A 1 Explain how a blue band appears at point C if the woman is pregnant. [3] 2 Explain why a blue band does not appear at point C if the woman is not pregnant. [3] 3 Explain the reasons for the use of immobilized monoclonal antibodies at point D, even though they do not indicate whether a woman is pregnant or not. [3]

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11.2 Movement Udertadig

Applicatio

 Bones and exoskeletons provide anchorage or      

 

muscles and act as levers. Movement o the body requires muscles to work in antagonistic pairs. Synovial joints allow certain movements but not others. Skeletal muscle fbres are multinucleate and contain specialized endoplasmic reticulum. Muscle fbres contain many myofbrils. Each myofbril is made up o contractile sarcomeres. The contraction o the skeletal muscle is achieved by the sliding o actin and myosin flaments. Calcium ions and the proteins tropomyosin and troponin control muscle contractions. ATP hydrolysis and cross-bridge ormation are necessary or the flaments to slide.

 Antagonistic pairs o muscles in an insect leg.

skill  Annotation o a diagram o the human elbow.  Drawing labelled diagrams o the structure o a

sarcomere.  Analysis o electron micrographs to fnd the state o contraction o muscle fbres.

nature of ciece  Fluorescence was used to study the cyclic

interactions in muscle contraction.

Boe ad exokeleto achor mucle Bones and exoskeletons provide anchorage or muscles and act as levers. E xoskeletons are external skeletons that surround and protect most o the body surace o animals such as crustaceans and insects. Figure 1 shows a scanning electron micrograph o a spider next to exoskeletons that have been moulted. B ones and exoskeletons acilitate movement by providing an anchorage or muscles and by acting as levers. Levers change the size and direction o orces. In a lever, there is an eort orce, a pivot point called a ulcrum and a resultant orce. The relative positions o these three determine the class o lever. In fgure 2 , the diagram shows that when a person nods their head backward, the spine acts as a frst- class lever, with the ulcrum ( F) being ound between the eort orce ( E) provided by the splenius capitis muscle and the resultant orce ( R) causing the chin to be extended. Figure 1

476

The grasshopper leg acts as a third-class lever as the ulcrum is at the body end and the eort orce is between the ulcrum and the resultant orce.

11 . 2 M o ve M e n t Muscles are attached to the insides o exoskeletons but to the outside o bones.

biceps contracted

F E R

triceps relaxed

E

E elbow extended

R

F

F E F

E

R

biceps relaxed

R

F

F

(a) First-class lever

humerus

E

R

scapula

R

radius

triceps contracted

(c) Third-class lever

(b) Second-class lever

Figure 2

ulna

skeletal mucle are antagonitic Movement of the body requires muscles to work in antagonistic pairs.

Figure 3 The biceps and triceps are antagonistic muscles

S keletal muscles occur in pairs that are antagonistic. This means that when one contracts, the other relaxes. Antagonistic muscles produce opposite movements at a j oint. For example, in the elbow, the triceps extends the orearm while the biceps fex the orearm.

daa-bas qusis: Flight muscles In one research project, pigeons (Columba livia) were trained to take o, fy 35 metres and land on a perch. During the fight the activity o two muscles, the sternobrachialis (SB) and the thoracobrachialis (TB) , was monitored using electromyography. The traces are shown in gure 4. The spikes show electrical activity in contracting muscles. Contraction o the sternobrachialis causes a downward movement o the wing. take o

fast ight

landing

1

2

3

[1 ]

C ompare the activity o the sternobrachialis muscle during the three phases o the fight.

[3 ]

D educe rom the data in the electromyograph how the thoracobrachialis is used.

[1 ]

4

Another muscle, the supracoracoideus, is antagonistic to the sternobrachialis. State the movement produced by a contraction o the supracoracoideus. [1 ]

5

Predict the pattern o the electromyograph trace or the supracoracoideus muscle during the 3 5 -metre fight. [2 ]

SB

TB

D educe the number o downstrokes o the wing during the whole fight.

400 ms

Figure 4 Electrical activity in the sternobrachialis (SB) and the thoracobrachialis (TB) muscles during fight o a pigeon

477

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An insect leg has antagonistic muscles Antagonistic pairs of muscles in an insect leg. The grasshopper, like all insects, has three pairs o appendages. The hindlimb o a grasshopper is specialized or j umping. It is a j ointed appendage with three main parts. B elow the j oint is reerred to as the tibia and at the base o the tibia is another j oint below which is ound the tarsus. Above the j oint is reerred to as the emur. Relatively massive muscles are ound on the emur. When the grasshopper prepares to j ump, the fexor muscles will contract bringing the tibia and tarsus into a position where they resemble the letter Z and the emur and tibia are brought closer together. This is reerred to as fexing. The extensor muscles relax during this phase. The extensor muscles will then contract extending the tibia and producing a powerul propelling orce. extensor muscle relaxes tibia exes

extensor muscle contracts

exor muscle contracts

Figure 6 Composite high-speed photograph of a grasshopper (Order Orthoptera) jumping from the head of a nail

tibia extends

exor muscle relaxes

Figure 5

The human elbow is an example of a synovial joint Annotation of a diagram of the human elbow. humerus bone  to which the biceps and triceps are attached triceps  extends biceps  exes the joint the joint

joint-capsule  seals the joint and helps to prevent dislocation synovial uid  lubricates the joint and prevents friction

radius bone  to which ulna bone  to which the biceps is attached the triceps is attached cartilage  covers the bones and prevents friction

Figure 7 The elbow joint

478

The point where bones meet is called a j oint. Most j oints allow the bones to move in relation to each other  this is called articulation. Most articulated j oints have a similar structure, including cartilage, synovial fuid and j oint capsule. 

C artilage is tough, smooth tissue that covers the regions o bone in the j oint. It prevents contact between regions o bone that might otherwise rub together and so helps to prevent riction. It also absorbs shocks that might cause bones to racture.



S ynovial fuid lls a cavity in the j oint between the cartilages on the ends o the bones. It lubricates the j oint and so helps to prevent the riction that would occur i the cartilages were dry and touching.



The joint capsule is a tough ligamentous covering to the joint. It seals the joint and holds in the synovial fuid and it helps to prevent dislocation.

11 . 2 M o ve M e n t

Diferent joints allow diferent ranges o movement Synovial joints allow certain movements but not others. The structure of a j oint, including the j oint capsule and the ligaments, determines the movements that are possible . The knee j oint can act as a hinge j oint, which allows only two movements: fle xion ( b ending) and extension ( straightening) . It can also act as a p ivo t j oint when flexed. The knee has a gre ater range of movement whe n it is flexed than when it is exte nded. The hip j oint, b etwee n the pelvis and the femur, is a ball and socket j oint. It has a greater range of movement than the knee j oint in that it can flex and e xtend, rotate, and move sideways and back. This latter type of movement is called abduction and adduction.

outward rotation

exion abduction

adduction

inward rotation hyperextension extension

Figure 8 Range of motion at the shoulder

exion

outward rotation

abduction

extension

adduction

inward rotation

Figure 9 Range of motion at the hip

479

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AN I M AL P H YS I O LO G Y ( AH L )

structure o ucle fbre Skeletal muscle fbres are multinucleate and contain specialized endoplasmic reticulum. The muscles that are used to move the body are attached to bones, so they are called skeletal muscles. When their structure is viewed using a microscope, stripes are visible. They are thereore also called striated muscle. The two other types o muscle are smooth and cardiac. S triated muscle is composed o bundles o muscle cells known as muscle fbres. Although a single plasma membrane called the sarcolemma surrounds each muscle fbre, there are many nuclei present and muscle fbres are much longer than typical cells. These eatures are due to the act that embryonic muscle cells use together to orm muscle fbres. Figure 1 0 shows a muscle fbre. sarcolemma nucleus

myobril

sarcoplasmic reticulum

Figure 10 A modifed version o the endoplasmic reticulum, called the sarcoplasmic reticulum, extends throughout the muscle fbre. It wraps around every myofbril, conveying the signal to contract to all parts o the muscle fbre at once. The sarcoplasmic reticulum stores calcium. Between the myofbrils are large numbers o mitochondria, which provide ATP needed or contractions. one sarcomere

myofbril Muscle fbres contain many myofbrils. light band

Z-line

dark band

Figure 11 The ultrastructure o the muscle fbre

480

Within each muscle fbre there are many parallel, elongated structures called myofbrils. These have alternating light and dark bands, which give striated muscle its stripes. In the centre o each light band is a discshaped structure, reerred to as the Z- line.

11 . 2 M o ve M e n t

structure o myofbril Each myofbril is made up o contractile sarcomeres. The micrograph in fgure 1 3 shows a longitudinal section through a myofbril. A number o repeating units that alternate between light and dark bands are visible. Through the centre o each light area is a line called the Z- line. The part o a myofbril between one Z-line and the next is called a sarcomere. It is the unctional unit o the myofbril. The pattern o light and dark bands in sarcomeres is due to a precise and regular arrangement o two types o protein flament  thin actin flaments and thick myosin flaments. Actin flaments are attached to a Z- line at one end. Myosin flaments are interdigitated with actin flaments at both ends and occupy the centre o the sarcomere. E ach myosin flament is surrounded by six actin flaments and orms crossbridges with them during muscle contraction.

The arcomere Drawing labelled diagrams o the structure o a sarcomere. light band

dark band

Figure 12 A transverse section through a skeletal muscle fbre showing numerous myofbrils. A nucleus is shown in the bottom let

light band

thick myosin laments

thin actin laments Z-line

sarcomere

Z-line

Figure 14 The structure o a sarcomere When constructing diagrams o a sarcomere, ensure to demonstrate understanding that it is between two Z- lines. Myosin flaments should be shown with heads. Actin flaments should be shown connected to Z- lines. Light bands should be labelled around the Z- line. The extent o the dark band should also be indicated.

Figure 13

daa-bas qusis: Transverse sections of striated muscle The drawings in fgure 1 5 show myofbrils in transverse section.

1

E xplain the dierence between a transverse and a longitudinal section o muscle. [2 ]

2

D educe what part o the myofbril is represented by the drawings as small dots. [2 ]

3

C ompare the pattern o dots in the three diagrams.

[3 ]

E xplain the dierences between the diagrams in the pattern o dots.

[3 ]

4

Figure 15

481

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AN I M AL P H YS I O LO G Y ( AH L )

mechanis of skeletal uscle contraction The contraction o the skeletal muscle is achieved by the sliding o actin and myosin flaments. During muscle contraction, the myosin flaments pull the actin flaments inwards towards the centre o the sarcomere. This shortens each sarcomere and thereore the overall length o the muscle fbre (see fgure 1 6) . The contraction o skeletal muscle occurs by the sliding o actin and myosin flaments. Myosin flaments cause this sliding. They have heads that can bind to special sites on actin flaments, creating cross-bridges, through which they can exert a orce, using energy rom ATP. The heads are regularly spaced along myosin flaments and the binding sites are regularly spaced along the actin flaments, so many cross-bridges can orm at once ( see fgure 1 7) .

cross-bridge detaches

(a) relaxed muscle binding site

actin

Z-line

light band

myosin head

light band dark band

Z-line

myosin

myosin lament

actin

formation of crossbridge in presence of calcium ions

light band shortens, indicating actin slides along myosin

dark band remains the same length

movement of actin cross-bridge moves actin along sarcomere contracts

shape of myosin head changes

(b) contracted muscle

Figure 17

Figure 16 Diagram of relaxed and contracted sarcomeres

Deterining the state of skeletal uscle contraction Analysis o electron micrographs to fnd the state o contraction o muscle fbres. relaxed sarcomere Relaxed muscle Contracted muscle contracted sarcomere

Figure 18 Electron micrograph of relaxed and contracted sarcomeres

482

In a relaxed sarcomere, the Z- lines are arther apart, the light bands are wider and overall the sarcomere is longer. In the centre o the sarcomere, there is another line called the M- line. In a relaxed sarcomere, there is a more visib le light b and on either side o the M- line.

11 . 2 M o ve M e n t

The control o skeletal muscle contraction Calcium ions and the proteins tropomyosin and troponin control muscle contractions. In relaxed muscle, a regulatory protein called tropomyosin blocks the binding sites on actin. When a motor neuron sends a signal to a muscle fbre to make it contract, the sarcoplasmic reticulum releases calcium ions. These calcium ions bind to a protein called troponin which causes tropomyosin to move, exposing actins binding sites. Myosin heads then bind and swivel towards the centre o the sarcomere, moving the actin flament a small distance.

The role o ATp in the sliding o flaments ATP hydrolysis and cross-bridge ormation are necessary or the flaments to slide.



ATP causes the breaking o the cross-bridges by attaching to the myosin heads, causing them to detach rom the binding sites on actin.



Hydrolysis o the ATP, to AD P and phosphate, provides energy or the myosin heads to swivel outwards away rom the centre o the sarcomere  this is sometimes called the cocking o the myosin head.



New cross- bridges are ormed by the binding o myosin heads to actin at binding sites adj acent to the ones previously occupied ( each head binds to a site one position urther rom the centre o the sarcomere) .



Energy stored in the myosin head when it was cocked causes it to swivel inwards towards the centre o the sarcomere, moving the actin flament a small distance. This sequence o stages continues until the motor neuron stops sending signals to the muscle fbre. C alcium ions are then pumped back into the sarcoplasmic reticulum, so the regulatory protein moves and covers the binding sites on actin. The muscle fbre thereore relaxes.

For signifcant contraction o the muscle, the myosin heads must carry out this action repeatedly. This occurs by a sequence o stages:

1 myosin laments have heads which form cross-bridges when they are attached to binding sites on actin laments.

2 ATP binds to the myosin heads and causes them to break the cross-bridges by detaching from the binding sites.

movement

5 the ADP and phosphate are released and the heads push the actin lament inwards towards the centre of the sarcomerethis is called the power stroke.

ADP + P

ADP + P 4 the heads attach to binding sites on actin that are further from the centre of the sarcomere than the previous sites.

ATP

3 ATP is hydrolysed to ADP and phosphate, causing the myosin heads to change their angle. ADP + P the heads are said to be cocked in their new position as they are storing potential energy from ATP.

Figure 19

483

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AN I M AL P H YS I O LO G Y ( AH L )

The use o fuorescence to study contraction Fluorescence has been used to study the cyclic interactions in muscle contraction. Fluoresence is the emission o electromagnetic radiation, oten visible light, by a substance ater it has been illuminated by electromagnetic radiation o a dierent wavelength. The fuorescence can oten be detected in a light microscope and captured on lm or later analysis. Some o the classic experiments in the history o muscle research have depended on fuorescence. The coelenterate Aequorea victoria (gure 20) produces a calcium-sensitive bioluminescent protein, aequorin. Scientists studied the contraction o giant single muscle bres o the acorn barnacle Balanus nubilus by injecting samples o the muscle with aequorin. When muscles were stimulated to contract in the study, initially there was strong bioluminescence coinciding with the release o Ca2+ rom the sarcoplasmic reticulum. The light intensity began to decrease immediately ater the cessation o the stimulus.

In another experiment, researchers cut apart Nitella axillaris cells. These cells are unique in that they have a network o actin laments underlying their membranes. Researchers attached fuorescent dye to myosin molecules in an eort to show that myosin can walk along actin laments. uorescent dye attached to myosin

bead

myosin

ATP ADP

actin lament from Nitella axillaris

actin

With this technique, the researchers were able to demonstrate the ATP-dependence o myosin- actin interaction. The graph in gure 2 1 shows the velocity o myosin molecules as a unction o ATP concentration.

lament velocity, m/s

5 4 3 2 1 0 0

Figure 21

Figure 20 Aequorea victoria

484

50

100

150 ATP, M

200 400 1000

1 1 . 3 t h e K i d n e y A n d o s M o r e g u l At i o n

11.3 t k a ma Udertadig  Animals are either osmoregulators or 

    

 

osmoconormers. The Malpighian tubule system in insects and the kidney carry out osmoregulation and removal o nitrogenous wastes. The composition o blood in the renal artery is dierent rom that in the renal vein. The ultrastructure o the glomerulus and Bowmans capsule acilitate ultrafltration. The proximal convoluted tubule selectively reabsorbs useul substances by active transport. The loop o Henl maintains hypertonic conditions in the medulla. The length o the loop o Henl is positively correlated with the need or water conservation in animals. ADH controls reabsorption o water in the collecting duct. The type o nitrogenous waste in animals is correlated with evolutionary history and habitat.

Applicatio  Consequences o dehydration and

overhydration.  Treatment o kidney ailure by hemodialysis or kidney transplant.  Blood cells, glucose, proteins and drugs are detected in urinary tests.

skill  Drawing and labelling a diagram o the human

kidney.  Annotation o diagrams o the nephron.

nature o ciece  Curiosity about particular phenomena:

investigations were carried out to determine how desert animals prevent water loss in their wastes.

Diferet repoe to chage i omolarity i the eviromet Animals are either osmoregulators or osmoconormers. O smolarity reers to the solute concentration o a solution. Many animals are known as osmoregulators because they maintain a constant internal solute concentration, even when living in marine environments with very dierent osmolarities. All terrestrial animals, reshwater animals and some marine organisms like bony fsh are osmoregulators. Typically these organisms maintain their solute concentration at about one third o the concentration o seawater and about 1 0 times that o resh water. O smoconormers are animals whose internal solute concentration tends to be the same as the concentration o solutes in the environment.

485

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AN I M AL P H YS I O LO G Y ( AH L )

data-base questions The striped shore crab Pachygrapsus crassipes (gure 1 ) is ound on rocky shores over the west coast o North and C entral America as well as in Korea and Japan. P. crassipes is oten exposed to dilute salinities in tide pools and reshwater rivulets, but it only rarely encounters salt concentrations much higher than that o the ocean. Samples o crabs were placed in water concentrations o varying osmolarity and samples o blood were taken to determine osmolarity o the blood. In this experiment, the unit o osmolarity is measured in units based on reezing point depression. When solutes are added to water they disrupt hydrogen bonding. Freezing requires additional hydrogen bonding so adding solute

lowers the reezing point. 2 delta is equivalent to about 1 00% ocean seawater, 0.2 delta is equivalent to about 1 0% ocean seawater, and 3 .4 delta is equivalent to about 1 70% seawater. 1

D etermine the solute concentration o crab blood at which the concentration o surrounding water is 1 delta.

(1 )

2

D etermine the range over which P. crassipes is able to keep its blood solute concentration airly stable. (1 )

3

Predict what the graph would look like i P. crassipes was not able to osmoregulate.

(1 )

D iscuss whether P. crassipes is an osmoconormer or an osmoregulator.

(3)

4

3.0

Pachygrapsus delta

line of isosmoticity 2.0

1.0 ocean seawater 0

Figure 1 The striped shore crab is exposed to varying salt concentrations in its habitat

0

1.0

2.0 water delta

3.0

Figure 2

The malpighian tubule syste The Malpighian tubule system in insects and the kidney carry out osmoregulation and removal of nitrogenous wastes. Arthropods have a circulating fuid, known as hemolymph, that combines the characteristics o tissue fuid and blood. Osmoregulation is a orm o homeostasis whereby the concentration o hemolymph, or blood in the case o animals with closed circulatory systems, is kept within a certain range. When animals break down amino acids, the nitrogenous waste product is toxic and needs to be excreted. In insects, the waste product is usually in the orm o uric acid and in mammals it is in the orm o urea. Insects have tubes that branch o rom their intestinal tract. These are known as Malpighian tubules. C ells lining the tubules actively transport ions and uric acid rom the hemolymph into the lumen o the tubules. This draws water by osmosis rom the hemolymph through the walls o the tubules into the lumen. The tubules empty their contents into the

486

1 1 . 3 t h e K i d n e y A n d o s M o r e g u l At i o n

gut. In the hindgut most of the water and salts are reabsorbed while the nitrogenous waste is excreted with the feces. 4 dehydrated uric acid paste is released with other waste

hindgut

2 the tubules empty into the gut

uric acid

midgut

midgut

semisolid wastes Na +

Malpighian tubule

K+ Malpighian tubules

H 20

3 some ions are actively reabsorbed in the hindgut and some water follows H20 Na +

hindgut

K+

1 uric acid, Na + and K+ are transported into the tubules and water follows by osmosis

uric acid H 20

Figure 3

Drawing the human kidney Drawing and labelling a diagram o the human kidney. When drawing a diagram of the kidney, the shape should be roughly oval with a concave side to which the renal artery and vein are attached. D rawings should clearly indicate the cortex shown at the edge of the kidney. It should be shown 1 with a thickness of about __ the entire width. The 5 medulla should be shown inside the cortex, with pyramids. The renal pelvis should be shown on the concave side of the kidney. The pelvis should drain into the ureter. The renal artery should have a smaller diameter than the renal vein.

cortex renal artery medulla

pelvis of kidney

renal vein

ureter (carries urine from the kidney)

Figure 4 Structure of the kidney

Comparing the composition of blood in the renal artery and the renal vein The composition o blood in the renal artery is diferent rom that in the renal vein. Kidneys function in both osmoregulation and excretion. The kidneys are responsible for removing substances from the blood that are not needed or are harmful. As a result, the composition of blood in the renal artery,

487

11

AN I M AL P H YS I O LO G Y ( AH L ) through which blood enters the kidney, is dierent rom that in the renal vein, through which blood leaves. S ubstances that are present in higher amounts in the renal artery than the renal vein include: 

Toxins and other substances that are ingested and absorbed but are not ully metabolized by the body, or example betain pigments in beets and also drugs.



Excretory waste products including nitrogenous waste products, mainly urea.

O ther things removed rom the blood by the kidney that are not excretory products include: 

Excess water, produced by cell respiration or absorbed rom ood in the gut.



Excess salt, absorbed rom ood in the gut.

These are not excretory products because they are not produced by body cells. Removal o excess water and salt is part o osmoregulation. While blood in the renal artery might contain a variable water or salt content, blood in the renal vein will have a more constant concentration because osmoregulation has occurred. The kidneys lter o about one th o the volume o plasma rom the blood fowing through them. This ltrate contains all o the substances in plasma apart rom large protein molecules. The kidneys then actively reabsorb the specic substances in the ltrate that the body needs. The result o this process is that unwanted substances pass out o the body in the urine. These substances are present in the renal artery but not the renal vein.

data-ase questins: Blood supply to the kidney Table 1 shows the fow rate o blood to the kidney and other organs, the rate o oxygen delivery and oxygen consumption. All o the values are given per 1 00 g o tissue or organ. The rates are or a person in a warm environment. 1

C ompare the rate o blood fow to the kidney with fow to the other organs.

Brain Skin Skeletal muscle (resting) Heart muscle Kidney Table 1

488

bl fw rate (ml min 1 100 g1 ) 54.0 13.0 2.7

oxygen elivery (ml min 1 100 g1 ) 10.8 2.6 0.5

87.0

17.4

420.0

84.0

2

3

In the brain, 3 4 per cent o the oxygen that is delivered is consumed. C alculate the same percentage or the other organs.

[4]

D iscuss the reasons or the dierence between the kidney and the other organs in the volume o blood fowing to the organ, and the percentage o oxygen in the blood that is consumed. [4]

5

S ome parts o the kidney have a high percentage rate o oxygen consumption, or example the outer part o the medulla. This is because active processes requiring energy are being carried out. S uggest one process in the kidney that requires energy. [1 ]

6

Predict, with a reason, one change in blood fow that would occur i the person were moved to a cold environment.

11.0 6.80

[2 ]

4 [2 ]

oxygen cnsumptin (ml min 1 100 g1 ) 3.70 0.38 0.18

C alculate the volume o oxygen delivered to the organs per litre o blood.

[2 ]

1 1 . 3 t h e K i d n e y A n d o s M o r e g u l At i o n A nal set o dierences between the composition o blood in the renal artery and the renal vein is due to the metabolic activity o the kidney itsel. B lood leaving the kidney through the renal vein is deoxygenated relative to the renal artery because kidney metabolism requires oxygen. It also has a higher partial pressure o carbon dioxide because this is a waste product o metabolism. E ven though glucose is normally ltered and then entirely reabsorbed, some glucose is used by the metabolism o the kidney and thereore the concentration is slightly lower in the renal vein compared to the renal artery. Plasma proteins are not ltered by the kidney so should be present in the same concentration in both blood vessels. Presence in the urine indicates abnormal unction. This is looked or during clinical examination o a urine sample.

The ultrastructure of the glomerulus The ultrastructure o the glomerulus and Bowmans capsule acilitate ultrafltration. B lood in capillaries is at high pressure in many o the tissues o the body, and the pressure orces some o the plasma out through the capillary wall, to orm tissue fuid. In the glomerulus o the kidney, the pressure in the capillaries is particularly high and the capillary wall is particularly permeable, so the volume o fuid orced out is about 1 00 times greater than in other tissues. The fuid orced out is called glomerular ltrate. The composition o blood plasma and ltrate is shown in table 2. The data in the table shows that most solutes are ltered out reely rom the blood plasma, but almost all proteins are retained in the capillaries o the glomerulus. This is separation o particles diering in size by a ew nanometres and so is called ultrafltration. All particles with a relative molecular mass below 65,000 atomic mass units can pass through. The permeability to larger molecules depends on their shape and charge. Almost all proteins are retained in the blood, along with all the blood cells.

Solutes

c (p m 3 f b pama) plasma fltrate

Na + ions (mol)

151

144

Cl - ions (mol)

110

114

glucose (mol) urea (mol) proteins (mg)

5 5 740

5 5 3.5

Table 2 The structure o a section o the lter unit is shown in gure 6 and gure 7. Figure 6 is a coloured transmission electron micrograph (TEM) o a section through a kidney glomerulus showing its basement membrane (brown line running rom top right to bottom let) . The basement membrane separates the capillaries (the white space at the let is the lumen o a capillary) . Note the gaps in the wall o the capillary which are reerred to as enestrations.

toK A  a a a b vp  jf   f ama  a? Figure 5 shows some o the techniques that have been used to investigate kidney unction. The animals used include rats, mice, cats, dogs and pigs. 1 What are the reasons or carrying out kidney research? 2 What criteria should be used to decide i a research technique is ethically acceptable or not? 3 Apply your criteria to the three techniques outlined in fgure 5 to determine whether they are ethically acceptable. 4 Who should make the decisions about the ethics o scientifc research?

Living animal is anaesthetized and its kidney is exposed by surgery. Fluid is sampled rom nephrons using micropipettes. Animal is then sacrifced so that the position o the sample point in the kidney can be located. 6 5 4 3 2 1

Animal is killed and kidneys are removed and rozen. Samples o tissue are cut rom regions o kidney that can be identifed. Temperature at which thawing occurs is ound, to give a measure o solute concentration. nephron

external uid Animal is killed and kidneys are dissected to obtain samples o nephron. Fluids are perused through nephron tissue, using experimental external uids to investigate the action o the wall o the nephron.

Figure 5

The smaller proj ections rom the membrane are podocyte oot processes, which attach the podocytes ( specialized epithelial cells) to the

489

11

AN I M AL P H YS I O LO G Y ( AH L ) membrane. The podocytes unction as a barrier through which waste products are ltered rom the blood. There are three parts to the ultraltration system.

Figure 6

1

Fenestrations between the cells in the wall o the capillaries. These are about 1 00 nm in diameter. They allow fuid to escape, but not blood cells.

2

The basement membrane that covers and supports the wall o the capillaries. It is made o negatively- charged glycoproteins, which orm a mesh. It prevents plasma proteins rom being ltered out, due to their size and negative charges.

3

Podocytes orming the inner wall o the B owmans capsule. These cells have extensions that wrap around the capillaries o the glomerulus and many short side branches called oot processes. Very narrow gaps between the oot processes help prevent small molecules rom being ltered out o blood in the glomerulus.

I particles pass through all three parts they become part o the glomerular ltrate. podocytes  strangely shaped cells with nger-like projections which wrap around capillaries in the glomerulus and provide support fenestrated wall of capillary

Figure 8 shows the relationship between the glomerulus and the B owmans capsule. aerent arteriole

basement membrane  the lter

podocytes basement membrane

fenestrated wall of capillary proximal convoluted tubule

eerent arteriole

blood plasma

red blood cell

nucleus of capillary wall cell

Figure 7 Structure o the flter unit o the kidney

lumen of Bowmans capsule

Figure 8

data-base questions: Ultrafltration o charged and uncharged dextrans D extrans are polymers o sucrose. D ierent sizes o dextran polymer can be synthesized, allowing their use to investigate the eect o particle size on ultraltration. Neutral dextran is uncharged, dextran sulphate has many negative charges, and D E AE is dextran with many positive charges. Figure 9 shows the relationship between particle size and the permeability o the lter

490

unit o rat glomeruli. Animal experiments like this can help us to understand how the kidney works and can be done without causing suering to the animals. 1

S tate the relationship between the size o particles and the permeability to them o the lter unit o the glomerulus.

[1 ]

2

a)

C ompare the permeability o the lter unit to the three types o dextran. [3 ]

b) Explain these dierences in permeability. 3

O ne o the main plasma proteins is albumin, which is negatively charged and has a particle size o approximately 4.4 nm. Using the data in the graph, explain the diagnosis that is made i albumin is detected in a rats urine.

[3 ]

relative ltration rate

1 1 . 3 t h e K i d n e y A n d o s M o r e g u l At i o n

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

[3 ]

DEAE neutral dextran dextran sulphate

2.0 2.4 2.8 3.2 3.6 4.0 4.4 particle size / nm

Figure 9 Relationship between particle size o dextrans and fltration rate

The role of the proximal convoluted tubule

microvilli

mitochondria

The proximal convoluted tubule selectively reabsorbs useul substances by active transport. The glomerular ltrate fows into the proximal convoluted tubule. The volume o glomerular ltrate produced per day is huge  about 1 8 0 dm 3 . This is several times the total volume o fuid in the body and it contains nearly 1 . 5 kg o salt and 5 . 5 kg o glucose. As the volume o urine produced per day is only about 1 . 5 dm 3 and it contains no glucose and ar less than 1 . 5 kg o salt, almost all o the ltrate must be reabsorbed into the blood. Most o this reabsorption happens in the rst part o the nephron  the proximal convoluted tubule. Figure 1 0 shows this structure in transverse section. The methods used to reabsorb substances in the proximal convoluted tubule are described in table 3 . B y the end o the proximal tubule all glucose and amino acids and 8 0 per cent o the water, sodium and other mineral ions have been absorbed.

sm : are moved by active transport rom ltrate to space outside the tubule. They then pass to the peritubular capillaries. Pump proteins are located in outer membrane o tubule cells. c : are attracted rom ltrate to space outside the tubule because o charge gradient set up by active transport o sodium ions. g: is co-transported out o ltrate and into fuid outside the tubule, by co-transporter proteins in outer membrane o tubule cells. Sodium ions move down concentration gradient rom outside tubule into tubule cells. This provides energy or glucose to move at the same time to fuid outside the tubule. The same process is used to reabsorb amino acids. Wa: pumping solutes out o ltrate and into the fuid outside the tubule creates a solute concentration gradient, causing water to be reabsorbed rom ltrate by osmosis.

invaginations of outer membrane

basement membrane

lumen containing ltrate

Figure 10 Transverse section o the proximal convoluted tubule

Av The drawing below shows the structure o a cell rom the wall o the proximal convoluted tubule. Explain how the structure o the proximal convoluted tubule cell, as shown in the diagram, is adapted to carry out selective reabsorption.

10 m

Table 3

491

11

AN I M AL P H YS I O LO G Y ( AH L )

Te nepron Annotation of diagrams of the nephron. The basic unctional unit o the kidney is the nephron. This is a tube with a wall consisting o one layer o cells. This wall is the last layer o cells that substances cross to leave the body  it is an epithelium. There are several dierent parts o the nephron, which have dierent unctions and structures ( see gure 1 1 ) : Bowmans capsule

aerent arteriole



Loop of Henl  a tube shaped like a hairpin, consisting o a descending limb that carries the ltrate deep into the medulla o the kidney, and an ascending limb that brings it back out to the cortex.



D istal convoluted tubule  another highly twisted section, but with ewer, shorter microvilli and ewer mitochondria.

distal convoluted tubule



venule peritubular capillaries

C ollecting duct  a wider tube that carries the ltrate back through the cortex and medulla to the renal pelvis.



B lood vessels  associated with the nephron are blood vessels. B lood fows though them in the ollowing sequence:

proximal convoluted tubule

eerent arteriole glomerulus collecting duct vasa recta

ascending limb of loop of Henl descending limb of loop of Henl

Figure 11 The nephron and associated blood vessels. The human kidney contains about a million nephrons 



B owmans cap sule  a cup- shaped structure with a highly porous inner wall, which collects the fuid ltered rom the blood. Proximal convoluted tubule  a highly twisted section o the nephron, with cells in the wall having many mitochondria and microvilli proj ecting into the lumen o the tube.



Afferent arteriole  brings blood rom the renal artery.



Glomerulus  a tight, knot- like, highpressure capillary bed that is the site o blood ltration.



E fferent arteriole  a narrow vessel that restricts blood fow, helping to generate high pressure in the glomerulus.



Peritubular capillaries  a low-pressure capillary bed that runs around the convoluted tubules, absorbing fuid rom them.



Vasa recta  unbranched capillaries that are similar in shape to the loops o Henl, with a descending limb that carries blood deep into the medulla and an ascending limb bringing it back to the cortex.



Venules  carry blood to the renal vein.

Te role of te loop of henl The loop of Henl maintains hypertonic conditions in the medulla. The overall eect o the loop o Henl is to create a gradient o solute concentration in the medulla. The energy to create the gradient is expended by wall cells in the ascending limb. Here sodium ions are pumped out o the ltrate to the fuid between the cells in the medulla  called the interstitial fuid. The wall o the ascending limb is unusual in that it is impermeable to water, so water is retained in the ltrate, even

492

1 1 . 3 t h e K i d n e y A n d o s M o r e g u l At i o n

This system or raising solute concentration is an example o a countercurrent multiplier system. It is a countercurrent system because o the fows o fuid in opposite directions. It is a countercurrent multiplier because it causes a steeper gradient o solute concentration to develop in the medulla than would be possible with a concurrent system. There is also a countercurrent system in the vasa recta. This prevents the blood fowing through this vessel rom diluting the solute concentration o the medulla, while still allowing the vasa recta to carry away the water removed rom ltrate in the descending limb, together with some sodium ions.

300

300

100

Na +

H 2O

600 Na +

600

H 2O

900

400

Na +

900 Na +

700

ascending limb

Normal body fuids have a concentration o 3 00 mO sm. The pump proteins that transer sodium ions out o the ltrate can create a gradient o up to 2 00 mO sm, so an interstitial concentration o 5 00 mO sm is clearly achievable. The cells in the wall o the descending limb are permeable to water, but are impermeable to sodium ions. As ltrate fows down the descending limb, the increased solute concentration o interstitial fuid in the medulla causes water to be drawn out o the ltrate until it reaches the same solute concentration as the interstitial fuid. I this was 5 00 mO sm, then ltrate entering the ascending limb would be at this concentration and the sodium pumps could raise the interstitial fuid to 700 mO sm. Fluid passing down the descending limb would thereore reach 700 mO sm, and the sodium pumps in the ascending limb could cause a urther 2 00 mO sm rise. The interstitial fuid concentration can thereore rise urther and urther, until a maximum is reached, which in humans is 1 , 2 00 mO sm.

from the proximal to the distal convoluted convoluted tubule tubule

descending limb

though the interstitial fuid is now hypertonic relative to the ltrate; i.e. , it has a higher solute concentration.

Na +

H 2O

1200

1200 Na +

1000

1200

Figure 12 Solute concentrations in the loop of Henl (in mOsm)

some animal ave relatively long loop of henl The length of the loop of Henl is positively correlated with the need for water conservation in animals. The longer the loop o Henl, the more water volume will be reclaimed. Animals adapted to dry habitats will oten have long loops o Henl. Loops o Henl are ound within the medulla. In order to accommodate long loops o Henl, the medulla must become relatively thicker.

daa-ba q: Medulla thickness and urine concentration Table 4 shows the relative medullary thickness ( RMT) and maximum solute concentration ( MS C ) o the urine in mO sm or 1 4 species o mammal. RMT is a measure o the thickness o the medulla in relation to the overall size o the kidney. All the species in the table that are shown with binomials are desert rodents. 1

2

D iscuss the relationship between maximum solute concentration o urine and the habitat o the mammal.

[3 ]

Plot a scattergraph o the data in the table, either by hand or using computer sotware.

[7]

493

11

AN I M AL P H YS I O LO G Y ( AH L )

3

a)

Using the scattergraph that you have plotted, state the relationship between RMT and the maximum solute concentration o the urine.

[1 ]

b) S uggest how the thickness o the medulla could aect the maximum solute concentration o the urine. [4]

speie

(a) low ADH

(b) high ADH

rMt

Msc (mom)

beaver

1.3

517

pig

1.6

1076

human

3.0

1399

dog

4.3

2465

cat

4.8

3122

rat

5.8

2465

Octomys mimax

6.1

2071

Dipodomys deserti

8.5

5597

Jaculus jaculus

9.3

6459

Tympanoctomys barrerae

9.4

7080

Psammomys obesus

10.7

4952

Eligmodontia typus

11.4

8612

Calomys mus

12.3

8773

Salinomys delicatus

14.0

7440

Table 4

interstitial uid

Function of ADh 125

300

300

ADH controls reabsorption of water in the collecting duct. 150

600

600

175

900

900

200

1200

1200

renal pelvis

Figure 13 Solute concentrations in the collecting duct

494

When ltrate enters the distal convoluted tubule rom the loop o Henl, its solute concentration is lower than that o normal body fuids  it is hypotonic. This is because proportionately more solutes than water have passed out o the ltrate as it fows through the loop o Henl in the medulla. I the solute concentration o the blood is too low, relatively little water is reabsorbed as the ltrate passes on through the distal convoluted tubule and the collecting duct. The wall o these parts o the nephron can have an unusually low permeability to water. A large volume o urine is thereore produced, with a low solute concentration, and as a result the solute concentration o the blood is increased ( see gure 1 3 a) . I the solute concentration o the blood is too high, the hypothalamus o the brain detects this and causes the pituitary gland to secrete a hormone  antidiuretic hormone or AD H. This hormone causes the walls o the distal convoluted tubule and collecting duct to become

1 1 . 3 t h e K i d n e y A n d o s M o r e g u l At i o n much more permeable to water, and most o the water in the ltrate is reabsorbed. This is helped by the solute concentration gradient o the medulla. As the ltrate passes down the collecting duct, it fows deep into the medulla, where the solute concentration o the interstitial fuid is high. Water continues to be reabsorbed along the whole length o the collecting duct and the kidney produces a small volume o concentrated urine ( gure 1 3 b) . As result the solute concentration o the blood is reduced. The action o the kidney thereore helps to keep the relative amounts o water and solutes in balance at an appropriate level. This is called osmoregulation.

daa-ba q: ADH release and feelings of thirst The plasma solute concentration, plasma antidiuretic hormone (ADH) concentration and eelings o thirst were tested in a group o volunteers. Figures 1 4 and 1 5 show the relationship between intensity o thirst, plasma ADH concentration and plasma solute concentration.

10 9 8 7 6 5 4 3 2 1 0

[1 ]

[1 ]

c) O utline what would happen to plasma solute concentration and AD H concentration i a person were to drink water to satisy his/her thirst.

[2 ]

d) S tate two reasons why a persons plasma solute concentration may increase.

[2 ]

plasma ADH/pmol dm -3

intensity of thirst/arbitrary units

a) Identiy the plasma AD H concentration at a plasma solute concentration o 3 00 mO smol kg - 1 using the line o best t.

b) C ompare intensity o thirst and plasma AD H concentration.

280 290 300 310 320 plasma solute concentration/mOsmol kg-1

Figure 14

20 18 16 14 12 10 8 6 4 2 0 280 290 300 310 320 plasma solute concentration/mOsmol kg-1

Figure 15

Ama va  m f  p f  wa  pc The type of nitrogenous waste in animals is correlated with evolutionary history and habitat. When animals break down amino acids and nucleic acids, nitrogenous waste in the orm o ammonia is produced. Ammonia is highly basic and can alter the pH balance. It is also toxic as it is a highly reactive chemical. I the organism lives in a marine or reshwater habitat, such as sh, echinoderms or coelenterates, they can release the waste directly as ammonia as it can be easily diluted within that environment. Terrestrial

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11

AN I M AL P H YS I O LO G Y ( AH L ) organisms will expend energy to convert ammonia to the less toxic orms o urea or uric acid depending on their habitats and evolutionary history. Marine mammals, despite their habitat, release urea because o their evolutionary history. Some organisms like amphibians release the waste as ammonia when they are larva and ater metamorphosis, release the waste as urea. C onverting ammonia to urea requires energy and converting it to uric acid requires even more energy. The advantage o uric acid is that it is not water-soluble and thereore does not require water to be released. B irds and insects release their nitrogenous waste as uric acid. For birds, not having to carry water or excretion means less energy needs to be expended on fight.

Figure 16 The white paste in bird droppings is uric acid

Uric acid is linked to adaptations or reproduction. Nitrogenous wastes are released by the developing organism within eggs. Uric acid is released as it is not soluble and crystallizes rather than building up to toxic concentrations within the egg.

Dehydration and overhydration Consequences of dehydration and overhydration. D ehydration is a condition that arises when more water leaves the body than comes in. It can arise rom a number o actors including exercise, insucient water intake or diarrhoea. It can lead to the disruption o metabolic processes. O ne sign o dehydration is darkened urine due to increased solute concentration. Water is necessary to remove metabolic wastes so dehydration can lead to tiredness and lethargy due to decreased eciency o muscle unction and increased tissue exposure to metabolic wastes. B lood pressure can all due to low blood volume. This can lead

to increases in heart rate. B ody temperature regulation may be aected because o an inability to sweat. Overhydration is less common and occurs when there is an over-consumption o water. The result is a dilution o blood solutes. It might occur when large amounts o water are consumed ater intense exercise without replacing the electrolytes lost at the same time. This makes body fuids hypotonic and could result in the swelling o cells due to osmosis. I this occurs, the most notable symptoms are headache and nerve unction disruption.

Treatment options for kidney failure Treatment of kidney failure by hemodialysis or kidney transplant.

496

Kidney ailure can occur or a number o reasons but most commonly occurs as a complication rom diabetes or chronic high blood pressure ( hypertension) as a result o diabetes.

blood pass through the membrane, but the larger blood cells and proteins cannot. The puried blood is then returned to the patient via a vein. This procedure takes several hours.

Figure 1 7 shows a patient undergoing renal dialysis (hemodialysis) . The dialysis machine (articial kidney) is on the let. Hemodialysis is required when the kidneys are no longer able to lter waste products rom the blood properly. D uring the procedure, a steady fow o blood passes over an articial semi-permeable membrane in the dialysis machine. The small waste products in the

An alternative to dialysis is a kidney transplant. In this treatment option, a kidney rom one person is placed in the body o a person whose kidneys arent unctioning. The donor can either be living or deceased. A living donor is possible because a person can survive with one unctional kidney. This approach can result in greater independence o movement and reedom to travel as compared

1 1 . 3 t h e K i d n e y A n d o s M o r e g u l At i o n

blood in tubing ows through dialysis uid blood pump

vein artery shunt

used dialysis uid

air detector

fresh dialysis uid

dialysis machine

compressed air

Figure 17 to dialysis. D ialysis also carries with it the risk o inection and other complications. A drawback to a transplant is that the recipients body can rej ect the organ. Figure 1 9 is o a light

Figure 18

micrograph through a transplanted kidney that has been rej ected by the recipients immune system. Numerous lymphocytes ( with small dots) have infltrated the kidney tissue.

Figure 19

Urinalysis Blood cells, glucose, proteins and drugs are detected in urinary tests. Urine is a product o osmoregulation, excretion and metabolism. These processes can be disrupted by illness or drug abuse. Urinalysis is a clinical procedure that examines urine or any deviation rom normal composition. Figure 2 0 shows a urine test strip being compared to the results chart on the testing kit bottle. This strip contains three test areas designed to change colour to indicate a positive or negative result ater being dipped in urine.

The colours displayed can then be compared to a results chart on the testing kit. This test indicates the pH, protein level and glucose level in the urine. High levels o glucose and protein in the urine can be an indication o diabetes. High protein levels can indicate damage to the kidneys as these do not get through ultrafltration in a healthy kidney. The strip in the picture is a normal negative result or protein and glucose.

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AN I M AL P H YS I O LO G Y ( AH L )

presence o traces o banned and controlled drugs in urine. Figure 2 1 shows a drug test card being dipped into a sample o urine. The card contains fve vertical strips that each test or a dierent drug. Here, the results are negative or all but the one second rom let. This indicates a positive test or opiates.

Figure 21

Figure 20 The panel drug test also uses test strips based on monoclonal antibody technology to look or the

Figure 22

498

Microscopic examination o urine is carried out to determine i cells are present, as under normal circumstances, these cells should not be present. Figure 2 2 shows white blood cells. The presence o 61 0 neutrophils ( white blood cells with a nucleus visible) can be a sign o urinary tract inection. Figure 2 3 indicates the presence o red blood cells ( erythrocytes) in the urine o this patient. This can be a sign that there is a kidney stone or a tumour in the urinary tract.

Figure 23

11 . 4 s e xu Al r e pro d u cti o n

11.4 sa  Udertadig  Spermatogenesis and oogenesis both involve



  

   

mitosis, cell growth, two divisions o meiosis and dierentiation. Processes in spermatogenesis and oogenesis result in dierent numbers o gametes with dierent amounts o cytoplasm. Fertilization involves mechanisms that prevent polyspermy. Fertilization in animals can be internal or external. Implantation o the blastocyst in the endometrium is essential or the continuation o pregnancy. hCG stimulates the ovary to secrete progesterone during early pregnancy. The placenta acilitates the exchange o materials between the mother and embryo. Estrogen and progesterone are secreted by the placenta once it has ormed. Birth is mediated by positive eedback involving estrogen and oxytocin.

Applicatio  The average 38-week pregnancy in humans

can be positioned on a graph showing the correlation between animal size and the development o the young at birth or other mammals.

skill  Annotation o diagrams o seminierous

tubule and ovary to show the stages o gametogenesis.  Annotation o diagrams o mature sperm and egg to indicate unctions.

nature of ciece  Assessing risks and benefts associated with

scientifc research: the risks to human male ertility were not adequately assessed beore steroids related to progesterone and estrogen were released into the environment as a result o the use o the emale contraceptive pill.

similaritie betwee oogeei ad permatogeei Spermatogenesis and oogenesis both involve mitosis, cell growth, two divisions o meiosis and dierentiation. O ogenesis is the production o egg cells in the ovaries. O ogenesis starts in the ovaries o a emale etus. Germ cells in the etal ovary divide by mitosis and the cells ormed move to distribute themselves through the cortex o the ovary. When the etus is our or fve months old, these cells grow and start to divide by meiosis. B y the seventh month, they are still in the frst division o meiosis and a single layer o cells, called ollicle cells, has ormed around them. No urther development takes place until ater puberty. The cell that has started to divide by meiosis, together with the surrounding ollicle cells, is called a p rimary follicle. There are about 400, 000 in the ovaries at birth. No more primary ollicles are produced, but at the start o each menstrual cycle a small batch are

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11

AN I M AL P H YS I O LO G Y ( AH L ) stimulated to develop by FS H. Usually only one goes on to become a mature follicle, containing a secondary oocyte. primary ollicle

maturing ollicle

Figure 1 Light micrograph o a section through tissue rom an ovary, showing a primary ollicle (let) and a maturing ollicle (centre) . Primary ollicles contain a central oocyte (emale germ cell, egg) surrounded by a single layer o ollicle cells. A mature ovarian ollicle has many more ollicle cells, outer and inner ollicle cells and cavities, and the oocyte is now more ully developed compared to the primordial and primary stages S permatogenesis is the production o sperm. It happens in the testes, which are composed o a mass o narrow tubes, called seminiferous tubules, with small groups o cells lling the gaps between the tubules. These gaps are called interstices, so the cells in them are interstitial cells. They are sometimes called Leydig cells. The seminierous tubules are also made o cells. The outer layer o cells is called the germinal ep ithelium. This is where the process o sperm production begins. C ells in various stages o sperm production are ound inside the germinal epithelium, with the most mature stages closest to the fuid-lled centre o the seminierous tubule. C ells that have developed tails are called sp ermatozoa, though this is almost always abbreviated to sperm. Also in the wall o the tubule are large nurse cells, called S ertoli cells. Figure 3 shows a small area o testis tissue, in which the structures described above can be seen.

Figure 2 Coloured scanning electron micrograph (SEM) o ovary tissue, showing two secondary ollicles. A secondary oocyte (pink) is seen at the centre o one ollicle. Follicles are surrounded by two types o ollicle cells (coloured blue and green) . Between the ollicle cells a space develops (at centre right, coloured brown) , into which ollicular uid is secreted. The amount o uid will increase signifcantly as the ollicle matures

500

spermatogonium

agella o spermatozoa

lumen o seminierous tubule

Figure 3 Transverse section through a seminierous tubule

11 . 4 s e xu Al r e pro d u cti o n

Diagrams of a seminiferous tubule and the ovary Annotation of diagrams of seminiferous tubule and ovary to show the stages of gametogenesis. basement membrane

12 An outer layer called germinal epithelium cells (2n) divide endlessly by mitosis to produce more diploid cells

spermatogonium

2 Diploid cells grow larger and are then called primary spermatocytes (2n)

primary spermatocyte

32 Each primary spermatocyte carries out the rst division of meiosis to produce two secondary spermatocytes (n)

secondary spermatocyte

42 Each secondary spermatocyte carries out the second division of meiosis to produce two spermatids (n)

62 Sperm detach from Sertoli cells and eventually are carried out of the testis by the uid in the centre of the seminiferous tubule

spermatids 52 Spermatids become associated with nurse cells, called Sertoli cells which help the spermatids to develop into spermatozoa (n) . This is an example of cell dierentiation

Figure 4

2 In a secondary follicle, the follicle cells proliferate, a uid-lled cavity develops and the oocyte starts the second division of meiosis

developing secondary oocyte follicles primary follicles follicle

1 Primary follicles consist of a central oocyte surrounded by a single layer of follicle cells. Every menstrual cycle, a few primary follicles start to develop and the oocyte completes the rst division of meiosis degenerating corpus luteum

mature follicle corpus luteum developing corpus luteum ovulated ovum

Figure 5

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AN I M AL P H YS I O LO G Y ( AH L )

Diagrams of sperm and egg Annotation of diagrams of mature sperm and egg to indicate functions. haploid nucleus cytoplasm (or yolk) containing droplets of fat

two centrioles

rst polar cell

Diameter of egg cell = 110 m

plasma membrane cortical granules layer of follicle cells (corona radiata)

layer of gel composed of glycoproteins zona ellucida

head (3 m wide and 4 m long)

Figure 6 Structure of the female gamete

haploid nucleus acrosome

mid-piece (7 m long)

microtubules in a 9+2 arrangement

centriole plasma membrane

helical mitochondria

Figure 7 Structure of the male gamete

502

tail (40 m long, two-thirds of it omitted from this drawing)

protein bres to strengthen the tail

11 . 4 s e xu Al r e pro d u cti o n

daa-ba q: Sizes of sperm S perm tails have a 9 + 2 arrangement o microtubules in the centre, with thicker protein fbres around. Table 1 shows the structure o sperm tails o eight animals in transverse section, with the tail lengths and the cross-sectional area o the protein fbres. 1

2

O utline the relationship between tail length and cross-sectional area o protein fbres. [2 ]

3

Explain reasons or the relationship.

4

D iscuss whether there is a relationship between the size o an animal and the size o its sperm. [2 ]

D raw a graph o tail length and cross-sectional area o protein fbres in the eight species o animal. [4]

[2 ]

h ham

a

ga g

ham

b

m

hma

a h

cross-sectional area o fbrous sheaths / m 2

0.22

0.16

0.13

0.11

0.08

0.04

0.02

0

length of sperm / m

258

187

107

187

54

123

58

45

Table 1

Diferences in the outcome o spermatogenesis and oogenesis Processes in spermatogenesis and oogenesis result in dierent numbers o gametes with dierent amounts o cytoplasm. While there are similarities in spermatogenesis and oogenesis, there are dierences that are necessary to prepare the gametes or their dierent roles. E ach mature sperm consists o a haploid nucleus, a system or movement and a system o enzymes and other proteins that enable the sperm to enter the egg. Each complete meiotic division results in our spermatids. The process o sperm dierentiation eliminates most o the cytoplasm, whereas the egg must increase its cytoplasm. All o the requirements or beginning the growth and development o the early embryo must be present in the egg. In emales, the frst division o meiosis produces one large cell and one very small cell (fgure 8) . The small cell is the frst polar body which eventually degenerates. The large cell goes on to the second division o meiosis, completing it ater ertilization. Again one large cell and one very small cell are produced. The small cell is the second polar body and it also degenerates and dies. Only the large cell, which is the emale gamete, survives. The result is that the egg is much larger than the sperm cell. Figures 6 and 7 show the dierences in structure. Note that the scale bars indicate that the sperm and egg are drawn to dierent scale and that the egg is much larger than the sperm.

Figure 8 The micrograph shows a primary oocyte split into two cells, known as the secondary oocyte (green) and the frst polar body (yellow)

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11

AN I M AL P H YS I O LO G Y ( AH L ) The process o egg ormation happens once per menstrual cycle in humans and usually only one egg cell per cycle is produced. D uring the years rom puberty to the menopause only a ew hundred emale gametes are likely to be produced.

sperm try to push through the layers of follicle cells around the egg

From puberty onwards, the testes produce sperm continuously. At any time, there are millions o sperm at all stages o development.

preventing olysermy

follicle cell

Fertilization involves mechanisms that prevent polyspermy. Fertilization is the union o a sperm and an egg to orm a zygote.

zona pellucida

The membranes o sperm have receptors that can detect chemicals released by the egg, allowing directional swimming towards the egg. Figure 9 illustrates that multiple sperm arrive at the egg. O nce the egg is reached, a number o events take place ( see fgure 1 0) . These events are designed to result in the union o a single sperm with the egg. The events are also designed to prevent multiple sperm entering, known as polyspermy.

plasma membrane of egg

acrosomal cap

tail and mitochondria usually remain outside

cortical granules

hardened zona pellucida

exocytosis of contents of cortical granules

sperm nucleus

Figure 9 Micrograph of egg surrounded by sperm

1 The acrosome reaction

two haploid nuclei from the sperm and the egg

Figure 10 Stages in fertilization

504

The zona p ellucida is a coat o glycoproteins that surrounds the egg. The acrosome is a large membrane-bound sac o enzymes in the head o the sperm. In mammals, the sperm binds to the zona pellucida and the contents o the acrosome are released. The enzymes rom it digest the zona pellucida.

11 . 4 s e xu Al r e pro d u cti o n

2 Penetration of the egg membrane The acrosome reaction exposes an area o membrane on the tip o the sperm that has proteins that can bind to the egg membrane. The rst sperm that gets through the zona pellucida thereore binds and the membranes o sperm and egg use together. The sperm nucleus enters the egg cell. This is the moment o ertilization.

3 The cortical reaction Not only does the sperm bring male genes, it also causes the activation o the egg. The rst eect o this is on the cortical granules  vesicles located near the egg membrane. There are thousands o these vesicles and when activation o the egg has taken place their contents are released rom the egg by exocytosis. In mammals, the cortical vesicle enzymes result in the digestion o binding proteins so that no urther sperm can bind. The enzymes also result in a general hardening o the zona pellucida.

FPO

Figure 11 Breeding pair o Anomalochromis thomasi cichlids. The emale (bottom) is laying eggs on a rock with the male in close proximity

Internal and external fertilization Fertilization in animals can be internal or external. Aquatic animals oten release their gametes directly into water in a process that will lead to ertilization outside o the emales body. S uch animals oten have behaviours that bring eggs into proximity with sperm ( see gure 1 1 ) . External ertilization has several risks including predation and the susceptibility to environmental variation such as temperature and pH fuctuations and more recently, pollution. Terrestrial animals are dependent on internal ertilization. O therwise, gametes would be at risk o drying out. Internal ertilization also ensures sperm and ova are placed in prolonged close proximity to each other. Marine mammals which have reinvaded aquatic habitats still use internal ertilization. O nce the eggs are ertilized, the developing embryo can be protected inside the emale.

Figure 12 Blastocyst

Implantation of the blastocyst Implantation of the blastocyst in the endometrium is essential for the continuation of pregnancy. Ater ertilization in humans, the ertilized ovum divides by mitosis to orm two diploid nuclei and the cytoplasm o the ertilized egg cell divides equally to orm a two- cell embryo. These two cells replicate their D NA, carry out mitosis and divide again to orm a our-cell embryo. The embryo is about 48 hours old at this point. Further cell divisions occur, but some o the divisions are unequal and there is also migration o cells, giving the embryo the shape o a hollow ball. It is called a blastocyst ( gure 1 2 ) . At 7 days old the blastocyst consists o about 1 2 5 cells and it has reached the uterus, having been moved down the oviduct by the cilia o cells in the oviduct wall. At this age the zona pellucida, which has surrounded and protected the embryo, breaks down. The blastocyst has used up the reserves o the egg cell and needs an external supply o ood. It obtains this by sinking into the endometrium or uterus

Figure 13 Implantation o the blastocyst

Figure 14 Growth and diferentiation o the early embryo

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AN I M AL P H YS I O LO G Y ( AH L ) lining in a process called imp lantation ( gure 1 3 ) . The outer layer o the blastocyst develops nger- like proj ections allowing the blastocyst to penetrate the uterus lining. They also exchange materials with the mothers blood, including absorbing oods and oxygen. The embryo grows and develops rapidly and by eight weeks has started to orm bone tissue. It is then considered to be a etus rather than an embryo. It is recognizably human and soon visibly either male or emale.

Role of hCG in early pregnancy hCG stimulates the ovary to secrete progesterone during early pregnancy. Pregnancy depends on the maintenance o the endometrium, which depends on the continued production o progesterone and estrogen. In part these hormones prevent the degeneration o the uterus lining which is required to support the developing etus. Early in pregnancy the embryo produces human chorionic gonadotropin  hC G. This hormone stimulates the corpus luteum in the ovary to continue to secrete progesterone and estrogen. These hormones stimulate the continued development o the uterus wall, which supplies the embryo with everything that it needs.

materials exchange by the placenta The placenta facilitates the exchange of materials between the mother and embryo. Humans are placental mammals. There are two other groups o mammals: the monotremes lay eggs and the marsupials give birth to relatively undeveloped ospring that develop inside a pouch. B y the stage when a marsupial would be born, a human etus has developed a relatively complex placenta and so can remain in the uterus or months longer. The placenta is needed because the body surace area to volume ratio becomes smaller as the etus grows larger. The placenta is made o etal tissues, in intimate contact with maternal tissues in the uterus wall. The etus also develops membranes that orm the amniotic sac. This contains amniotic fuid, which supports and protects the developing etus. The basic unctional unit o the placenta is a nger- like piece o etal tissue called a placental villus. These villi increase in number during pregnancy to cope with the increasing demands o the etus or the exchange o materials with the mother. Maternal blood fows in the inter-villous spaces around the villi ( gure 1 5 ) . This is a very unusual type o circulation as elsewhere blood is almost always conned in blood vessels. Fetal blood circulates in blood capillaries, close to the surace o each villus. The distance between etal and maternal blood is thereore very small  as little as 5 m. The cells that separate maternal and etal blood orm the p lacental barrier. This must be selectively permeable, allowing some substances to pass, but not others ( gure 1 6) .

506

11 . 4 s e xu Al r e pro d u cti o n

maternal venule maternal blood pools

fetal blood carbon dioxide

maternal arteriole

fetal capillaries

placental barrier diusion diusion facilitated diusion

umbilical cord umbilical vein umbilical arteries

maternal blood

oxygen glucose

urea endocytosis

water

antibodies

osmosis water

Figure 16 Exchange processes in the placenta fetal portion of placenta (chorion)

maternal portion of placenta

Figure 15

Release of hormones by the placenta Estrogen and progesterone are secreted by the placenta once it has formed. B y about the ninth week o pregnancy, the placenta has started to secrete estrogen and progesterone in large enough quantities to sustain the pregnancy, and the corpus luteum is no longer needed or this role. There is a danger o miscarriage at this stage o pregnancy i this switchover ails.

daa-ba q: Electron micrograph of placenta Figure 1 7 shows a small region at the edge o a placental villus. The magnifcation is  1 7, 000. 1

a) Identiy the structures that are visible in the upper part o the micrograph. [1 ] b) Explain the unctions o these structures.

[3 ]

2

In much o the area o the electron micrograph there are rounded structures, surrounded by a single membrane. These are parts o a system o tubules called the smooth endoplasmic reticulum (sER) . Its unction is the synthesis o lipids, including steroids. Suggest a unction or the sER in the placenta. [3]

3

Identiy, with reasons, the structure in the lower let part o the micrograph. [3 ]

Figure 17 Small region at the edge of a placental villus

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AN I M AL P H YS I O LO G Y ( AH L )

Assessing risks of estrogen pollution Assessing risks and benefts o scientifc research: the risks to human male ertility were not adequately assessed beore steroids related to progesterone and estrogen were released into the environment as a result o the use o the emale contraceptive pill. High levels o estrogen are present in pregnant women and inhibit FS H release. I women consume pills containing estrogen, then this would mimic pregnancy and inhibit the development o mature ollicles thus preventing pregnancy. Ethinyl estradiol is a synthetic orm o estrogen that was frst introduced as a contraceptive in 1 943 . At the time, little thought was given to the idea that i a large number o women used this orm o contraception, then levels o estrogen in bodies o water might be raised through sewage. It wasnt until the mid- 1 980s that the frst reports o elevated contraceptive pill hormones present in water were reported. Since then, a number o problems have been attributed to estrogen pollution. In 1 992 , a review article summarizing 61 dierent studies concluded that human male sperm counts have declined by 5 0% over the past 5 0 years. In one o the largest studies o the problem, the UK governments Environment Agency ound in

2 004 that 86% o male fsh sampled at 5 1 sites around the country were intersex, that is male fsh showed signs o eminization. However, there is limited scientifc consensus that pollution with steroids related to estrogen and progesterone is the causative agent behind reduced male ertility. In 2 01 2 the E uropean C ommission proposed a policy which would limit the concentrations in water o a widely used contraceptive drug. This has sparked intense lobbying by the water and pharmaceutical industries, which say that the science is uncertain and the costs too high. Upgrading the technology or wastewater treatment could eliminate most o the pollution. Researchers and policy experts suggest sharing the costs among all responsible parties, including the water and drug industries, and that some expense would be passed on to the public. The drugs are widely used in livestock, so preventing animals rom urinating close to rivers could urther reduce the amount o drugs leaking into surace waters.

35

Rivers vary in terms o the quantities o synthetic estrogen (E 2 ) ound. A study was conducted to investigate the relationship between concentrations o synthetic estrogen in water and impacts on male fsh rom the genus Rutilus (roach) (see fgure 1 8) .

30

a) S tate the relationship between synthetic estrogen ( E 2 ) and the appearance o oocytes in testes.

percent of sh

data-base questions: Estrogen pollution

oocytes in testes feminized reproductive ducts

25 20 15 10 5

[1 ]

b) D etermine the mean percentage o male fsh with oocytes in their testes at concentrations o estrogen greater than 1 0 ng/L. [2 ]

0

10

Figure 18 Source: Jobling et al, Environ Health Perspect. 2006 April; 114( S-1) : 3239 .

The role of hormones in parturition Birth is mediated by positive eedback involving estrogen and oxytocin. D uring pregnancy, progesterone inhibits secretion o oxytocin by the pituitary gland and also inhibits contractions o the muscular outer wall o the uterus  the myometrium. At the end o pregnancy,

508

11 . 4 s e xu Al r e pro d u cti o n hormones produced by the etus signal to the placenta to stop secreting progesterone, and oxytocin is thereore secreted. O xytocin stimulates contractions o the muscle bres in the myometrium. These contractions are detected by stretch receptors, which signal to the pituitary gland to increase oxytocin secretion. Increased oxytocin makes the contractions more requent and more vigorous, causing more oxytocin secretion. This is an example o a positive eedback system  a very unusual control system in human physiology. In this case it has the advantage o causing a gradual increase in the myometrial contractions, allowing the baby to be born with the minimum intensity o contraction. Relaxation o muscle bres in the cervix causes it to dilate. Uterine contraction then bursts the amniotic sac and the amniotic fuid passes out. Further uterine contractions, usually over hours rather than minutes, nally push the baby out through the cervix and vagina. The umbilical cord is broken and the baby takes its rst breath and achieves physiological independence rom its mother.

1 Baby positions itself before birth so that its head rests close to the cervix uterus wall

mucus plug (pushed down front of into vagina) pelvis

bladder (compressed)

rectum

placenta umbilical spine cord

2 Baby passes into vagina and amniotic uid is released

daa-ba q: Hormone levels during pregnancy In the graph ( gure 2 0) , the thickness o the arrows indicates relative quantities. corpus luteum 3 Baby is pushed out of mothers body

30 days harmone levels

120 days full term 4 Placenta and umbilical cord are expelled from body

ESTROGEN

placenta becoming detached from uterus wall

hCG PROGESTERONE 0 1 conception

2

3

4 5 6 months of pregnancy

7

8 9 delivery

Figure 20 1

2 3

D escribe the changes over the course o a pregnancy in relative amounts and source o:

umbilical cord

Figure 19 Stages in childbirth

a) hC G

[2 ]

b) estrogen

[2 ]

c) progesterone

[2 ]

S uggest reasons or the drop in hC G concentration ater the second month o the pregnancy.

[2 ]

Predict the consequences o the placenta ailing to secrete estrogen and progesterone during a pregnancy.

[2 ]

509

11

AN I M AL P H YS I O LO G Y ( AH L )

Gestation times, mass and growth, and development strategies The average 38-week pregnancy in humans can be positioned on a graph showing the correlation between animal size and the development of the young at birth for other mammals. Mammals differ in their growth and development strategies. Altricial species give birth to relatively helpless, incompletely developed offspring. Their newly- born young are relatively immobile, lack hair and are unable to obtain food on their own. At the opposite end of the spectrum are precocial

mammals in which the offspring have open eyes, hair, are immediately mobile and are somewhat able to defend themselves against predators. Mammals with a large body size are more likely to be precocial. This is correlated with a long gestation period.

data-base questions: Gestation length and body mass Figure 2 1 shows the relationship between gestation period and body mass for 42 9 placental mammal species subdivided into whether the species is described as altricial or precocial.

log10 gestation period

3

2

1 0

1

2

3 4 5 log10 body mass

6

7

8

Figure 21 1

The solid dots and open dots are representative of two different growth and development strategies. D educe which circles are used to represent precocial mammals. [2 ]

2

O utline the relationship between adult body mass and gestation period. [1 ]

3

E xplain the relationship between body mass and the length of gestation. [3 ]

4

The mean length of human gestation is 283 days (log1 0 283 = 2.45) The mean body mass of an adult human is 65 kg (log1 0 65 = 1 .8) . (i) D etermine the approximate location of humans on the graph. [1 ] (ii) S uggest reasons for humans being an outlier on this graph. [3 ]

510

Figure 22 Laboratory mice are altricial. They have a gestation period of about 19 days

Figure 23 Elephant calves are born after a 22-month gestation period and they nurse for around three years. They are categorized as precocial. The African elephant is the largest and heaviest land animal alive today

Question s

Questions 1

Figure 2 4 shows how the surace pH o human skin varies between dierent areas o the body. It also shows dierences between adults and newborn inants ( neonates) . S kin pH protects the skin rom colonization by certain microorganisms.

b) S uggest reasons or calves that have endured a long and dicult birth being more likely to suer rom inection.

c) Predict how the concentration o antibodies might vary in the cows colostrum over the rst 2 4 hours ater birth. [2 ] d) D educe the reasons or vaccinating sheep against pulpy kidney and other liethreatening diseases three weeks beore lambs are due to be born. [2 ]

soles back abdomen

e) Explain which method o transport across membranes is likely to be used or absorption o antibodies in the stomach o newborn mammals. [2 ]

palms forearm forehead 5

6

7

8

3

pH neonates

adults

Figure 24 How the surace pH o human skin varies between diferent areas o the body a) C ompare the skin pH o neonates and adults.

[2 ]

c) S uggest why the use o soaps ( which are basic) might have a more irritating eect on the skin o a neonate. [2 ]

b) Explain why glucose is not all reabsorbed rom the glomerular ltrate o diabetic patients. [4]

d) D educe how basic soaps might undermine the skins deensive unction. [2 ]

c) S uggest why untreated diabetics tend to pass large volumes o urine and oten eel thirsty. [3 ]

Figure 2 5 shows the ability o a cal ( Bos taurus) to absorb antibodies ater birth. antibodies absorbed/%

4 100 75 50 25 0 0

6

12

18

24

30

36

42

calfs age at rst feeding/hours

Figure 25 The ability o a cal (Bos taurus) to absorb antibodies a) D escribe how the ability o a cal to absorb antibodies changes over the initial hours ater birth. [2 ]

The blood glucose concentration o a person with untreated diabetes oten rises to 300500 mg per 1 00 ml o blood. It can even rise to concentrations above 1 ,000 mg per 1 00 ml. When the blood glucose level rises above 225 mg per 1 00 ml, glucose starts to appear in the urine. The volumes o urine produced become larger than normal, making the person dehydrated and thirsty. a) Explain how glucose is completely reabsorbed rom the glomerular ltrate o people who do not have diabetes. [3 ]

b) S uggest how the adult skin pH might be established. [1 ]

2

[2 ]

Muscles oten increase in mass i the amount that they are used increases. An experiment was perormed to examine the eect o fight on muscle mass in European starlings ( Sturnus vulgaris) . S tudy birds were randomly assigned to three groups. O ver 6 weeks, each group was subj ected to 3 4 1 - hour study periods. The exercise group was trained to fy or 1 hour by receiving ood rewards. C ontrol group 1 was allowed to eed reely but placed into cages that prevented fying. C ontrol group 2 was ed the same ood rewards at the same time as the exercise group, but was also placed into cages that prevented fying. B ody mass was monitored beore and during the experiment ( see gure 2 6) . At the end o the experiment,

511

11 3

An i M Al p h ys i o lo g y ( Ah l ) the mean mass o the birds pectoralis muscles was compared ( fgure 2 6) .

(a)

85 80

[3 ]

c) Suggest how the mass o the birds pectoralis muscle could be determined.

[2 ]

d) O ne hyp othesis that might be ge ne rated rom this e xperiment would be that reducing mo tion in birds might le ad to greate r muscle mass per bird. S uch knowle dge might be used in the arming o poultry. Greate r meat production per bird would result rom the motion o the birds b eing restricte d. D iscuss the ethics o de signing and carrying out experiments to test this hypothe sis. [3 ]

75 70 control 1 control 2 exercise group

65 60 before

(b)

2 weeks

4 weeks

6 weeks

7.5 pectoralis mass (g)

b) Evaluate the claim that preventing exercise increases pectoralis muscle mass.

body mass (g)

a) C ompare the changes in body mass in control group 2 and the exercise group. [2 ]

7 6.5 6 5.5 5

control 1

control 2

exercise

Figure 26 The efect o exercise on body mass and muscle mass in starlings

512

AC E LNEUROBIOLOGY AND BEHAVIOUR L B I O LO GY Introduction

Neurobiology is the scientifc study o the nervous system. Living organisms use their nervous system to detect and respond to changes in the environment. C ommunication between neurons can be altered through the manipulation o the release and reception o chemical messengers. Modifcation o neurons

starts in the earliest stages o embryogenesis and continues to the fnal years o lie. The parts o the brain specialize in dierent unctions. B ehaviour patterns can be inherited or learned. Natural selection avours types o behaviour that increase the chance o survival and reproduction.

A.1 Neural development Understanding  The neural tube o embryonic chordates is

       

ormed by inolding o ectoderm ollowed by elongation o the tube. Neurons are initially produced by diferentiation in the neural tube. Immature neurons migrate to a nal location. An axon grows rom each immature neuron in response to chemical stimuli. Some axons extend beyond the neural tube to reach other parts o the body. A developing neuron orms multiple synapses. Synapses that are not used do not persist. Neural pruning involves the loss o unused neurons. The plasticity o the nervous system allows it to change with experience.

Applications  Incomplete closure o the embryonic neural

tube can cause spina bida.  Events such as strokes may promote reorganization o brain unction.

Skills  Annotation o a diagram o embryonic tissues

in Xenopus, used as an animal model, during neurulation.

Nature of science  Use models as representations o the real

world: developmental neuroscience uses a variety o animal models.

513

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N E U R O B I O LO G Y AN D B E H AVI O U R

Animal models in neuroscience Use models as representations o the real world: developmental neuroscience uses a variety o animal models. Neuroscience is the branch o biology concerned with neurons and nervous systems. The aim o research in developmental neuroscience is to discover how nervous systems are ormed as animals grow rom embryo into adult. The aim o many neuroscientists is to understand and development treatments or diseases o the nervous system, but many experiments are impossible to perorm in humans or ethical reasons. Also, research into other animal species is usually easier because the nervous system develops more rapidly, is less complex and is easier to observe because the embryo develops externally rather than in a uterus.

number o species is used or most o this research and these species are known as animal models:

For these reasons, even when researchers are trying to make discoveries about humans, they work with other species. A relatively small

neural plate dorsal surface



Caenorhabditis elegans ( fatworm) because they have a low xed number o cells as adults and mature very quickly.



Drosophila melanogaster ( ruit fy) because they breed readily, have only 4 pairs o chromosomes and mature very quickly.



Danio rerio ( zebrash) because the tissues are almost transparent.



Xenopus laevis ( Arican clawed rog) because the eggs are large and easily manipulated.



Mus musculus ( house mouse) because ater millennia living near people and their ood, it shares many human diseases.

Development of the neural tube The neural tube o embryonic chordates is ormed by inolding o ectoderm ollowed by elongation o the tube.

gut cavity

neural groove

lateral edges of neural plate join together forming a tube

All chordates develop a dorsal nerve cord at an early stage in their development. The process is called neurulation and in humans it occurs during the rst month o gestation. An area o ectoderm cells on the dorsal surace o the embryo develops into the neural plate. The cells in the neural plate change shape, causing the plate to old inwards orming a groove along the back o the embryo and then separate rom the rest o the ectoderm. This orms the neural tube, which elongates as the embryo grows. The channel inside the neural tube persists as a narrow canal in the centre o the spinal cord.

Development of neurons neural tube

ectoderm

mesoderm

endoderm  Figure 1

514

Stages in neurulation

Neurons are initially produced by diferentiation in the neural tube. There are billions o neurons in the central nervous system ( C NS ) , most o them in the brain. The origins o these neurons can be traced back to the early stages o embryonic development, when part o the ectoderm develops into neuro- ectodermal cells in the neural plate. Although not yet neurons, the developmental ate o these cells is now determined and it is rom them that the nervous system is ormed.

A.1 N e u rAl d e ve lo pm e N t

The neural plate develops into the neural tube, with continued prolieration o cells by mitosis and dierentiation along the pathways leading to the cells becoming unctioning neurons. The mature C NS has ar more neurons than are initially present in the embryonic neural tube, so cell prolieration continues in both the developing spinal cord and brain. Although cell division ceases beore birth in most parts o the nervous system, there are many parts o the brain where new neurons are produced during adulthood.

Neurulation in Xenopus Annotation o a diagram o embryonic tissues in Xenopus, used as an animal model, during neurulation. 13 22 The diagrams in igure 2 sho w ive stage s in the de ve lo p me nt o  a Xen opus e mb ryo , inclu ding the de ve lo p me nt o  the ne u ral tub e . The y sho w the no to cho rd, a su p p o rtive structu re that is p re se nt in all cho rdate s du ring the e arly stage s o  e mb ryo nic de ve lo p me nt b ut which de ve lo p s into the ve rte b ral co lu mn in ve rte b rate s. The no to cho rd is p art o  the me so de rm o  the e mb ryo .

1 18 2

36

Make copies o the diagrams and annotate them to show these structures or stages: 0 

ectoderm, mesoderm and endoderm



development o the neural tube



wall o developing gut and gut cavity



notochord



developing dorsal fn.

neurulation in xenopus  Figure 2

Five stages of embryonic development in Xenopus from day 13 to day 36

Spina bifda Incomplete closure o the embryonic neural tube can cause spina bifda. In vertebrates, including all mammals, the spine comprises a series o bones called vertebrae. Each has a strong centrum that provides support and a thinner vertebral arch, which encloses and protects the spinal cord. The centrum develops on the ventral side o the neural tube at an early stage in embryonic development. Tissue migrates rom both sides o the centrum around the neural tube and normally meets up to orm the vertebral arch.

In some cases the two parts o the arch never become properly used together, leaving a gap. This condition is called spina bifda. It is probably caused by the embryonic neural tube not closing up completely when it is ormed rom the neural groove. S pina bifda is commonest in the lower back. It varies in severity rom very mild with no symptoms, to severe and debilitating.

515

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toK Can reasn n is wn, independen f sense percepin, ever give us knwledge? In the 16th century, both Descartes and Harvey believed that the nerves were hollow conducting tubes through which the Animal spirits do rather beam than are transported. The analogy o messages being beamed like light, or alternatively, fowing like a fuid through tubes is a reasonable hypothesis explaining how our movements could be smooth, sudden and coordinated quickly. It also provided an explanation or how the refexive response to a stimulus could work. Despite Descartes insistence on the hollow nerve, contemporaries noted that nerves have no perceptible cavity internally, as the veins and arteries have. In other words, the theory based on reason was contravened by the empirical evidence.

Migration of neurons Immature neurons migrate to a nal location. Neuronal migration is a distinctive eature o the development o the nervous system. The movement o the unicellular organism Amoeba is easy to observe under a microscope. Neural migration can occur by a similar mechanism. The cytoplasm and organelles in it are moved rom the trailing end o the neuron to the leading edge by contractile actin flaments. Migration o neurons is particularly important in brain development. S ome neurons that are produced in one part o the developing brain migrate to another part where they fnd their fnal position. Mature, unctional neurons do not normally move, though their axons and dendrites can oten regrow i damaged.

Development of axons An axon grows rom each immature neuron in response to chemical stimuli. An immature neuron consists o a cell body with cytoplasm and a nucleus. An axon is a long narrow outgrowth rom the cell body that carries signals to other neurons. O nly one axon develops on each neuron, but it may be highly branched. Many smaller dendrites that bring impulses rom other neurons to the cell body may also develop. C hemical stimuli determine neuron dierentiation when the axon grows out rom the cell body and also the direction in which it grows in the developing embryo.

Growth of axons Some axons extend beyond the neural tube to reach other parts o the body. Axons grow at their tips. In some cases they are relatively short and make connections between neurons within the central nervous system, but other neurons develop very long axons which can reach any part o the body. D espite only being outgrowths o a single cell, axons can be more than a metre long in humans and many metres long in larger mammals such as blue whales. Axons carry impulses to other neurons or to cells that act as eectors  either muscle or gland cells. As long as the cell body o its neuron remains intact, its axon may be able to regrow i severed or damaged in other ways outside the central nervous system. Regrowth rates can be as rapid as fve millimetres per day so sensation or control o muscles can sometimes return over time ater damage. O  course this recovery depends on the correct connections being re- established between an axon and the cells with which it should be communicating.

516

A.1 N e u rAl d e ve lo pm e N t

Development of synapses A developing neuron forms multiple synapses. The growth o an axon or dendrite is directed so that it reaches a cell with which it interacts. A synapse is then developed between the neuron and the other cell. The axons o motor neurons develop synapses with striated muscle fbres or gland cells or example. S ynapse development involves special structures being assembled in the membranes on either side o the synapse and in the synaptic clet between them. The smallest number o synapses that a neuron could theoretically have is two  one to bring impulses rom another cell and another to pass them on. In practice most neurons develop multiple synapses and some neurons in the brain develop hundreds, allowing complex patterns o communication.

Elimination of synapses Synapses that are not used do not persist. Many synapses are ormed at an early stage o development, but new synapses can be ormed at any stage o lie. Synapses oten disappear i they are not used. When transmission occurs at a synapse, chemical markers are let that cause the synapse to be strengthened. Synapses that are inactive do not have these markers so become weaker and are eventually eliminated. The maxim use it or lose it thereore describes synapses very well.

Neural pruning Neural pruning involves the loss of unused neurons. Measurements o the number o neurons have shown that there are more neurons in at least some parts o newborn babies brains than in adults, which indicates that some neurons are lost during childhood. There is also evidence or the removal o dendrites and axon branches rom some neurons. Neurons that are not used destroy themselves by the process o apoptosis. The elimination o part o a neuron or the whole cell is known as neural pruning.

cell body of post-synaptic neuron

nerve endings of pre-synaptic neurons orming synapses

 Figure 3

Drawing based on an electron micrograph showing multiple synapses between pre-synaptic neurons and one post-synaptic neuron. Only the nerve endings of the pre-synaptic neurons are shown

Aciiy Na ning in h isa haas Newborn babies were found to have an estimated 11.2 million neurons in the mediodorsal nucleus of the thalamus, but in adult brains the estimated number was only 6.43 million. Assuming that no extra neurons were produced during childhood, what percentage of neurons disappears by neural pruning?

Plasticity of the nervous system The plasticity of the nervous system allows it to change with experience. C onnections between neurons can be changed by growth o axons and dendrites, by the establishment o new synapses and also by the elimination o synapses and pruning o dendrites, branches o axons or even whole neurons. This ability o the nervous system to rewire its connections is known as plasticity. It continues throughout lie, but there is a much higher degree o plasticity up to the age o six than later. The stimulus or a change in the connections between neurons comes rom the experiences o a person and thus how their nervous system is used. Plasticity is the basis or orming new memories and also or certain orms o reasoning. It is also very important in repairing damage to the brain and spinal cord.

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Strokes Events such as strokes may promote reorganization o brain unction. An ischemic stroke is a disruption o the supply o blood to a part o the brain. Most strokes are caused by a blood clot blocking one o the small vessels in the brain, but bleeding rom a blood vessel is another cause. D uring a stroke part o the brain is deprived o sufcient oxygen and glucose. I cell respiration ceases in neurons, they become irreparably damaged and die.  Figure 4 Angiogram

o the brain o a 48-year-old patient who had sufered a massive stroke. A middle cerebral artery has become blocked by a blood clot

Strokes vary greatly in severity. Many are so minor that the patient hardly notices. About one third o suerers rom major strokes make a ull recovery and another third survive but are let with disability. In many cases recovery rom strokes involves parts o the brain taking on new unctions to supplement the damaged areas. Most recovery happens over the frst six months ater a major stroke and may involve relearning aspects o speech and writing, regaining spatial awareness and the ability to carry out skilled physical activities such as dressing or preparing ood.

A.2 The human brain Understanding  The anterior part o the neural tube expands to  





 



518



orm the brain. Diferent parts o the brain have specic roles. The autonomic nervous system controls involuntary processes in the body using centres located in the medulla oblongata. The cerebral cortex orms a larger proportion o the brain and is more highly developed in humans than other animals. The human cerebral cortex has become enlarged principally by an increase in total area with extensive olding to accommodate it within the cranium. The cerebral hemispheres are responsible or higher order unctions. The let cerebral hemisphere receives sensory input rom sensory receptors in the right side o the body and the right side o the visual eld in both eyes and vice versa or the right hemisphere. The let cerebral hemisphere controls muscle activity in the right side o the body and vice versa or the right hemisphere. Brain metabolism requires large energy inputs.

Applications  Visual cortex, Brocas area, nucleus accumbens

as areas o the brain with specic unctions.  Swallowing, breathing and heart rate as examples o activities coordinated by the medulla.  Use o the pupil reex to evaluate brain damage.  Use o animal experiments, autopsy, lesions and MRI to identiy the role o diferent brain parts.

Skills  Identication o parts o the brain in a

photograph, diagram or scan o the brain.  Analysis o correlations between body size and brain size in diferent animals.

Nature of science  Use models as representations o the real

world: the sensory homunculus and motor homunculus are models o the relative space human body parts occupy on the somatosensory cortex and the motor cortex.

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Development of the brain The anterior part o the neural tube expands to orm the brain. D uring the development o vertebrate embryos a neural tube orms along the whole o the dorsal side, above the gut, near the surace. Most o the neural tube becomes the spinal cord, but the anterior end expands and develops into the brain as part o a process called cephalization, the development o a head. The human brain contains approximately 86 billion neurons ( 8.6  1 0 1 0 ) . The brain acts as the central control centre or the whole body, both directly rom cranial nerves and indirectly via the spinal cord and numerous signal molecules carried by the blood. The advantage o having a brain is that communication between the billions o neurons involved can be more rapid than i control centres were more dispersed. The major sensory organs are located at the anterior end o vertebrates: the eyes, ears, nose and tongue.

Structure of the brain Identication o parts o the brain in a photograph, diagram or scan o the brain. Figure 1 is a diagram showing the main parts o the human brain. Use it to identiy the parts o the brain visible in the photo o the brain and the MRI and C AT scans. These three images are in the electronic resources that accompany this book. skull cerebral hemisphere pineal gland

Roles of the parts of the brain

hypothalamus cerebellum medulla oblongata

Diferent parts o the brain have specic roles. The brain has regions that are distinguishable by their shape, colour or by microscopic structure. These regions have dierent roles, identifed by physiological research in humans and other mammals. The medulla oblongata is used in autonomic control o gut muscles, breathing, blood vessels and heart muscle.

spinal cord pituitary gland  Figure 1

vertebra

Diagram of the brain

The cerebellum coordinates unconscious unctions, such as posture, non- voluntary movement and balance. The hyp othalamus is the interace between the brain and the pituitary gland, synthesizing the hormones secreted by the posterior pituitary, and releasing actors that regulate the secretion o hormones by the anterior pituitary. The p ituitary gland: the posterior lobe stores and releases hormones produced by the hypothalamus and the anterior lobe produces and secretes hormones that regulate many body unctions. The cerebral hemisp heres act as the integrating centre or high complex unctions such as learning, memory and emotions.

Methods of brain research Use o animal experiments, autopsy, lesions and MRI to identiy the role o diferent brain parts. Lesion studies gave the frst useul inormation about brain unctions. For example, in the 1 9th century, ater the death and autopsy o a patient who could only say the word Tan, the French neurologist Charcot ound a single large tumour damaging the lower let side o the patients brain. He deduced

that this part o the brain is involved with speech. Another amous case was the railway construction worker Phineas Gage, who suered severe damage to the rontal lobes o his brain in 1 848 when an accident with explosives caused a large metal rod to pass through his orehead. He recovered rom

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the wound but the brain damage radically and permanently altered his personality and particularly his capacity or social interaction. Many lesions due to tumours, strokes or accidental damage have been investigated by carrying out an autopsy and relating the position o the lesion to observed changes in behaviour and capacities, but rather than wait or these ortuitous opportunities, some neuroscientists have studied experimental animals. Removal o parts o the skull gives access to the brain and allows experimental procedures to be perormed. The brain itsel does not eel pain  even today some orms o neurosurgery are perormed on ully conscious patients. The eects o local stimulation in an animals brain can be observed, as can long- term changes in the animals temperament and capacities. There are widespread obj ections to such research, because o the suering they may cause to the animal and because at the end the animal is oten sacriced, but the inormation obtained is useul to understanding, and thereore treating, conditions such as epilepsy, Parkinsons disease and multiple sclerosis. Increasingly genetic mutants and selective inactivation o genes, which are technically possible only in mice, are used to achieve similar experimental modication o brain structure and behaviour.

 Figure 2

Image of brain lesion

that are activated by specic thought processes to be identied. Active parts o the brain receive increased blood fow, oten made visible by inj ecting a harmless dye, which MRI records. The subj ect is placed in the scanner and a highresolution scan o the brain is taken. A series o low- resolution scans is then taken while the subj ect is being given a stimulus. These scans show which parts o the brain are activated during the response to the stimulus.

Magnetic resonance imaging ( MRI) is a more modern and less controversial technique. B asic MRI is used to investigate the internal structure o the body, including looking or tumours or other abnormalities in patients. Figure 2 shows the results o an MRI scan o the upper part o a patients body, including the head and brain. A specialized version o MRI, called unctional magnetic resonance imaging ( MRI) has been developed, which allows the parts o the brain

 Figure 3

fMRI scan of endometriosis pain

Examples of brain functions Visual cortex, Brocas area, nucleus accumbens as areas o the brain with specifc unctions. E ach o the two cerebral hemispheres has a visual cortex in which neural signals originating rom light sensitive rod and cone cells in the retina o the eyes are processed. Although

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there is an initial stage in which a map o visual inormation is proj ected in a region called V1 , the inormation is then analysed by multiple pathways in regions V2 to V5 o the visual

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cortex. This analysis includes pattern recognition and j udging the speed and direction o moving obj ects. B rocas area is a part o the let cerebral hemisphere that controls the production o speech. I there is damage to this area an individual knows what they want to say and can produce sounds, but they cannot articulate meaningul words and sentences. For example, i we see a horse- like animal with black and white stripes, B rocas area allows us to say zebra, but a

person with a damaged B rocas area knows that it is a zebra but cannot say the word. There is a nucleus accumbens in each o the cerebral hemispheres. It is the pleasure or reward centre o the brain. A variety o stimuli including ood and sex cause the release o the neurotransmitter dopamine in the nucleus accumbens, which causes eelings o well-being, pleasure and satisaction. C ocaine, heroin and nicotine are addictive because they articially cause release o dopamine in the nucleus accumbens.

The autonomic nervous system The autonomic nervous system controls involuntary processes in the body using centres located in the medulla oblongata. The peripheral nervous system comprises all o the nerves outside the central nervous system. It is divided into two parts: the voluntary and the autonomic nervous systems. Involuntary processes are controlled by the autonomic nervous system, using centres in the medulla oblongata. The autonomic nervous system has two parts: sympathetic and parasympathetic. These oten have contrary eects on an involuntary process. For example, parasympathetic nerves cause an increase in blood fow to the gut wall during digestion and absorption o ood. S ympathetic nerves cause a decrease in blood fow during asting or when blood is needed elsewhere.

Activities coordinated by the medulla Swallowing, breathing and heart rate as examples of activities coordinated by the medulla. The rst phase o swallowing, in which ood is passed rom the mouth cavity to the pharynx, is voluntary and so is controlled by the cerebral cortex. The remaining phases in which the ood passes rom the pharynx to the stomach via the esophagus, are involuntary and are coordinated by the swallowing centre o the medulla oblongata. Two centres in the medulla control breathing: one controls the timing o inspiration; the other controls the orce o inspiration and also active, voluntary expiration. There are chemoreceptors in the medulla that monitor blood pH. The carbon dioxide concentration in the blood is very important in controlling breathing rate, even

more than oxygen concentration. I blood pH alls, indicating an increase in carbon dioxide concentration, breathing becomes deeper and/or more requent. The cardiovascular centre o the medulla regulates the rate at which the heart beats. B lood pH and pressure are monitored by receptor cells in blood vessels and in the medulla. In response to this inormation, the cardiovascular centre can increase or decrease the heart rate by sending signals to the hearts pacemaker. S ignals carried rom the sympathetic system speed up the heart rate; signals carried by the parasympathetic system in the vagus nerve slow the rate down.

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The pupil refex and brain damage Use o the pupil refex to evaluate brain damage. Muscles in the iris control the size o the pupil o the eye. Impulses carried to radial muscle bres by neurons o the sympathetic system cause them to contract and dilate the pupil; impulses carried to circular muscle bres by neurons o the parasympathetic system cause the pupil to constrict. The pupil refex occurs when bright light suddenly shines into the eye. Photoreceptive ganglion cells in the retina perceive the bright light, sending signals through the optic nerve to the mid- brain, immediately activating the parasympathetic system that stimulates circular

muscle in the iris, constricting the pupil and reducing the amount o light entering the eye, protecting the delicate retina rom damage. D octors sometimes use the pupil refex to test a patients brain unction. A light is shone into each eye. I the pupils do not constrict at once, the medulla oblongata is probably damaged. I this and other tests o brain stem unction repeatedly ail, the patient is said to have suered brain death. It may be possible to sustain other parts o the patients body on a lie support machine, but ull recovery is extremely unlikely.

The cerebral cortex The cerebral cortex orms a larger proportion o the brain and is more highly developed in humans than other animals. The cerebral cortex is the outer layer o the cerebral hemispheres. Although it is only two to our millimetres thick, up to six distinctively dierent layers o neurons can be identied in sections studied under a microscope. It is has a highly complex architecture o neurons and processes the most complex tasks in the brain. Only mammals have a cerebral cortex. B irds and reptiles have regions o the brain that perorm a similar range o unctions but they are structurally dierent, with cells arranged in clusters rather than layers. Among the mammals the cerebral cortex varies in size considerably. In humans it orms a larger proportion o the brain than in any other mammal.

The evolution o the cerebral cortex frontal lobe

parietal lobe occipital lobe

temporal lobe

medulla oblongata

 Figure 4 The folded

cerebellum

structure of the cerebral cortex, viewed from the left side. The four lobes are indicated

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The human cerebral cortex has become enlarged principally by an increase in total area with extensive olding to accommodate it within the cranium. The cerebral cortex has become greatly enlarged during human evolution, and now contains more neurons than that o any other animal. There has been a modest increase in thickness, but the cortex is still only a ew millimetres thick. The increase is due principally to an increase in total area and that necessitates the cortex becoming extensively olded during development. It is hard to measure, but the area is estimated to be about 1 80, 000 mm 2 or 0.1 8 m 2 . This is so large that the brain can only be accommodated inside a greatly enlarged cranium, orming the distinctive shape o the human skull. Most o the surace area o the cerebral cortex is in the olds rather than on the outer surace. In contrast, mice and rats have an unolded smooth cortex, but in cats there are some olds and elephants and dolphins have

A. 2 th e h u m AN b r Ai N more. Among the primates, monkeys and apes show a range o cortex size and degree o olding, with larger sizes in primates that are more closely related to humans.

Comparing brain size Analysis o correlations between body size and brain size in diferent animals.

elephant 4.8 kg human 1.4 kg chimp 0.42 kg

S cattergraphs show a positive correlation between body size and brain size in animals, but that the relationship is not directly proportional. The databased questions below can be used to develop your skill in analysing this type o data.

daa-as qsons: Brain and body size in mammals

mass of brain/g (log scale)

10 4 10 3

monotremes marsupials placentals

elephant human

1

hump-backed whale

dolphin chimpanzee fox sheep 10 2 cat echidna grey kangaroo squirrel monkey quokka 10 1 platypus brush-tailed possum opossum bandicoot 10 0 rat hedgehog shrew 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 mass of body/g (log scale)

The scattergraph in fgure 5 shows the relationship between brain and body mass in species o placental, marsupial and monotreme mammal.

[1 ]

2

E xplain how the points on the scattergraph would have been arranged i brain mass was directly proportional to body mass. [2 ]

3

S tate which mammals have ( a) the largest and ( b) the smallest brain mass. [2 ]

4

D iscuss the evidence provided by the scattergraph or the hypothesis that humans have the largest relative brain mass. [2 ]

5

E valuate the hypothesis that marsupials have relatively small brains compared with other mammals.

10 8

 Figure 5

S tate the relationship between brain and body mass.

6

[2 ]

S uggest a reason or the researchers not including more data or monotremes in the scattergraph. [1 ]

Functions of the cerebral hemispheres The cerebral hemispheres are responsible or higher order unctions. The cerebral hemispheres carry out the most complex o the brains tasks. These are known as higher order unctions and include learning, memory, speech and emotions. These higher order unctions involve association o stimuli rom dierent sources including the eye and ear and also rom memories. They rely on very complex networks o neurons that are still only partially understood by neurobiologists. The most sophisticated thought processes such as reasoning, decision- making and planning occur in the rontal and prerontal lobes o the cerebral cortex. Using these parts o the brain we can organize our actions in a

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Sensory inputs to the cerebral hemispheres The let cerebral hemisphere receives sensory input rom sensory receptors in the right side o the body and the right side o the visual feld in both eyes and vice versa or the right hemisphere. The cerebral hemispheres receive sensory inputs rom all the sense organs o the body. For example, signals rom the ear pass to the auditory area in the temporal lobe. Signals rom the let ear pass to the let hemisphere and rom the right ear to the right hemisphere. Inputs rom the skin, muscles and other internal organs pass via the spinal cord to the somatosensory area o the parietal lobe. Perhaps surprisingly, the impulses rom each side cross in the base o the brain so that the let hemisphere receives impulses rom the right side o the body and vice versa. Inputs rom the eye pass to the visual area in the occipital lobe, known as the visual cortex. Impulses rom the right side o the feld o vision in each eye are passed to the visual cortex in the let hemisphere, while impulses rom the let side o the feld o vision in each eye pass to the right hemisphere. This integration o inputs enables the brain to judge distance and perspective.

Motor control by the cerebral hemispheres The let cerebral hemisphere controls muscle activity in the right side o the body and vice versa or the right hemisphere. Regions in each o the cerebral hemispheres control striated (voluntary) muscles. The main region is in the posterior part o the rontal lobe and is called the primary motor cortex. In this region there is a series o overlapping areas that control muscles throughout the body, rom the mouth at one end o the primary motor cortex to the toes at the other end. The primary motor cortex in the let hemisphere controls muscles in the right side o the body and that in the right side controls muscles in the let side o the body. So a stroke ( or other brain damage) in the let side o the brain can cause paralysis in the right side o the body and vice versa.

Homunculi Use models as representations o the real world: the sensory homunculus and motor homunculus are models o the relative space human body parts occupy on the somatosensory cortex and the motor cortex. Neurobiologists have constructed models o the body in which the size o each part corresponds to the proportion o the somatosensory cortex

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devoted to sensory inputs rom that part. This type o model is called a sensory homunculus. S imilar models have been constructed to show the

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r lip

primary somatosensory cortex

lip s lower lip teeth, gums tongue

d

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l i tt l m i ri n e dd g i th n d e l e um x b

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of the relative importance given to sensory inputs from different parts of the body and to control of muscles in different parts.

ha n

proportion of the motor cortex that is devoted to control of muscles in each part of the body. These models are useful as they give a good impression

li p s

primary motor cortex

ja w sw

pharynx

allo

ton g wi n

ue

g

int ra ab do mi na l

 Figure 6 Sensory

homunculus (left) and motor homunculus ( right)

Energy and the brain Brain metabolism requires large energy inputs. E nergy released by cell respiration is needed to maintain the resting potential in neurons and to re- establish it after an action potential, as well as for synthesis of neurotransmitters and other signal molecules. The brain contains a huge number of neurons so it needs much oxygen and glucose to generate this energy by aerobic cell respiration. In most vertebrates the brain uses less than 1 0% of the energy consumed by basal metabolism but in the adult human brain it is over 2 0% and an even higher proportion in infants and small children.

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A.3 percetion of stimuli Understanding

Applications

 Receptors detect changes in the environment.

 Red-green colour-blindness as a variant o

 Rods and cones are photoreceptors located in

normal trichromatic vision.  Detection o chemicals in the air by the many diferent olactory receptors.  Use o cochlear implants in dea patients.

   

   

the retina. Rods and cones difer in their sensitivities to light intensities and wavelengths. Bipolar cells send the impulses rom rods and cones to ganglion cells. Ganglion cells send messages to the brain via the optic nerve. The inormation rom the right eld o vision rom both eyes is sent to the let part o the visual cortex and vice versa. Structures in the middle ear transmit and ampliy sound. Sensory hairs o the cochlea detect sounds o specic wavelengths. Impulses caused by sound perception are transmitted to the brain via the auditory nerve. Hair cells in the semicircular canals detect movement o the head.

Skills  Labelling a diagram o the structure o the

human eye.  Annotation o a diagram o the retina to show the cell types and the direction o the light source.  Labelling a diagram o the structure o the human ear.

Nature of science  Understanding o the underlying science is

the basis or technological developments: the discovery that electrical stimulation in the auditory system can create a perception o sound resulted in the development o electrical hearing aids and ultimately cochlear implants.

Sensory receptors Receptors detect changes in the environment. The environment, particularly its changes, stimulate the nervous system via sensory receptors. The nerve endings of sensory neurons act as receptors, for example touch receptors. In other cases there are specialized receptor cells that pass impulses to sensory neurons, as with the light-sensitive rod and cone cells of the eye. Humans have the following types of specialized receptor.

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

Mechanoreceptors respond to mechanical forces and movements.



C hemoreceptors respond to chemical substances.



Thermoreceptors respond to heat.



Photoreceptors respond to light.

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Olfactory receptors Detection o chemicals in the air by the many diferent olactory receptors. O lfaction is the sense of smell. O lfactory receptor cells are located in the epithelium inside the upper part of the nose. These cells have cilia which proj ect into the air in the nose. Their membrane contains odorant receptor molecules, proteins which detect chemicals in the air. O nly volatile chemicals can be smelled in air within the nose. O dorants from food in the mouth can pass through mouth and nasal cavities to reach the nasal epithelium. There are many different odorant receptor proteins, each encoded by a different gene. In some mammals such as mice there are over a thousand different odorant receptors, each of which detects a different chemical or group of chemicals ( though the exact mechanisms are still unclear in spite of intensive study) . Each olfactory receptor cell has j ust one type of odorant receptor in its membrane, but there are many receptor cells with each type of odorant receptor, distributed though the nasal epithelium. Using these receptor cells most animals, including mammals, can distinguish a large number of chemicals in the air, or in water in the case of aquatic animals. In many cases the chemical can be detected in extremely low concentrations but the human

sense of smell is very insensitive and imprecise compared to that of other animals.

 Figure 1

Olfactory receptor cell (centre) with two of its cilia visible and also cilia in adjacent cells in the nasal epithelium

Structure of the eye Labelling a diagram o the structure o the human eye. sclera lens aqueous humour pupil

choroid retina fovea

iris conjunctiva blind spot cornea optic nerve vitreous humour  Figure 2

A diagram of the human eye in horizontal section

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Photoreceptors

toK if ur senses can be fled by llusns, wha are he mplcans fr knwledge clams based n emprcal evdence? Scientists argue that because the visual sense is dominant, illusions can arise when conficting inormation is received rom visual inormation and the other senses. Food dyed with colouring to make it appear odd becomes unpalatable. In the McGurk eect, seeing mouth movements corresponding to one sound paired with the auditory inormation o another sound causes the subject to hear the sound corresponding to the mouth movements. In the rubber hand illusion, experimenters can eect a sensation in subjects by stroking a rubber hand that they have stroked in the same way as their real hand.

Acvy

Rods and cones are photoreceptors located in the retina. Light entering the eye is ocused by the cornea and the lens onto the retina, the thin layer o light- sensitive tissue at the back o the eye. Figure 5 shows the cell types in the retina. Two main types o photoreceptor are present in the human retina, rods and cones. Many nocturnal mammals have only rods and cannot distinguish colours. Rods and cones are stimulated by light and so together detect the image ocused on the retina and convert it into neural signals.

Diferences between rods and cones Rods and cones dier in their sensitivities to light intensities and wavelengths. Rods are very sensitive to light, so work well in dim light. In very bright light the pigment in them is temporarily bleached so or a ew seconds they do not work. Rod cells absorb a wide range o visible wavelengths o light ( see fgure 3 ) but cannot respond selectively to dierent colours, so they give us black and white vision. There are three types o cone, which absorb dierent ranges o wavelengths o light. They are named according to the colour that they absorb most strongly: red, blue or green. When light reaches the retina, the red, blue and green cones are selectively stimulated. B y analysing the relative stimulation o each o the three cone types, the colour o light can be very precisely determined, though experiments show that peoples perception o colour diers quite a lot. C ones are only stimulated by bright light and thereore colour vision ades in dim light.

Caarac surgery 420

498

534 564

100 normalized absorbance

Accumulation o metabolic wastes in the cells o the eyes lens gradually turns them yellow so blues ade. The dierence in colour perception ater a cataract operation is startling. Talk to a person, probably elderly, who has had cataract surgery to nd out how it changed their colour perception.

50

S

R

M

L

0 400 violet

500 blue

cyan

green

600 yellow

700 red

wavelength (nm)  Figure 3 Absorption spectra for blue (short, S) , green (medium, M)

wavelength-sensitive cones and for rods (dotted line)

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and red (long, L)

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Red-green colour-blindness Red-green colour-blindness as a variant of normal trichromatic vision. Red-green colour- blindness is a common inherited condition in humans and some other mammals. It is due to the absence o, or a deect in, the gene or photoreceptor pigments essential to either red or green cone cells. B oth genes are located on the human X chromosome so it is a sex- linked condition. The normal alleles o both genes are dominant and the alleles that cause red- green colour- blindness are recessive. Red-green colourblindness is thereore much commoner among males, who have only one X chromosome, than emales, and males inherit the allele that causes the condition rom their mother.

 Figure 4 Red

and green colours cannot easily be distinguished by some males and fewer females

Structure of the retina Annotation of a diagram of the retina to show the cell types and the direction of the light source. The arrangement o the layers o cells in the retina may seem surprising. The light passes frst through a layer o transparent nerve axons that carry impulses rom the retina to the brain through the optic nerve, then through a layer o specialized bipolar neurons that process signals beore they reach the optic nerve, and only then does the light reach the rod and cone cells. This is shown in fgure 5 .

ganglion cell

direction of light nerve bres of ganglion cells bipolar neuron

rod cell cone cell

layer of pigmented cells  Figure 5 Arrangement of cell

types in the retina

Bipolar cells Bipolar cells send the impulses from rods and cones to ganglion cells. Rod and cone cells synapse with neurons called bipolar cells in the retina. I rod or cone cells are not stimulated by light they depolarize and release an inhibitory neurotransmitter onto a bipolar cell, causing it to become hyperpolarized and not transmit impulses to its associated retinal ganglion cell. When light is absorbed by a rod or cone cell it becomes hyperpolarized and stops sending inhibitory neurotransmitter to the bipolar cell. The bipolar cell can thereore depolarize, activating the adj acent ganglion cell. Groups o rod cells send signals to the brain via a single bipolar cell, so the brain cannot distinguish which rod absorbed the light. The images

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N E U R O B I O LO G Y AN D B E H AVI O U R transmitted to the brain by rods alone are lower resolution, like a grainy photograph, whereas those based on the cones are sharper because each cone cell sends signals to the brain via its own bipolar cell.

Ganglion cells Ganglion cells send messages to the brain via the optic nerve. Retinal ganglion cells have cell bodies in the retina with dendrites that orm synapses with bipolar cells. Ganglion cells also have long axons along which impulses pass to the brain. Impulses are passed at a low requency when the ganglion cell is not being stimulated and at an increased rate in response to stimuli rom bipolar cells. The axons o ganglion cells pass across the ront o the retina to orm a central bundle at the blind spot, so called because their presence makes a gap in the layer o rods and cones. The axons o the ganglion cells pass via the optic nerve to the optic chiasma in the brain.

Vision in the right and let felds The inormation rom the right feld o vision rom both eyes is sent to the let part o the visual cortex and vice versa.

visual eld

right eye right optic nerve optic chiasma thalamus visual cortex  Figure 6 The optic chiasma

Simple experiments comparing vision with one eye or with both eyes show the distance and relative size o objects can be judged most precisely when observed by two eyes simultaneously. Stimuli rom both eyes are integrated by the axons o some retinal ganglion cells crossing rom one side to the other between eye and brain while other axons stay on the same side. The crossing over o axons between let and right sides happens in the optic chiasma, shown in fgure 6. As a result, the visual cortex in the right cerebral hemisphere processes visual stimuli rom the let side o the visual feld o both eyes, and vice versa or stimuli rom the right side o the feld o vision.

Structure o the ear Labelling a diagram o the structure o the human ear. pinna

bones of skull

incus malleus stapes semicircular canals

muscle attached

auditory nerve

oval window ear drum cochlea

round window

 Figure 7

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The structure of the ear

A. 3 per Ce pti o N o f sti m u li

The middle ear Structures in the middle ear transmit and ampliy sound. The middle ear is an air-lled chamber between the outer ear and the inner ear. A thin, taut sheet o fexible tissue called the eardrum separates the middle ear rom the outer ear. Two other thin sheets o tissue called the oval and round windows separate the middle ear rom the inner ear. Three tiny bones are in the middle ear, the malleus ( hammer) , incus ( anvil) and stapes ( stirrup) , which articulate with each other to orm a connection between the eardrum and the oval window. These bones, also called ossicles, transmit vibrations rom the eardrum to the oval window, ampliying sound twentyold because the oval window has a smaller area than the eardrum. D uring very loud sounds, the delicate sound- reception components o the ear are protected by contraction o the muscles attached to the bones in the middle ear, which weakens the connections between the ossicles and so damps the vibrations.

The cochlea Sensory hairs o the cochlea detect sounds o specic wavelengths. The cochlea is the part o the inner ear where vibrations are transduced into neural signals. It is a tubular, coiled, fuid-lled structure. Within the cochlea are layers o tissue (membranes) to which sensory cells are attached. Each o these cells has a bundle o hairs, stretching rom one membrane to another. When vibrations are transmitted rom the oval window into the cochlea, they resonate with the hair bundles o particular hair cells, stimulating these cells. Selective activation o dierent hair cells enables us to distinguish between sounds o dierent pitch. The round window is another thin sheet o fexible tissue, located between the middle and inner ear. I it was sti and indeormable, the oval window would not be able to vibrate, because the incompressible fuid in the cochlea would prevent it rom moving. When vibrations o the oval window push the fuid in the cochlea inwards, the round window moves outwards, and when the oval window moves outwards, the round window moves inwards, enabling the oval window to transmit vibrations through the fuid in the cochlea.

i han a nv ny  can ang  , wha cnqnc  an gh h hav  h acqn  knwdg? Figure 8 shows the requency sensitivity o six land mammals. The solid area shows where requency sensitivity is best, while the lines indicate how much louder other requencies need to be in order to be heard. 1 Does the world sound the same to any o the animals? 2 Which is the real world  the one we perceive or the world perceived by the bat? 3 Animals also difer considerably in their visual perception. Is what each animal sees what is really there, is it a construction o reality, or is reality a alse concept? 0 dB +20 dB +40 dB +60 dB human cat guinea pig monkey bat rat 10

100

1000

10000

100000 frequency (Hz)

 Figure 8 Sensitivity

of mammals to frequencies of sound

The auditory nerve Impulses caused by sound perception are transmitted to the brain via the auditory nerve. When a hair cell in the cochlea is depolarized by the vibrations that constitute sounds, it releases neurotransmitter across a synapse, stimulating an adj acent sensory neuron. This triggers an action potential in the sensory neuron which propagates to the brain along the auditory nerve. The auditory nerve is one o the cranial nerves that serve the brain.

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Cochlear implants Use of cochlear implants in deaf patients. Deaness has a variety o causes and in many cases a hearing aid that amplifes sounds can overcome the problem. However, i the hair cells in the cochlea are deective, such hearing aids do not help. In this case the best option, as long as the auditory nerve is unctioning properly, is a cochlear implant. More than a quarter o a million people have had these devices implanted and although they do not ully restore normal hearing, they improve it and usually allow recognition o speech.

these signals into electrical impulses and an array o electrodes that carry these impulses to the cochlea. The electrodes stimulate the auditory nerve directly and so bypass the nonunctional hair cells.

transmitter receiver and stimulator microphone

C ochlear implants consist o external and internal parts. 



The external parts are a microphone to detect sounds, a speech processor that selects the requencies used in speech and flters out other requencies, and a transmitter that sends the processed sounds to the internal parts. The internal parts are implanted in the mastoid bone behind the ear. They consist o a receiver that picks up sound signals rom the transmitter, a stimulator that converts

electrode array

 Figure 8 Cochlear implant with

microphone behind the ear connected to the transmitter and adjacent to this the internal receiver and stimulator, with electrodes leading to the auditory nerve that arises in the cochlea

The science behind cochlear implants Understanding of the underlying science is the basis for technological developments: the discovery that electrical stimulation in the auditory system can create a perception of sound resulted in the development of electrical hearing aids and ultimately cochlear implants. Research into artifcial electrical stimulation o the cochlea began as early as the 1 9 5 0s. E arly attempts showed that it was possible to give some perception o sound to people who were severely or prooundly dea due to non- unctioning hair cells. E xperiments with humans showed that electrical stimulation could be used to give perception o dierent requencies o sound, as in music. Research continued and involved electronic engineers, neurophysiologists and clinical audiologists. An understanding o which requencies are used to understand speech was used to develop speech processors or example.

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D uring the 1 970s early versions o cochlear implants were ftted to over a thousand patients. S ince then research has led to huge technological developments in these devices with greatly improved outcomes or the increasing number o people that have had them ftted. Further improvements can be expected and although cochlear implants can never give severely or prooundly dea people normal hearing, they can allow ar better hearing than without this technology.

A . 4 i N N At e A N d l e A r N e d b e h Av i o u r ( A h l )

Detecting head movements

1

Hair cells in the semicircular canals detect movement o the head. There are three fuid-lled semicircular canals in the inner ear. Each has a swelling at one end in which there is a group o sensory hair cells, with their hairs embedded in gel to orm a structure called the cupula. When the head moves in the plane o one o the semicircular canals, the sti wall o the canal moves with the head, but due to inertia the fuid inside the canal lags behind. There is thereore a fow o fuid past the cupula. This is detected by the hair cells, which send impulses to the brain. The three semicircular canals are at right angles to each other, so each is in a dierent plane. They can thereore detect movements o the head in any direction. The brain can deduce the direction o movement by the relative amount o stimulation o the hair cells in each o the semicircular canals.

2

 Figure 9

Inner ear with cochlea (left) and semicircular canals (right) : superior (1) , lateral (2) and posterior (3)

A.4 inna an an a (Ahl) Understanding  Innate behaviour is inherited rom parents

    

  

and so develops independently o the environment. Autonomic and involuntary responses are reerred to as refexes. Refex arcs comprise the neurons that mediate refexes. Learned behaviour develops as result o experience. Refex conditioning involves orming new associations. Imprinting is learning occurring at a particular lie stage and is independent o the consequences o behaviour. Operant conditioning is a orm o learning which consists o trial and error experiences. Learning is the acquisition o skill or knowledge. Memory is the process o encoding, storing and accessing inormation.

Applications  Withdrawal refex o the hand rom a painul

stimulus.  Pavlovs experiments into refex conditioning in dogs.  The role o inheritance and learning in the development o birdsong.

Skills  Analysis o data rom invertebrate behaviour

experiments in terms o the eect on chances o survival and reproduction.  Drawing and labelling a diagram o a refex arc or a pain withdrawal refex.

Nature of science  Looking or patterns, trends and discrepancies:

laboratory experiments and eld investigations helped in the understanding o dierent types o behaviour and learning.

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Innate behaviour Innate behaviour is inherited rom parents and so develops independently o the environment. Animal behaviour is divided into two broad categories, innate and learned. The orm o innate behaviour is unaected by external infuences that an animal experiences. It develops independently o the environment. For example, i an obj ect touches the skin in the palm o a babys hand, the baby grips the obj ect by closing its ngers around it. This innate behaviour pattern, called the palmar grasp refex, is seen in babies rom birth until they are about six months old, whatever experiences the baby has. Innate behaviour is genetically programmed, so it is inherited. It can change through evolution i there is genetically determined variation in behaviour and natural selection avours one behaviour pattern over others, but the rate o change is much slower than with learned behaviour.

Research methods in animal behaviour Looking or patterns, trends and discrepancies: laboratory experiments and eld investigations helped in the understanding o diferent types o behaviour and learning. The scientic study o animal behaviour became established as a signicant branch o biology in the 1 93 0s. B eore then naturalists observed the behaviour o animals in natural habitats but had rarely analysed it scientically. Two general types o methodology have since been used: laboratory experiments and eld investigations.

The advantage o laboratory experiments is that variables can be controlled more eectively and innate behaviour in particular can be investigated rigorously. The disadvantage is that animal behaviour is an adaptation to the natural environment o the species and animals oten do not behave normally when removed rom that environment, especially with learned behaviour.

Invertebrate behaviour experiments Analysis o data rom invertebrate behaviour experiments in terms o the efect on chances o survival and reproduction. Many invertebrates have relatively simple behaviour patterns, so they can be studied more easily than mammals, birds or other vertebrates. A stimulus can be given and the response to it observed. Repeating the stimulus with a number o individuals allows quantitative data to be obtained and tests o statistical signicance to be done. O nce the response to a stimulus has been discovered, it may be possible to deduce how the response improves animals chances o survival

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and reproduction and thus how it evolved by natural selection as an innate behaviour pattern. Many dierent invertebrates can b e used in experiments. Planarian latworms, woodlice, b lowly larvae, snails and b eetles are oten used. S ome species can b e purchased rom suppliers b ut it is also possible to use invertebrates rom local hab itats. These should be kept or a short time only, protected rom suering during the

A . 4 i N N At e A N d l e A r N e d b e h Av i o u r ( A h l )

experiments and then returned to their habitat. E ndangered species should not be used. Two types o behaviour involving movement could be investigated: 



Taxis is movement towards or away rom a directional stimulus. An example is movement o a woodlouse or slater away rom light. Kinesis also involves movement as a response, but the direction o movement is not infuenced by the stimulus. Instead, the speed o movement or the number o times the animal turns is varied. An example is slower movement, with more requent turning, when woodlice are transerred rom drier to more damp conditions.

S tages in designing an investigation: 1

Place the animals in conditions that are similar to the natural habitat.

2

O bserve the behaviour and see what stimuli aect movement.

3

C hoose one stimulus that appears to cause a taxis or kinesis.

4

D evise an experiment to test responses to the stimulus.

5

E nsure that other actors do not have an eect on the movement.

6

D ecide how to measure the movement o the invertebrates.

Refexes Autonomic and involuntary responses are reerred to as refexes. A stimulus is a change in the environment, either internal or external, that is detected by a receptor and elicits a response. A response is a change in an organism, oten carried out by a muscle or a gland. S ome responses happen without conscious thought and are thereore called involuntary responses. Many o these are controlled by the autonomic nervous system. These autonomic and involuntary responses are known as refexes. A refex is a rapid unconscious response to a stimulus. The pupil refex is an example: in response to the stimulus o bright light, the radial muscles in the iris o the eye contract, constricting the pupil. This involuntary response is carried out by the autonomic nervous system.

Refex arcs Refex arcs comprise the neurons that mediate refexes. All refexes start with a receptor that perceives the stimulus and ends with an eector, usually a muscle or gland, which carries out the response. Linking the receptor to the eector is a sequence o neurons, with synapses between them. The sequence o neurons is known as a refex arc. In the simplest refex arcs there are two neurons: a sensory neuron to carry impulses rom the receptor to a synapse with a motor neuron in the spinal cord and a motor neuron to carry impulses on to the eector. Most refex arcs contain more than two neurons, as there are one or more relay neurons connecting the sensory neuron to the motor neuron.

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The withdrawal refex

Activity refex speed

Withdrawal refex o the hand rom a painul stimulus.

The withdrawal refex takes less than a tenth o a second. Reaction times that involve more complex processing take longer. Use online tests i you want to assess your reaction time, using the search term refex test to nd them.

The pain withdrawal refex is an innate response to a pain stimulus. For example i we touch a hot obj ect with the hand, pain receptors in the skin o the nger detect the heat and activate sensory neurons, which carry impulses rom the nger to the spinal cord via the dorsal root o a spinal nerve. The impulses travel to the ends o the sensory neurons in the grey matter o the spinal cord where there are synapses with relay neurons. The relay neurons have synapses with motor neurons, which carry impulses out o the spinal cord via the ventral root and to muscles in the arm. Messages are passed across synapses rom motor neurons to muscle bres, which contract and pull the arm away rom the hot obj ect.

Neural pathways in a refex arc Drawing and labelling a diagram o a refex arc or a pain withdrawal refex. Figure 1 shows the refex arc or the pain withdrawal refex. receptor cells or nerve endings sensing pain relay neuron cell body of sensory neuron in the dorsal root ganglion dorsal root of spinal nerve

nerve bre of sensory neuron

central canal

spinal nerve

nerve bre of motor neuron eector (muscle that pulls hand away from pain when it contracts)

ventral root of spinal nerve cell body of motor neuron

white matter grey matter spinal cord

 Figure 1

Components o a refex arc

Learned behaviour Learned behaviour develops as result o experience. O spring inherit the capacity or propensity to acquire new patterns o behaviour during their lie, as a result o experience. This is known as learned behaviour. O spring learn behaviour patterns rom their parents, rom other individuals and rom their experience o the

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A . 4 i N N At e A N d l e A r N e d b e h Av i o u r ( A h l ) environment. For example, human ospring inherit the capacity to learn a language. The language that they learn is usually that o their biological parents, but not i they are adopted by adults who speak a dierent language. The ability to make sense o vocal patterns and then make them onesel is innate but the specifc language spoken is learned.

Development of birdsong The role of inheritance and learning in the development of birdsong. B irdsong has been investigated intensively in some species and evidence has been ound or it being partly innate and partly learned. All members o a bird species share innate aspects o song, allowing each individual to recognize other members o the species. In many species,

including all passerines, males learn mating calls rom their ather. The learned aspects introduce dierences, allowing males to be recognized by their song and in some species mates to be chosen by the quality o their singing.

daa-as qsns: Birdsong  innate or learned? The sonograms in fgure 2 are a visual representation o birdsong, with time on the x-axis and requency or pitch on the y- axis. 1

C ompare sonograms I and II, which are rom two populations o white- crowned sparrows (Zonotrichia leucophrys). [2 ]

2

S onogram III is rom a white- crowned sparrow that was reared in a place where it could not hear any other birdsong. a) C ompare sonogram III with sonograms I and II. [2 ]

c)

S uggest two reasons why birds rarely imitate other species.

[2 ]

d) D iscuss whether Morton and B aptistas observation is evidence or innate or learned development o birdsong. [2 ] I

II

b) Discuss whether the song o white-crowned sparrows is innate, learned or due to both innate actors and learning. [3 ] 3

In 1 981 Martin Morton and Luis B aptista published a very unusual discovery  a whitecrowned sparrow had learned to imitate the song o another species. S onogram IV is rom a strawberry fnch (Amandava amandava). Sonogram V is rom a white- crowned sparrow that had been hand- reared by itsel until it was 46 days old and then placed in an aviary with other white-crowned sparrows and a strawberry fnch. a)

III

IV

V

Compare sonogram V with sonogram IV. [2 ]

b) C ompare sonogram V with sonograms I and II. [2 ]  Figure 2

Sonograms of birdsong

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N E U R O B I O LO G Y AN D B E H AVI O U R Innate and learned behaviour thus both depend on genes, but whereas the development o learned behaviour develops as a result o experience, innate behaviour is independent o it.

Refex conditioning Refex conditioning involves orming new associations. S everal dierent types o learning have been dened. O ne o these, called refex conditioning, was investigated by the Russian physiologist Ivan Pavlov, using dogs. Refex conditioning involves orming new associations by establishing new neural pathways in the brain. C onditioned refexes are used extensively in animal behaviour and can greatly increase survival chances.

 Figure 3

Monarch butterfy caterpillars ingest toxins (cardenolide aglycones) rom the milkweed plants that they eat, making them distasteul to birds

For example, birds have an innate refex to avoid oods with a bitter tastethis is an unconditioned refex, but they have to learn which insects are likely to have that taste. I a bird tries to eat an insect with warning coloration o black and yellow stripes, or example, and nds that the insect tastes unpleasant, it develops an association between black and yellow stripes and bitter taste and thereore avoids all insects with such a colour pattern. In some cases the smell o the distasteul insect has to be combined with its coloration to cause avoidance.

Pavlovs experiments Pavlovs experiments into refex conditioning in dogs. The 1 9th century Russian physiologist Pavlov developed apparatus to collect saliva rom the mouth o his experimental dogs. He ound that saliva was secreted in response to the sight or smell o ood. These types o stimulus, to which all dogs respond without learning, are called unconditioned stimuli and the secretion o saliva that results is the unconditioned response. Pavlov observed that ater a while the dogs were starting to secrete saliva beore they received the unconditioned stimulus. S omething else had become a stimulus that allowed the dogs to anticipate the arrival o ood. He ound that the dogs could learn to use a variety o signals in this way, including the ringing o a bell, the fashing o a light, a metronome ticking or a musical box playing. These are examples o conditioned stimuli and the secretion o saliva that these stimuli elicit is the conditioned response. Pet dogs and children also quickly learn indicators that they will soon be ed.

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 Figure 4 Pavlov's dogs

A . 4 i N N At e A N d l e A r N e d b e h Av i o u r ( A h l )

Imprinting Imprinting is learning occurring at a particular life stage and is independent of the consequences of behaviour. The word imprinting was rst used in the 1 93 0s by Konrad Lorenz to describe a type o learning. Imprinting can only occur at a particular stage o lie and is the indelible establishment o a preerence or stimulus that elicits behaviour patterns, oten but not always, o trust and recognition. The example that was made amous by Lorenz was in greylag geese. Eggs are normally incubated by their mother so that she is the rst large moving obj ect that the hatchlings see. The young birds then ollow their mother around during the rst ew weeks o lie. S he leads them to ood and protects them. Lorenz showed that young geese that are hatched in an incubator and who do not encounter their mother attach themselves to another large moving obj ect and ollow it around. This can be a bird o another species, Lorenzs boots or even an inanimate moving obj ect. This attachment is what Lorenz called imprinting. The critical period in greylag geese when imprinting occurs is 1 3 1 6 hours ater hatching. A distinctive eature o imprinting is that it is independent o the consequences o the behaviour  in experiments animals remain imprinted on something even i it does not increase their chance o survival.

 Figure 5 Young geese imprinted

on

their mother

Operant conditioning Operant conditioning is a form of learning which consists of trial and error experiences. O perant conditioning is sometimes explained in simple terms as learning by trial and error. It is a dierent orm o learning rom refex conditioning. Whereas refex conditioning is initiated by the environment imposing a stimulus on an animal, operant conditioning is initiated by an animal spontaneously testing out a behaviour pattern and nding out what its consequences are. D epending on whether the consequences are positive or negative or the animal or its environment, the behaviour pattern is either reinorced or inhibited. Lambs learn not to touch electric encing by operant conditioning. They explore their environment and i electric encing is used to enclose their fock, lambs sooner or later touch it, probably with their nose. They receive a painul electric shock and through operant conditioning they avoid touching the ence in the uture.

Learning Learning is the acquisition of skill or knowledge. The behaviour o animals changes during the ir lie time. In a ew cases be haviour patterns are lost, or example the palmar grasp rele x and o ther primitive relexes in human b abies. Far more commonly animals acquire typ es o be haviour patte rn during their

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N E U R O B I O LO G Y AN D B E H AVI O U R lives. In some cases the se b ehavio ur changes are a natural part o growth and maturation, such as the b e haviour change s that o ccur during pub e rty in humans. In othe r case s the modiication o be haviour is acquired b y learning  the b e haviour do es not de ve lo p unle ss it is learned. Motor skills such as walking, talking or playing the violin are learned. Knowledge also has to be learned. For example the rainorest tribes learn the types o tree that can provide ood or other useul materials and they also learn the location in the orest o individual trees o the useul types. Learning is a higher order unction o the brain and humans have a greater capacity to learn than any other species. The degree o learning during an animals lietime is dependent on their longevity as well as their neural capacity. S ocial animals are more likely to learn rom each other.

Memory  Figure 6 Learning starts in

children but is a lifelong process due to neural plasticity

Memory is the process of encoding, storing and accessing information. Memory is one o the higher order unctions o the brain. Encoding is the process o converting inormation into a orm in which it can be stored by the brain. Short- term memory lasts up to about a minute and may or may not lead to long- term memory, which can be retained or indefnite periods o time. Accessing is the recall o inormation so that it can be used actively in the thought processes o the brain. D ierent parts o the brain have a role in the encoding, storage and accessing o memory. The importance o the hippocampus was strikingly demonstrated in 1 95 3 when a patient called Henry Molaison had the amygdala and a section o hippocampus rom both o his cerebral hemispheres removed in an experimental attempt to cure epilepsy. He immediately became incapable o making new memories unless they were procedural and his recall o memories ormed during the eleven years beore the surgery was also impaired. Recent research into the role o the hippocampus has shown that experiences cause large numbers o new synapses to be ormed, which are then gradually pruned to refne the memory o the experience and allow it to be recalled when it is relevant and not at other times.

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A.5 Naac (Ahl) Understanding  Some neurotransmitters excite nerve impulses in 

 

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post-synaptic neurons and others inhibit them. Nerve impulses are initiated or inhibited in postsynaptic neurons as a result o summation o all excitatory and inhibitory neurotransmitters received rom pre-synaptic neurons. Many diferent slow-acting neurotransmitters modulate ast synaptic transmission in the brain. Memory and learning involve changes in neurons caused by slow-acting neurotransmitters. Psychoactive drugs afect the brain by either increasing or decreasing post-synaptic transmission. Anaesthetics act by interering with neural transmission between areas o sensory perception and the CNS. Stimulant drugs mimic the stimulation provided by the sympathetic nervous system. Addiction can be afected by genetic predisposition, social environment and dopamine secretion.

Applications  Efects on the nervous system o two stimulants

and two sedatives.  The efect o anaesthetics on awareness.  Endorphins can act as painkillers.

Skills  Evaluation o data showing the impact o

MDMA (ecstasy) on serotonin and dopamine metabolism in the brain.

Nature of science  Assessing risk associated with scientic

research: patient advocates will oten press or the speeding up o drug approval processes, encouraging more tolerance o risk.

Excitatory and inhibitory neurotransmitters Some neurotransmitters excite nerve impulses in postsynaptic neurons and others inhibit them. The basic principles o synaptic transmission were described in sub-topic 6.5: neurotransmitter is released into the pre-synaptic neuron when a depolarization o the pre-synaptic neuron reaches the synapse. The neurotransmitter depolarizes the post- synaptic neuron by binding to receptors in its membrane. Excitatory neurotransmitters excite the post-synaptic neuron or periods ranging rom a ew milliseconds to many seconds, producing depolarization that may be sufcient to trigger action potentials. Some neurotransmitters have a dierent eect  they inhibit the ormation o action potentials in the post-synaptic neuron because the membrane potential becomes more negative when the neurotransmitter binds to the post-synaptic membrane. This hyperpolarization makes it more difcult or

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N E U R O B I O LO G Y AN D B E H AVI O U R the post-synaptic neuron to reach the threshold potential so nerve impulses are inhibited. Inhibitory neurotransmitters are small molecules that are inactivated by specic enzymes in the membrane o the post-synaptic neuron.

IPSP

EPSP

Summation Nerve impulses are initiated or inhibited in post-synaptic neurons as a result o summation o all excitatory and inhibitory neurotransmitters received rom pre-synaptic neurons.

EPSP plus IPSP

action potential EPSPs

action potential IPSP

EPSPs 100 ms  Figure 1

Excitatory post-synaptic potentials (EPSP) , inhibitory postsynaptic potentials (IPSP)

More than one pre-synaptic neuron can orm a synapse with the same post-synaptic neuron. Especially in the brain, as there are hundreds or even thousands o pre-synaptic neurons! Usually a single release o excitatory neurotransmitter rom one pre-synaptic neuron is insucient to trigger an action potential. Either one pre-synaptic neuron must repeatedly release neurotransmitter, or several adjacent pre-synaptic neurons must release neurotransmitter more or less simultaneously. The additive eect rom multiple releases o excitatory neurotransmitter is called summation. S ome pre-synaptic neurons release an inhibitory rather than an excitatory neurotransmitter. S ummation involves combining the eects o excitatory and inhibitory neurotransmitters. Whether or not action potentials orm in a post-synaptic neuron depends on the balance between the eects o the synapses that release excitatory and inhibitory neurotransmitters and thereore whether the threshold potential is reached. This integration o signals rom many dierent sources is the basis o decision-making processes in the central nervous system.

Slow and fast neurotransmitters Many diferent slow-acting neurotransmitters modulate ast synaptic transmission in the brain. The neurotransmitters so ar described have all been ast- acting, with the neurotransmitter crossing the synapse binding to receptors less than a millisecond ater an action potential has arrived at the pre- synaptic membrane. The receptors are gated ion- channels, which open or close in response to the binding o the neurotransmitter, causing an almost immediate but very brie change in post- synaptic membrane potential. Another class o neurotransmitter is slow-acting neurotransmitters or neuromodulators which take hundreds o milliseconds to have eects on post- synaptic neurons. Rather than having an eect on a single postsynaptic neuron they may diuse through the surrounding fuid and aect groups o neurons. Noradrenalin/norepinephrine, dopamine and serotonin are slow- acting neurotransmitters. S low acting neurotransmitters do not aect ion movement across postsynaptic membranes directly, but instead cause the release o secondary messengers inside post- synaptic neurons, which set o sequences o intracellular processes that regulate ast synaptic transmission. S low

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A. 5 N e u ro ph Arm ACo lo g y ( Ah l) acting neurotransmitters can modulate ast synaptic transmission or relatively long periods o time.

Memory and learning Memory and learning involve changes in neurons caused by slow-acting neurotransmitters. Psychologists have studied learning and memory or decades but it is only relatively recently that neurobiologists have been able to study these processes at the level o the synapse. S low- acting neurotransmitters ( neuromodulators) have a role in memory and learning. They cause the release o secondary messengers inside post-synaptic neurons that can promote synaptic transmission by mechanisms such as an increase in the number o receptors in the post-synaptic membrane or chemical modifcation o these receptors to increase the rate o ion movements when neurotransmitter binds. The secondary messengers can persist or days and cause what is known as long-term potentiation ( LTP) . This may be central to the synaptic plasticity that is necessary or memory and learning. E ven longer-term memories may be due to a remodelling o the synaptic connections between neurons. The learning o new skills has been shown to be linked to the ormation o new synapses in the hippocampus and elsewhere in the brain.

Endorphins Endorphins can act as painkillers. Pain receptors in the skin and other parts o the body detect stimuli such as the chemical substances in a bees sting, excessive heat or the puncturing o skin by a hypodermic needle. These receptors are the endings o sensory neurons that convey impulses to the central nervous system. When impulses reach sensory areas o the cerebral cortex we experience the sensation o pain. E ndorphins are oligopeptides that are secreted by the pituitary gland and act as natural painkillers, blocking eelings o pain. They bind to receptors in synapses in the pathways used in the perception o pain, inhibiting synaptic transmission and preventing the pain being elt.

Psychoactive drugs Psychoactive drugs afect the brain by either increasing or decreasing post-synaptic transmission. The brain has many synapses, perhaps as many as 1 0 1 6 in children. These synapses vary in their organization and use a wide variety o neurotransmitters. O ver a hundred dierent brain neurotransmitters are known. Psychoactive drugs aect the brain and personality by altering the unctioning o some o these synapses. S ome drugs are excitatory, because they increase post- synaptic transmission. O thers are inhibitory because they decrease it. E xamples o excitatory drugs: 

Nicotine contained in cigarettes and other orms o tobacco, derived rom the plant Nicotiana tabacum.

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C ocaine extracted rom the leaves o a Peruvian plant, Erythroxylon coca.

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Amphetamines, a group o artifcially synthesized compounds.

E xamples o inhibitory drugs: 

B enzodiazepines, a group o compounds including Valium that are synthesized artifcially.

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Alcohol in the orm o ethanol, obtained by ermentation using yeast.

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Tetrahydrocannabinol ( THC ) obtained rom the leaves o the Cannabis sativa plant.

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Ecstasy Evaluation o data showing the impact o MDMA (ecstasy) on serotonin and dopamine metabolism in the brain. data-base questions: Efects o ecstasy on the striatum The graphs in fgure 2 show the results o an experiment in which mice were treated with MD MA ( ecstasy) and levels o dopamine and serotonin were measured in the striatum o their brains. Two doses o MD MA were used and also saline ( no MD MA) . Wild-type mice were used and also three strains o knockout mice that lacked genes or making the dopamine transporter protein ( D AT-KO) , the serotonin transporter ( SERT-KO) or both transporters ( D AT/SERT-KO ) . The graphs show the levels o dopamine and serotonin in the three-hour period ater MD MA had been administered.

D escribe the trends in dopamine level wild- type mice in the three hours ater administration o 1 0 mg o MD MA.

a) Distinguish between the results or the wild-type mice and the DAT-KO mice. [2]

D istinguish between the results or the D AT-KO mice and the S ERT-KO mice.

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Explain the results or the D AT/S ERT- KO mice. [2 ]

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Suggest one beneft o using knockout mice in this experiment. [1 ]

Wild, 3 mg DAT-KO, 3 mg SERT-KO, 3 mg DAT/SERT-KO, 3 mg Wild, 10 mg DAT-KO, 10 mg SERT-KO, 10 mg DAT/SERT-KO, 10 mg

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400 300 200 100

[2 ]

[3 ]

serotonin (% of basal level)

dopamine (% of basal level)

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4

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D iscuss the evidence rom the data or the hypothesis that MD MA has a greater eect on serotonin level than dopamine level in the wild- type mice. [3 ]

b) D iscuss whether these dierences are statistically signifcant or not. [2 ]

Questions 1

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Reference: Hagino et al, Efects o MDMA on Extracellular Dopamine and Serotonin Levels in Mice Lacking Dopamine and/or Serotonin Transporters, Curr. Neuropharmacol. 2011 March; 9(1) : 9195.

Anaesthetics Anaesthetics act by interering with neural transmission between areas o sensory perception and the CNS. Anaesthetics cause a reversible loss o sensation in part or all o the body. Local anaesthetics cause an area o the body to become numb, or example the gums and teeth during a dental procedure. General anaesthetics cause unconsciousness and thereore a total lack o sensation.

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A. 5 N e u ro ph Arm ACo lo g y ( Ah l) Anaesthetics are chemically varied and work in a variety of ways. Many of them affect more than just the sense organs and can also inhibit signals to motor neurons and other parts of the nervous system so they should only ever be administered by highly trained medical practitioners.

Anaesthetics and awareness The efect o anaesthetics on awareness. A patient who has been given a general anaesthetic normally has no awareness of the surgical or other procedures that they are undergoing because they are totally unconscious. There are some procedures where it is either not necessary or is undesirable for the patient to be unconscious. For example patients are kept partially conscious during some operations to remove brain tumours, so that the effects on the brain can be monitored. There have been some cases of patients retaining some awareness during operations, when

they have not been given a high enough dose of anaesthetic. The patient may or may not feel pain. The risk of awareness is highest in operations such as emergency caesarean sections in which it is best for the mother and child for the dose of anaesthetic to be minimized, although in these procedures a spinal block is almost always now used rather than a general anaesthetic, so the patient is awake and breathing is normal, but pain sensation cannot get beyond the spinal cord.

Drug testing Assessing risk associated with scientic research: patient advocates will oten press or the speeding up o drug approval processes, encouraging more tolerance o risk. There are strict protocols for testing new drugs with several phases that establish two things  an appropriate dose and route of administration that make the drug effective, and that its side-effects are minor and infrequent enough for the drug to be regarded as safe. These tests take many years to complete but approval for the introduction of a new drug is only given once the tests have been rigorously carried out. There have been some trials where the difference between the control group of patients given a placebo and the group given the new drug is so great that it seems unethical to deny the control group the treatment. It therefore seems reasonable to abandon the trials and introduce the drug immediately. The danger of this policy is that harmful side-effects may only then be discovered when large numbers of patients have been given the new drug. There have been some cases where groups of patients have campaigned for a new drug to be introduced before it has been fully tested. This may be acceptable with terminal diseases such as AID S or certain forms of heart disease where the patient may regard any level of risk acceptable given the certainty of death without treatment. It is unlikely to be acceptable with non- critical illnesses where the risks from using a drug that has not been fully tested are too great compared with the risks associated with the disease remaining untreated.

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Stimulant drugs Stimulant drugs mimic the stimulation provided by the sympathetic nervous system. S timulants are drugs that promote the activity o the nervous system. They make a person more alert, energetic and sel- confdent. They also increase heart rate, blood pressure and body temperature. The eects o stimulant drugs match those o the sympathetic nervous system. This is because stimulant drugs act by a variety o mechanisms to make the body respond as though it had been naturally stimulated by the sympathetic nervous system.

 Figure 3

Drug enforcement measures near a school

S ome mild stimulants are present in oods and drinks, or example caeine in tea and coee and theobromine in chocolate. D octors sometimes prescribe stronger stimulants to treat conditions such as clinical depression and narcolepsy. S timulant drugs are also sometimes used against medical advice. E xamples include cocaine, amphetamines and nicotine in cigarettes.

Examples of stimulants and sedatives Efects on the nervous system o two stimulants and two sedatives. Pramipexole mimics dopamine and binds to dopamine receptors in post-synaptic membranes at dopaminergic synapses. Whereas some drugs that mimic neurotransmitters are antagonists because they block synaptic transmission, pramipexole is an agonist because it has the same eects as dopamine when it binds. Pramipexole is used during the early stages o Parkinsons disease to help to reduce the eects o insufcient dopamine secretion that characterize this disease. It has also sometimes been used as an anti-depressant.

GAB A ( - amino butyric acid) is an inhibitory neurotransmitter and when it binds to its receptor a chloride channel opens, causing hyperpolarization o the post- synaptic neuron by entry o chloride ions. When diazepam is bound to the receptor the chloride ions enter at a greater rate, inhibiting nerve impulses in the post- synaptic neuron. D iazapam is thereore a sedative. It can reduce anxiety, panic attacks and insomnia and it is also sometimes used as a muscle relaxant.

C ocaine also acts at synapses that use dopamine as a neurotransmitter. It binds to dopamine reuptake transporters, which are membrane proteins that pump dopamine back into the pre- synaptic neuron. B ecause cocaine blocks these transporters, dopamine builds up in the synaptic clet and the post- synaptic neuron is continuously excited. C ocaine is thereore an excitatory psychoactive drug that gives eelings o euphoria that are not related to any particular activity.

THC ( Tetrahydrocannabinol) is present in cannabis. It binds to cannabinoid receptors in pre- synaptic membranes. B inding inhibits the release o neurotransmitters that cause excitation o post- synaptic neurons. THC is thereore an inhibitory psychoactive drug and a sedative. C annabinoid receptors are ound in synapses in various parts o the brain, including the cerebellum, hippocampus and cerebral hemispheres. The main eects o THC are disruption o psychomotor behaviour, shortterm memory impairment, intoxication and stimulation o appetite.

D iazepam ( Valium) binds to an allosteric site on GAB A receptors in post- synaptic membranes.

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Drug addiction Addiction can be afected by genetic predisposition, social environment and dopamine secretion. The American Psychiatric Association has defned addiction as: a chronically relapsing disorder that is characterized by three main elements: (a) compulsion to seek and take the drug, (b) loss of control in limiting intake and (c) emergence of a negative emotional state when access to the drug is prevented. O nly certain drugs cause addiction and usually repeated use over a prolonged period o time is needed. With a ew drugs, addiction can develop more rapidly. The causes o addiction are clearly not simple and three areas need to be considered. 1

Some people seem much more vulnerable to addiction than others because o their genes. This is known as genetic predisposition. O ne example is the gene, D RD 2 , which codes or the dopamine receptor protein. There are multiple alleles o this gene and a recent study showed that people with one or more copies o the A1 allele consumed less alcohol than those homozygous or the A2 allele.

2

Addiction is more prevalent in some parts o society than others because the social environment greatly aects the likelihood o taking drugs and becoming addicted. Peer pressure, poverty and social deprivation, traumatic lie experiences and mental health problems all contribute. C ultural traditions are very important and help to explain why dierent drugs cause problems in dierent parts o the world.

3

Many addictive drugs, including opiates, cocaine, nicotine and alcohol aect dopamine secreting synapses. D opamine secretion is associated with eelings o well- being and pleasure. Addictive drugs cause prolonged periods with high dopamine levels in the brain. This is so attractive to the drug user that they fnd it very difcult to abstain.

 Figure 4 Alcohol

is an addictive drug but is legal in many counties

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A.6 etoogy (Ahl) Understanding  Ethology is the study o animal behaviour in

natural conditions.  Natural selection can change the requency o observed animal behaviour.  Behaviour that increases the chances o survival and reproduction will become more prevalent in a population.  Learned behaviour can spread through a population or be lost rom it more rapidly than innate behaviour.

Applications  Migratory behaviour in blackcaps as an example

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

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Nature of science  Testing a hypothesis: experiments to test

hypotheses on the migratory behaviour o blackcaps have been carried out.

 

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o the genetic basis o behaviour and its change by natural selection. Blood sharing in vampire bats as an example o the evolution o altruistic behaviour by natural selection. Foraging behaviour in shore crabs as an example o increasing chances o survival by optimal prey choice. Breeding strategies o hooknoses and jacks in coho salmon populations as an example o behaviour afecting chances o survival and reproduction. Courtship in birds o paradise as an example o mate selection. Synchronized oestrus in emale lions in a pride as an example o innate behaviour that increases the chances o survival and reproduction o ofspring. Feeding on cream rom milk bottles in blue tits as an example o the development and loss o learned behaviour.

Ethology Ethology is the study o animal behaviour in natural conditions. Animals are adapted to their natural habitat in their behaviour. I we remove them rom this habitat and place them in a zoo or laboratory, animals may not behave normally because they may not receive the same stimuli as in their natural habitat. For this reason it is best whenever possible to carry out research into animal behaviour in their natural habitat rather than in an artifcial environment. The study o the actions and habits o animals in their natural environment is called ethology.

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Natural selection and animal behaviour Natural selection can change the requency o observed animal behaviour. Natural selection is the theme that runs through the whole o modern biology, including ethology. It adapts species to all aspects o their environment. Adaptation extends over the whole range o animal characteristics, rom the structure o a single molecule such as hemoglobin to the patterns o behaviour in a species. Animal behaviour has been observed to change rapidly in some cases. House fnches Carpodacus mexicanus are an example. In C aliornia the native population is sedentary  the birds remain in the same area throughout the year. A small number were illegally released in the 1 940s in New York C ity and spread though the eastern United S tates, and within twenty years migratory behaviour was observed. The requency o this behaviour rose to more than 5 0% o the population, presumably as a result o natural selection.

The mechanism of natural selection Behaviour that increases the chances o survival and reproduction will become more prevalent in a population. Natural selection works in same way or animal behaviour as or other biological characteristics. Individuals with the best- adapted actions and responses to the environment are most likely to survive and produce ospring. I behaviour is genetically determined, rather than learned, it can be inherited by ospring. The breeding season o the great tit Parus major illustrates how behaviour evolves by natural selection, oten as a response to environmental changes. This bird lives in woodland and eeds its young on caterpillars and other insects. The availability o this ood rises to a peak in spring soon ater the new leaves on trees have grown. D ue to global warming, the time o peak availability has become earlier. The timing o nesting and egg laying varies within narrow limits within the population. Researchers have shown that birds that lay their eggs a ew days earlier than the mean date have more success in rearing young. According to natural selection, the mean date o egg laying should evolve to be earlier and researchers ound this prediction ulflled.

Breeding strategies in salmon Breeding strategies o hooknoses and jacks in coho salmon populations as an example o behaviour afecting chances o survival and reproduction. C oho salmon Oncorhynchus kisutch breed in rivers that discharge into the North Pacifc Ocean, including those on the west coast o North America.

The adults die ater breeding and the young live or about a year in the river and then migrate to the ocean where they remain or several years

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beore returning to spawn. There are two breeding strategies among males. Hooknoses fght each other or access to emales laying eggs, with the winner shedding sperm over the eggs to ertilize them. Jacks usually avoid fghts and instead sneak up on emales and attempt to shed sperm over their eggs beore being noticed. O bervations on individually identifed fsh, usually identifed by a tag, show that whether a male becomes a j ack or a hooknose depends on his growth rate. Males that grow rapidly are able to return to breed two years ater they were spawned and are j acks. Males that grow less rapidly remain in the ocean or one year longer, but are then signifcantly larger and are hooknoses. The smaller j acks are more likely to reproduce by the sneaking strategy than by fghting the larger hooknoses. The larger

hooknoses are unlikely to sneak up on a emale without being noticed so they must fght other hooknoses and end o j acks i they are to be successul in breeding.

 Figure 1

Brown bear catching salmon as they swim upstream to breed

Synchronized oestrus Synchronized oestrus in emale lions in a pride as an example o innate behaviour that increases the chances o survival and reproduction o ofspring. Female lions remain in the group ( pride) into which they were born, but male lions are expelled rom the pride when they are about three years old. Males can only breed i, when ully grown adults, they overcome the dominant male in another pride by fghting. Within two or three years o taking over a pride o emales, the breeding male is likely to be replaced by a younger rival. When a new dominant male takes over a pride he may kill all the suckling cubs, thus making the emales come into oestrus more quickly so the male can then mate with them to ather his own cubs. Females protect their cubs rom marauding males, sometimes leading to ferce fghts, but accept his sexual advances ater he has taken over the pride. Sometimes two or more closely related young males together fght or dominance o another group. This increases their chance o success, especially i they are fghting a single dominant male. Females can only breed when they come into oestrus. All emales in a pride tend to come into

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oestrus at the same time. This behaviour has several advantages: the emales have their cubs at the same time so are all lactating while the cubs are suckling, so they can suckle each others cubs when they are hunting, increasing the cubs chance o survival. Also a group o male cubs o the same age are ready to leave the pride at the same time so can compete or dominance o another pride more eectively.

 Figure 2

Lions in a group known as a pride

A. 6 e th o lo g y ( Ah l)

Blackcap migration Migratory behaviour in blackcaps as an example of the genetic basis of behaviour and its change by natural selection. The blackcap Sylvia atricapilla breeds during the northern summer. Until relatively recently, populations o blackcaps that breed in C entral Europe including Germany almost all migrated to S pain and Portugal or the winter, where the weather is warmer and the availability o ood is greater. D uring the second hal o the 2 0th century a ew blackcaps rom the population in Germany were ound to be migrating to B ritain and Ireland instead. The numbers o blackcaps overwintering in B ritain rose rapidly to more than 1 0% .

There are several possible reasons or this change in migration behaviour. Global warming has led to winters being warmer in B ritain so the long migration to S pain is not necessary. Many people in B ritain eed wild birds in winter which may acilitate survival o overwintering blackcaps more than in S pain. In winter the minimum day length in B ritain is shorter than in S pain, which may prompt earlier migration to breeding grounds. B lackcaps that arrive earlier take the best territories  another advantage o overwintering in B ritain.

Experiments with migrating blackcaps Testing a hypothesis: experiments to test hypotheses on the migratory behaviour of blackcaps have been carried out. In ethology as in other branches o science it is essential to test hypotheses and either obtain evidence or them or prove them to be alse. The adaptive value o behaviour patterns have sometimes been assumed without evidence. These accounts o evolution are known as  Just S o S tories ater Rudyard Kiplings childrens story book. However intuitively obvious a hypothesis about the evolution o a behaviour pattern, it is only a j ust so story until tested. Hypotheses about evolutionary changes in blackcap migration have been rigorously tested. For example, the hypothesis that the direction o migration is genetically determined has been tested. E ggs were collected in Germany rom parent birds that had migrated to B ritain in the previous winter and rom parents who had migrated to S pain. The young were reared without their parents so that they could not learn rom them and when they migrated the direction was recorded. B irds whose parents had migrated to B ritain tended to fy west, wherever they were reared, and birds whose parents had migrated to S pain tended to fy south- west. They thereore responded to

migratory stimuli in the same way as their parents, indicating that the direction o migration is genetically determined, and can thus be subj ect to long- term evolutionary change under natural selection.

FPO Britain (winter) Germany (summer)

Spain (winter)

 Figure 3

Migration of blackcaps

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Vampire bats Blood sharing in vampire bats as an example of the evolution of altruistic behaviour by natural selection. Female vampire bats Desmodus rotundus live in colonies o 81 2 individuals, with the same individuals roosting together or several years. Their diet is about 2 5 ml o vertebrate blood, usually mammalian, each night. I a bat ails to eed or two or three consecutive nights they risk death rom starvation. However, this rarely happens because when the bats return to the roost at the end o the night, those that have ed regurgitate blood or those that have not.

on o genes o the altruistic animal. B lood sharing is an example o reciprocal altruism. Individual A gains a beneft rom giving blood to Individual B because Individual B survives and can share blood on a later night i Individual A ails to eed. It only occurs in stable groups o emales that roost together regularly and as it aids the chances o survival and reproduction o all o the members o such groups, natural selection avours it.

This behaviour pattern is a rare example o altruism. It ulfls two necessary criteria: 

there may be siblings or mothers with daughters in a group but tests have shown that there are also unrelated emales who also share blood, so blood sharing is not kin-selection;



giving blood to an individual who has not ed incurs a cost to the giver because some o their daily diet is lost, so blood sharing is not merely cooperation  it is genuine altruism.

The evolution o altruism is an interesting conundrum: we might not expect natural selection to promote the evolution o behaviour that incurs a cost, because it should reduce the chances o survival, reproduction and the passing

 Figure 4 Vampire bats show reciprocal

altruism by

blood sharing

Foraging in shore crabs Foraging behaviour in shore crabs as an example of increasing chances of survival by optimal prey choice.

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per cent of diet

protability/J s -1

Foraging is searching or ood. Animals (a) 1.5 must decide what type o prey to search (b) 50 or and how to fnd it. Studies have shown 40 1.0 that the prey chosen by animals tends to 30 be the type that gives the highest rate o 20 energy return. For example, the shore crab 0.5 Carcinus moenas preers to eat mussels o 10 intermediate size when presented in an 0 1-0 1-5 2-0 2-5 3-0 aquarium with equal numbers o each size, 1-0 2-0 3-0 4-0 size of mussel/cm as shown in the bar chart in fgure 5 . The size of mussel/cm graph in fgure 5 shows that mussels o  Figure 5 Proftability in Joules per second and percentage in diet o mussels o dierent size intermediate size are the most proftable in terms o the energy yield per second o time spent breaking open the shells.

A. 6 e th o lo g y ( Ah l)

Courtship in birds of paradise Courtship in birds o paradise as an example o mate selection. S ome animals have anatomical eatures that seem to the human eye to be excessive, or example the tail eathers o the peacock. O ther animals have behaviour patterns that seem bizarre. The plumage and courtship displays o male birds o paradise are examples o both o these types o exaggerated trait. There are about orty species o bird o paradise living on New Guinea and other nearby islands. The males have very showy plumage with bright coloration and elongated or elaborate tail eathers that are o no use in fying. The emales, which build the nest, incubate the eggs and rear the young, are relatively drab. Males in many species o bird o paradise have a complicated and eye-catching courtship dance that they use to try to attract emales. In some species the males all gather at a site called a lek and emales select a mate rom among the males displaying. The coloured plumage and courtship dances o birds help to avoid interspecic hybridization by allowing emales to determine i a male belongs to their species, but this could be achieved in much more subtle ways than those

used by birds o paradise and biologists have long speculated on the reasons or exaggerated traits. D arwin explained them in terms o mate selection  emales preer to mate with males that have exaggerated traits. The reason may be that these traits indicate overall tness. I a bird o paradise has enough energy to grow and maintain the elaborate plumage and repeatedly to carry out very vigorous courtship displays it indicates that the male must have ed eciently. I it can survive in the rainorest with the encumbrance o its tail eathers and with bright plumage that makes it visible to predators, it is probably well adapted in other ways and is thereore a good mate to choose. O ver the generations emales that selected males with showier plumage and more spectacular courtship dances have produced ospring athered by males with greater overall tness. Natural selection has thereore caused these traits to become exaggerated. An example o a male bird o paradise can be seen in sub-topic 4.1 .

Changing learned and innate behaviour

toK

Learned behaviour can spread through a population or be lost rom it more rapidly than innate behaviour.

W ar sciniss smims suspicius f vidnc basd n amaur bsrvains rar an n numrica daa frm cnrd xprimns?

S ome patterns o behaviour are entirely innate, or example the withdrawal refex, so are programmed into an animals genes. They can happen immediately in an individual without any period o learning. However, they can only be modied by natural selection relatively slowly because there must be variation in the alleles that aect the behaviour and a change in allele requencies in the population due to one behaviour pattern increasing chances o survival and reproduction over the other patterns o behaviour. Other patterns o behaviour are either partially or entirely learned  although these take longer to develop in an individual, they do not involve changes in allele requency and can spread in a population relatively rapidly as one individual learns rom another. C himpanzees show many examples o tool use that are learned, with considerable variation between groups o chimpanzees in the types o tool used. I one individual discovers a new use o an object as a tool, others can learn it quickly. However, learned behaviour can also disappear rom a population rapidly. An example is blue tits eeding on cream rom milk bottles.

With respect to the observations o the changes in the behaviour o blue tits and milk bottles, an article appeared in 1952 in the journal Nature: Although no experimental analysis o the behaviour involved in the opening o milk bottles has yet been made, urther observations in the feld enable the discussion to be carried urther.

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Blue tits and cream Feeding on cream from milk bottles in blue tits as an example of the development and loss of learned behaviour. B lue tits Cyanistes caeruleus were rst observed pecking through the aluminium oil caps o milk bottles let outside houses, to drink the cream, in the 1 92 0s in Southampton, England. This behaviour was observed soon aterwards 1 5 0 kilometres away  ar urther than blue tits normally fy. Amateur birdwatchers ollowed the rapid spread o the behaviour, in both blue tits and great tits, across Europe to the Netherlands, Sweden and D enmark.

Much less milk is now delivered to doorsteps because milk in supermarkets is cheaper. Also skimmed milk, without cream at the top, has become popular with humans. This may explain why blue tits have not recently been observed pecking through bottle tops.

German occupation o the Netherlands during the Second World War stopped deliveries o milk or eight years  ve years longer than the maximum lie o a blue tit. However, within months o the resumption o deliveries, blue tits throughout the Netherlands were pecking through the bottle tops. The rapid spread o this behaviour pattern shows that it must be due to learned rather than innate behaviour. Newspaper articles recently reported that blue tits had stopped eeding on cream rom milk bottles.

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 Figure 6 Blue tit pecking through

milk bottle cap

QuestioN s

Questions When birds are in danger of attack by predators, they sometimes sleep with one eye open and one eye closed. Neurobiologists investigated this behaviour pattern using mallard ducks ( Anas platyrhynchos) . Video recordings were made of groups of four sleeping birds, arranged in a row. The birds at the ends of the row were more vulnerable to predator attacks and kept one eye open 1 5 0% more of the time than the two birds in the centre of the row.

c) S uggest two advantages to birds of keeping one eye open during sleep.

2

 AD patients  pre-AD patients with plaques but no dementia  a control group with no plaques and no dementia.

0

both eyes closed

both eyes left eye left eye open closed right open right eye open eye closed

Source: Rattenborg, et al. , Nature, 1999, 397, pages 397398

a) State the effect of opening both eyes on activity in the region of the brain that was being monitored. [1 ] b) ( i)

( ii)

Using the data in the bar chart, deduce the effect on the two cerebral hemispheres of opening only the right eye. [2] D etermine which hemisphere is more awake when the right eye is open. [1 ]

( iii) Using the data in the bar chart, deduce how the left and right eyes and left and right hemispheres are connected. [1 ]

% NGF of the control temporal cortex

left hemisphere right hemisphere

75

Alzheimers disease ( AD ) is characterized by increasing dementia ( mental and emotional deterioration) in affected persons.

A study was carried out to measure the postmortem NGF concentrations in two regions of the cortex, the temporal cortex and the frontal cortex. Three groups of people were compared:

125

100

[2 ]

Evidence from the post-mortem (after death) analysis of the brains of affected patients has revealed two abnormalities. Affected persons show a change in the concentration of nerve growth factor (NGF) in a region of the brain known as the cortex. The brains of affected patients also have plaques. These are accumulations of insoluble material in and around cells.

Electroencephalograph ( E EG) recordings were made to monitor the brain state of the birds at the ends of the rows. A region of the brain which indicates whether the bird is asleep or awake was monitored in each of the left and right cerebral hemispheres. E EG recordings were made when the birds were sleeping with both eyes closed, when the birds had both eyes open and also when they had one eye open. These results are shown in the bar chart below, as a percentage of the activity of the brain region when the birds were sleeping with both eyes closed. activity of the brain region (% of activity with both eyes closed)

1

140 120 100 80 60 40 20 0

frontal AD patients

temporal pre-AD patients

controls

Source: R Hellweg et al., (1999) , International Journal of Development Neuroscience, 16, (7/8) , pages 787794

a) C ompare the data for the two regions of the cortex.

[3 ]

b) C alculate the increase in percentage NGF in the frontal cortex of AD patients compared to the control group. [1 ] c) S uggest what happens to the quantity of NGF in the cortex as the disease progresses. [2 ]

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Many animal species use long- range calls to establish their use of space and their relationships with me mbers of their own and other species. Most of the calls of the African S avanna elephant ( Loxodonta africana) are below the range of human hearing. The area in which the elephants can detect the calls is known as the calling area. O n any given day, the calling area undergoes expansions and contractions. The diagrams on the right show the calling area ( solid line) of elephants in the E tosha National Park at different times of the day. The position of the calling elephants is the centre of the diagram. C ircular rings depict distance ( in km) . The wind speed ( in m s - 1 ) and direction are shown with an arrow. If there is no arrow on the diagram it sho ws there was no wind. a) Identify the time of the day when the calling area was greatest.

[1 ]

b) Identify the wind speed at 08:00h.

[1 ]

N

E 2 4 6 8 10

W

17:00h S

S

N

N

E 2 4 6 8 10

W

19:00h

E 2 4 6 8 10

W

20:08h S

S

N

N

E 2 4 6 8 10

W

06:05h

[3 ]

E 2 4 6 8 10

W

18:00h

c) C ompare the calling area at 1 7: 00h with 1 8:00h. [2 ] d) D iscuss the relationship between the wind and the calling area.

N

E 2 4 6 8 10

W

08:00h S

S

Source: D Larom, et al. , Journal of Experimental Biology (1997) , 200, page 421431. Reprinted with the permission of the Company of Biologists

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B I O TE CH N O LO GY AN D B I O I N F O R M AT I CS CE LL B I O LO GY Introduction B iotechnology is the use o organisms, especially microorganisms to perorm industrial processes. The organisms used may be genetically modifed to make them more suitable. C rops can be modifed to increase yields and to obtain novel products. B iotechnology can

be used in the prevention and mitigation o contamination rom industrial, agricultural and municipal wastes. B iotechnology can also be used in the diagnosis and treatment o disease. B ioinormatics is the use o computers to analyse sequence data in biological research.

B.1 Microbiology: organisms in industry Understanding  Microorganisms are metabolically diverse.  Microorganisms are used in industry because       

they are small and have a ast growth rate. Pathway engineering optimizes genetic and regulatory processes within microorganisms. Pathway engineering is used industrially to produce metabolites o interest. Fermenters allow large-scale production o metabolites by microorganisms. Fermentation is carried out by batch or continuous culture. Microorganisms in ermenters become limited by their own waste products. Probes are used to monitor conditions within ermenters. Conditions are maintained at optimal levels or the growth o the microorganisms being cultured.

Applications  Deep-tank batch ermentation in the mass

production o penicillin.  Production o citric acid in a continuous ermenter by Aspergillus niger and its use as a preservative and avouring.  Biogas is produced by bacteria and archaeans rom organic matter in ermenters.

Skills  Gram staining o Gram-positive and Gram-

negative bacteria.  Experiments showing zone o inhibition o bacterial growth by bactericides in sterile bacterial cultures.  Production o biogas in a small-scale ermenter.

Nature of science  Serendipity has led to scientifc discoveries:

the discovery o penicillin by Alexander Fleming could be viewed as a chance occurrence.

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Metabolic diversity Microorganisms are metabolically diverse. Microorganisms occupy a number o niches in ecosystems. In order to serve their ecological role, they require certain metabolic pathways that correspond to their role.

 Figure 1

Penicillium mold growing on an orange. The antibiotic penicillin is derived from this microorganism

S aprotrophs release nutrients trapped in detritus and make it available to ecosystems. As saprotrophs, bacteria and ungi compete with one another or ood sources. Many ungi release anti- bacterial antibiotics into the environment in an eort to limit interspecifc competition. O ther microorganisms act as producers. C yanobacteria ( blue- green algae) and protoctists such as algae and Euglena are photosynthetic. They produce carbohydrates by fxing carbon dioxide in the C alvin cycle. O ther microorgansims act as heterotrophs. Yeast such as Saccharomyces cerevisiae carry out anaerobic respiration producing alcohol and carbon dioxide by a pathway known as alcoholic ermentation. The bacteria Rhizobium and Azotobacter can fx nitrogen and convert it to a orm that living things can use. B acteria such as Nitrobacter and Nitrosomonas can use inorganic chemicals as energy sources. They are known as chemoautotrophs. Humans have been able to take advantage o the metabolic pathways o microorganisms in biotechnology applications.

 Figure 2

Microalgae production for biofuels. Ponds being used to culture Chlorella vulgaris microalgae as a source of biofuel. The carbon dioxide is pumped into ponds (seen here) to promote photosynthesis and therefore growth of the algae

The advantages of using microorganisms in biotechnology Microorganisms are used in industry because they are small and have a fast growth rate. Humans have been exploiting the metabolism o microorganisms throughout history or example in the production o ood such as yogurt, bread, wine and cheese. More recently, industrial biotechnology has increased the number o metabolic pathways exploited or drug and uel production as well as additional applications involving genetically modifed microbes. Industrial biotechnology takes advantage o the acts that microorganisms are small and reproduce at a ast rate. They can be grown on a range o nutrient substrates and can produce a range o products. C onditions can be easily monitored in an industrial setting and maintained at optimum levels.

Pathway engineering Pathway engineering is used industrially to produce metabolites of interest. Traditionally either through selective breeding or genetic modifcation, microorganisms used in biotechnology applications were selected because they were the variants that provided the maximum yield o a

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desired metabolite. What this didnt take into account was the possibility that there were points in the metabolic pathway that constrained yields to the point where actual yields were much lower than theoretical yields. What distinguishes pathway engineering rom traditional methods is the use o detailed knowledge and analysis o the cellular system o metabolic reactions. This allows scientists to direct changes at multiple points to improve yields o metabolites o interest. This can include extending the range o substrates, elimination o by-products that slow the process down and extension o the range o products.

Pathway engineering uses knowledge of metabolic pathways to increase yields Pathway engineering optimizes genetic and regulatory processes within microorganisms. Pathway engineering is a technique that analyses the metabolic pathway o a particular microorganism to determine the bottleneck points o the pathway that constrain the production o the desired compound. Researchers can then address the constraint using genetic modifcation. For example, the yeast Saccharomyces cerevisiae occurs naturally on the skin o grapes. The ermentation o grapes is carried out by S. cerevisiae with the desired end product being ethanol. Maintaining the correct pH is important in wine production. Malate is a metabolite that appears during wine making. Its degradation is essential or the deacidifcation o grapes. However, malate permease, a membrane protein necessary or the transport o malate into cells is not present in S. cerevisiae. Further, while S. cerevisiae has an enzyme that can degrade malate, it was ound to be relatively inefcient. The gene or MAE 2 , a highly efcient malate degrading enzyme rom Lactococcus lactis was inserted into S. cerevisiae along with the gene or malate permease rom the yeast Schizosaccharomyces pombe. The ability o transgenic S. cerevisiae to undertake more efcient malate degradation was successully achieved.

Fermenters in industry Fermenters allow large-scale production of metabolites by microorganisms.

 Figure 3

Coloured scanning electron micrograph (SEM) of naturally occuring yeast cells (red) on the skin of a grape. In the processing of the grapes to make wine, the presence of the yeast is essential for the fermentation of the grapes that is part of the wine making process

Technically, ermentation reers to the anaerobic generation o ATP rom glucose that generates characteristic end products such as alcohol and lactic acid. With respect to biotechnology, microbiologists have a broad interpretation o the term ermentation; i.e., the word reers to the processes involved in the large- scale culture o microorganisms to produce metabolites o interest. A ermenter is oten a large stainless steel vessel flled with sterile nutrient medium. The medium is inoculated with the desired microorganism. An impeller is a rotating set o paddles that mixes the medium preventing sedimentation. Gas is bubbled through i the desired metabolic process is aerobic. A pressure gauge detects gas build-up and

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toK t wha exen is scienifc develpmen dependen n lucky accidens? In 1897, Hans and Eduard Buchner were investigating yeast extracts as a source of medicine. They ground up yeast cells with silica and sand and used a hydraulic press to create a yeast extract. They applied high concentrations of sugar to serve as a preservative. What surprised them was this cell-free system began to ferment the sugar. Eduard Buchner received the Nobel Prize for his discovery of cellless fermentation. This had actually been discovered in 1878 by Wilhelm Kuhne, but he had not been successfully able to isolate the chemical element in the way that the Buchners did. Kuhne did provide the name for the element that was causing the cell-less ferementation contained in the yeast. He created the term enzyme from the Greek words en (in) and zume (yeast) .

allows waste gases to escape. C onditions within the vessel are monitored by probes. B ecause heat can build up as a waste product o metabolism, a cooling j acket surrounds the reaction vessel with cooling water fowing through it. O nce the medium is used up, new medium can be added. Product removal may also occur leading out o the vessel. antifoam

motor

steam

acid/base pressure guage

nutrient or inoculant ltered waste gases cold-water outlet

sterile nutrient medium impeller

pH probe temperature probe

oxygen concentration probe

cooling jacket

cold-water inlet

sparger compressed air steam harvest pipe

 Figure 4 A fermenter

There are two approaches to industrial fermentation Fermentation is carried out by batch or continuous culture. Mass culture o microorganisms is carried out in two ways in industry. B atch culture is used or producing secondary metabolites; i.e., those which are not essential or the growth o the culture. In this case, inoculation o the medium is ollowed by the culture passing through all o the stages o the sigmoid growth curve. To begin the process, a xed volume o medium is added to the closed ermentation vat. Ater inoculation, no urther nutrients or microorganisms are added during the incubation period. The products are extracted only when they reach a high enough concentration. In continuous culture, nutrients are added and products harvested at a constant rate. Conditions are monitored closely and eorts are made to keep conditions at a constant level so the process can continue over a long period.

Factors limiting industrial fermentation Microorganisms in fermenters become limited by their own waste products. A number o abiotic actors set limits to the activity o microorganisms in ermentation tanks. These can be due to the consumption o raw materials by the microorganism or by the production o waste products due to their activities.

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

C arbon dioxide production can lower the pH aecting enzyme activity.



Gas production can lead to pressure build- up possibly aecting reaction rates.



Alcoholic ermentation can yield levels o alcohol which have an osmotic eect on cells.



O xygen levels can be depleted due to cellular respiration.



Heat as a waste product o metabolism can raise the temperature o the reaction vessel.

Probes monitor conditions within fermenters Probes are used to monitor conditions within fermenters. In gure 5 , oxygen concentration, pH, volume, oam levels and temperature probes are shown. These are the most commonly monitored variables in ermentation tanks. C omputer-based probes gather data on these conditions. In batch ermentation, they can provide an indication o the stage o the production process. In continuous ermentation, they can signal to a technician actions to be taken to keep conditions within the avourable range.

Maintaining optimum conditions within fermenters

 Figure 5 A system

of probes is connected to the fermenter to monitor conditions within the vessel

Conditions are maintained at optimal levels for the growth of the microorganisms being cultured. C onditions are more likely to be monitored and kept at optimal levels in continuous culture. S uch conditions include water content, temperature, pH, macro- and micro- nutrient levels, levels o waste products, cell density, dissolved oxygen content, dissolved carbon dioxide content, culture volume and culture mixing. The optimum level o each variable depends on the species. The level o a variable is oten infuenced by a number o variables and is thereore important to monitor constantly. C onsider the example o oxygen. S pecies dier in their tolerance o low oxygen. Penicillium is less tolerant o low oxygen than Saccharomyces. When concentrations o dissolved oxygen go below a critical value, then it becomes limiting. D issolved oxygen is aected by the temperature and the nutrients being oxidized by the organism. Adding oxygen by aeration to a culture is not a simple matter as it generates oam which can limit production. Antioaming agents are oten added to the reaction vessel.

Deep-tank fermentation Deep-tank batch fermentation in the mass production of penicillin. In the early 2 0th century, eorts were concerted to nd ways to mass produce penicillin. Initial experiments showed that Penicillium notatum grew best in shallow pans due to the need or aeration.

However, this did not produce signicant enough yields to meet the demands or treatment o the casualties o World War II. Large- scale production was acilitated by deep- tank ermentation. This

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employed both a source o oxygen bubbled in to the tank and paddles to distribute the oxygen. The nutrient source or the Penicillium is corn steep liquor. This is the liquid produced by warming a vat o corn in water near 5 0 C or two days. Antibiotics are secondary metabolites in the sense that they are produced at a certain point in the lie cycle o the microbe under certain conditions.

In the case o penicillin, optimum conditions are about 2 4 C , slightly basic pH and a good oxygen supply. The product typically starts being ormed about 3 0 hours ater the start o the batch culture as nutrient concentrations begin to decline and continues or about six days ater which the ermenter has to be drained and the liquid ltered. Using solvents, a crystalline precipitate is generated rom the ltered liquid.

Industrial production of citric acid Production o citric acid in a continuous ermenter by Aspergillus niger and its use as a preservative and favouring. C itric acid is an important ood additive, both as a favour enhancer and a preservative. Industrial production o citric acid relies on the ungus Aspergillus niger. While the greatest raction o industrially produced citric acid is produced by batch ermentation, continuous ermentation has also been attempted. The optimal conditions or citric acid production are high dissolved oxygen concentration, high sugar concentration, an acidic pH and a temperature o about 3 0 C . C itric acid is

produced in the Krebs cycle and so is reerred to as a primary metabolite. I the culture medium is under- supplied with certain minerals such as iron, citric acid builds up in the reaction vessel. Ater the contents o the ermentation vessel are ltered out, calcium hydroxide is added to the ltrate and solid calcium citrate precipitates out o solution. It can then be urther treated chemically to yield citric acid.

Gram staining Gram staining o Gram-positive and Gram-negative bacteria. A traditional test used to classiy bacteria is whether they are Gram-negative or Gram-positive, based on how they react to Gram-staining. The cell wall o Gram-positive bacteria consists o a thick layer o

acidic polysaccharides

peptidoglycan (a polymer consisting o amino acids and sugars) . The greatest raction o the Grampositive cell wall is composed o peptidoglycan.

lipopolysacchariderich outer envelope

thick peptidoglycan layer thin peptidoglycan layer plasma membrane

(a) gram-positive: thick cell wall, no outer envelope  Figure 6

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plasma membrane

(b) gram-negative: thinner cell wall, with outer envelope

B . 1 M i c r o B i o l o g y: o r g a n i s M s i n i n d u s t r y

The cell wall o Gram-negative bacteria is much thinner  only about 20% peptidoglycan (see fgure 6) . Crystal violet binds to the outer membrane in Gram-negative bacteria and when alcohol is added, it washes away the outer membrane and the

crystal violet stain with it. In contrast, the crystal violet binds to the multiple layers within the thick peptidoglycan layer which is not washed away by the alcohol and thus the colour persists.

av: gm- pee 1 Prepare smears of Bacillus cereus, Streptococcus fecalis, Escherichia coli and Micrococcus luteus. Fix these preparations by heating over a bunsen burner.

gram positive

gram negative xation

2 Stain with crystal violet for about 30 seconds. 3 Rinse with water, then cover with Grams iodine. Allow stain to act for about 30 s.

crystal violet

4 Rinse with water, then decolorize with 95% alcohol for 1020 s.

iodine treatment

5 Rinse with water, then counterstain with safranin for 2030 s. decolorization

6 Rinse with water and blot dry. Gram-negative bacteria will be pink. Gram-positive bacteria will be blue or violet.

counter stain safranin

7 Depending on local restrictions, you might choose to examine prepared slides of Gram-negative and Gram-positive bacteria.

Biogas production Biogas is produced by bacteria and archaeans from organic matter in fermenters. B iogas reers to the combustible gas produced rom the anaerobic breakdown o organic matter such as manure, waste plant matter rom crops and household organic waste. D epending on the construction o the ermenter, biogas is mostly methane with some carbon dioxide, though other gases may be present. Three dierent communities o anaerobic microbes are required. The frst group convert the raw organic waste into a mixture o organic acids, alcohol, hydrogen and carbon dioxide. The second group use the organic acids and alcohol rom the frst stage to produce acetate, carbon dioxide and hydrogen. These frst two communities are Eubacteria. The last group are Archaea called methanogens. The methanogens produce methane by one o the ollowing two reactions: C O 2 + 4H 2  C H 4 + 2 H 2 O ( reduction o carbon dioxide to methane)

C H3C O O H  C H4 + C O 2 ( splitting ethanoic acid to orm methane and carbon dioxide) IN  sewage from people  manure from animals  farm waste  garden waste

OUT  methane for cooking, heating or refrigeration

OUT  Slurry, which can be used as a fertilizer

 Figure 7

Methane generator. Conditions inside must be anaerobic

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Producing biogas in the classroom Production o biogas in a small-scale ermenter. Figure 8 shows an example o a set- up o a biogas generator. Mylar balloons are the ones that are commonly flled with helium as party balloons. The eedstock bottle should be plastic rather than glass due to the risk o explosion. The tube clamps can be used to prevent gas leakage when the balloon is to be disconnected rom the set- up. The balloon should be sealed to the glass tube with insulating tape. The rate o biogas generated by dierent eedstocks could be compared. Relative quantities

o organic waste and water could be compared in terms o rate o biogas production. rubber tube

the end of this tube must be ABOVE the level of the feedstock

tube clamps fe ed sto c k

mylar balloon glass connector tubes seal mylar balloon to glass tube with insulating tape  Figure 8

Serendipity and the discovery o penicillin Serendipity has led to scientifc discoveries: the discovery o penicillin by Alexander Fleming could be viewed as a chance occurrence. Serendipity is defned as a lucky accident or the situation where something good or useul is revealed when it was not being specifcally searched or. However, it is only useul i the observer recognizes its value. Alexander Fleming was a S cottish medical doctor and scientist who spent the early part o his career searching or anti- bacterial agents. In 1 92 8, he was investigating the properties o the bacterium Staphylococcus. Ater returning rom an extended holiday, he noticed that one

o his bacterial plates was contaminated with ungus and the zone around the ungus on the plate appeared to have no bacteria while urther away rom the ungus, bacteria grew on the plate. Fleming was wise enough to connect the unexpected observation to his earlier studies o anti- bacterial agents. He proceeded to grow the mold in pure culture and then test it on a number o pathogenic bacteria and discovered that it had an antibiotic eect on several species.

Zones o inhibition as a measure o bactericide efectiveness Experiments showing zone o inhibition o bacterial growth by bactericides in sterile bacterial cultures. B acteria are oten grown on nutrient media in glass or plastic plates called Petri dishes. The plates are incubated under laboratory conditions. Lids are kept on the plates in order to prevent contamination. Individual bacteria divide and orm colonies, but i the entire nutrient surace is exposed to the bacterium, then a lawn o bacteria is grown. What Fleming observed is known as a zone o inhibition; that is, a region on a bacterial lawn

564

 Figure 9

B . 2 B i o t e c h n o l o g y i n a g r i c u lt u r e

where an anti-bacterial eect prevents the growth o bacteria. The consequence is an oten circularshaped disc region. The diameter o the zone o inhibition is a measure o the strength o the anti- bacterial agent. In fgure 9 a plate inoculated with Pseudomonas aeruginosa bacteria had various types o antibiotic discs placed on the surace to determine which is the most eective. This is a species o bacteria that rarely inects healthy

individuals but is a maj or cause o inections acquired by people in hospitals. This technique can be modifed by students to investigate the eectiveness o various antibacterial agents. Absorbent flter paper can be cut into disc shapes by a hole puncher. The discs can be soaked in disinectants, or example, and placed on to a plate that has been inoculated with bacteria.

B.2 B   Understanding  Transgenic organisms produce proteins that were       

 

not previously part o their species proteome. Genetic modifcation can be used to overcome environmental resistance to increase crop yields. Genetically modifed crop plants can be used to produce novel products. Bioinormatics plays a role in identiying target genes. The target gene is linked to other sequences that control its expression. An open reading rame is a signifcant length o DNA rom a start codon to a stop codon. Marker genes are used to indicate successul uptake. Recombinant DNA must be inserted into the plant cell and taken up by its chromosome or chloroplast DNA. Recombinant DNA can be introduced into whole plants, lea discs or protoplasts. Recombinant DNA can be introduced by direct physical and chemical methods or indirectly by vectors.

Applications  Use o tumour-inducing (Ti) plasmid o

Agrobacterium tumefaciens to introduce glyphosate resistance into soybean crops.  Genetic modifcation o tobacco mosaic virus to allow bulk production o Hepatitis B vaccine in tobacco plants.  Production o Amora potato (Solanum tuberosum) or paper and adhesive industries.

Skills  Evaluation o data on the environmental impact

o glyphosate-tolerant soybeans.  Identifcation o an open reading rame (ORF) .

Nature of science  Assessing risks and benefts associated with

scientifc research: scientists need to evaluate the potential o herbicide resistant genes escaping into the wild population.

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Transgenic organisms Transgenic organisms produce proteins that were not previously part o their species proteome. The complete set o proteins that a cell or organism can make is reerred to as its proteome. Proteins are key components o a cells structure and carry out most cellular unctions. S ometimes genetic engineers seek to extend the proteome o an organism or the purposes o a biotechnological application. I the addition to the proteome is due to the addition o a gene rom a dierent organism, then the modied organism is said to be transgenic.  Figure 1

Figure 1 shows glo-sh  , the rst genetically modied organism to be sold as a pet. These transgenic sh have had the gene or the production o green fuorescent protein introduced into their genome. The original organism that was the source o the gene was Aequorea victoria, a j ellysh. The protein SRY is a transcription actor which triggers the expression o genes that lead to the development o male characteristics. Figure 2 shows a transgenic emale mouse ( on the right) that has been genetically modied to express the protein SRY within its proteome. It has caused the emale mouse to develop the same genetalia as the male on the let.

Genetically modifed crop plants Genetically modifed crop plants can be used to produce novel products.  Figure 2

A novel product reers to the presence o a protein or phenotype that was not previously ound in the species. The production o golden rice involved the introduction into rice o three genes, two rom daodil plants and one rom a bacterium, so that the orange pigment - carotene is produced in the rice grains. - carotene is a precursor to vitamin A. The development o golden rice was intended as a solution to the problem o vitamin A deciency, which is a signicant cause o blindness among children globally. C orn has been genetically modied to produce the C RY toxin due to the insertion o a gene rom Bacillus thuringiensis. As a consequence, the plant becomes unpalatable to the European corn borer, an insect pest that signicantly reduces crop yields.

Overcoming environmental resistance in crops Genetic modifcation can be used to overcome environmental resistance to increase crop yields. Limiting actors aecting crop plant growth can be biological or non- biological. B iotic actors include competition rom weed species, predation by insects and inection by pathogens.

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Resistance to the herbicide glyphosate has been introduced to crop plants such as soybeans as part o a strategy or reducing competition with weeds. The introduction o genes or the production o B t toxin into corn is part o a strategy or reducing predation by insects such as the western corn rootworm. Non- transgenic roots will suer considerable damage rom pests, but transgenic roots suer little damage as they have resistance to the rootworm due to the expression o the B t toxin. In Hawaii, researchers genetically modifed papaya plants to be resistant to papaya ringspot virus by leading the plant to express the gene or the virus coat triggering a protective response to the virus. Abiotic actors that limit crop growth include such actors as drought, rost, low soil nitrogen and high soil salinity. D roughtGard  maize contains the gene or cold shock protein B  ( cspB) rom the bacterium Bacillis subtilis that enables it to retain water during drought conditions. A gene rom Thale cress ( Arabidopsis) , AtNHXI, codes or the production o a membrane protein that captures excess sodium into plant vacuoles. Peanut plants have been genetically modifed to express this gene allowing them to grow in saline soils that would otherwise limit crop output.

Components of the gene construct The target gene is linked to other sequences that control its expression. To carry out genetic modifcation, more than the gene must be inserted. Additional sequences are necessary to control the expression o the gene. Most commonly, sequences such as a eukaryotic promoter must be added upstream in the construct and a eukaryotic terminator sequence must be included downstream in the construct. The construct also oten contains a second gene called a recognition sequence which allows engineers to confrm that the construct has been taken up by the host D NA and is being expressed. In some cases, specifc additional sequences have to be added. C onsider the example o genetically modiying sheep to express human proteins such as alpha- 1 - antitrypsin in the sheeps milk. In this case, a specifc promoter sequence that will ensure that the gene is expressed in milk is necessary in creating the gene construct. In addition, a signal sequence has to be added to ensure that the protein is produced by ribosomes on the endoplasmic reticulum rather than by ribosomes that are ree in the cytoplasm. This is to ensure that the alpha- 1 - antitrypsin protein is secreted by the mammary cells rather than released intracellularly.

 Figure 3

Transgenic sheep, awaiting milking. The sheep are ospring o ewes which have a human gene responsible or the production o the protein alpha-1-antitrypsin (A1AT) incorporated into their DNA. A1AT is produced in mammary cells, and secreted in the sheep's milk. The A1AT can then be isolated and used to treat hereditary A1AT defciency in humans, which leads to the lung disease emphysema

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Marker genes Marker genes are used to indicate successful uptake. In addition to the target gene, an additional gene is oten added to provide some way to indicate that uptake o the target gene has occured. Some markers are based on articial selection. In this case, the gene is called a selectable marker. The marker gene oten coners antibiotic resistance. Those bacteria that have taken up the marker gene and the target gene construct will survive exposure to antibiotic. These can then be cultured separately.

 Figure 4

The gene or the production o green fuorescent protein ( GFP) is also used as a marker. Figure 4 shows mosquitos that have been genetically modied to resist being hosts or the malarial parasite. The donor gene has been linked to the gene or GFP so that the transgenic mosquitos can be detected under a microscope.

Recombinant DNA Recombinant DNA must be inserted into the plant cell and taken up by its chromosome or chloroplast DNA. Recombinant D NA is a molecule that has been manipulated so that it contains sequences rom two or more sources. In order to create a transgenic organism, the recombinant D NA must be taken up by the host cell. In order or the gene to be expressed, it has to be taken up into a chromosome. In plant cells, it can also be taken up by a chloroplast. This process o uptake and expression o the donor D NA is called transormation. The new genes can be inserted into the D NA o the chloroplasts. The maj or advantage o inserting into chloroplasts is that the chloroplast D NA is not transmitted through pollen which prevents gene fow rom the genetically modied plant to other plants. Transormation usually requires the use o a vector.

Diferent targets or genetic transormation Recombinant DNA can be introduced into whole plants, leaf discs or protoplasts. O nce the transgene has been introduced into the host cell, the production o a whole plant rom the transormed cell has to be perormed. Protoplasts are plant cells that have had their cell walls removed. Transormation by Agrobacterium was initially attempted on protoplasts. While this was somewhat successul, the diculty o obtaining sucient high quality protoplasts combined with the diculty o growing whole plants rom protoplasts led to the search or other methods. The lea disc methods involves incubating lea cut- outs with Agrobacterium containing a plasmid with the target gene along with an antibiotic resistance gene. The lea discs are then transerred to a plate containing two dierent antibiotics which ensures that only transormed cells will grow. The transormed cells are then cultured and treated in such a way so that roots and shoots develop rom the discs.

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Diferent methods o genetic transormation Recombinant DNA can be introduced by direct physical and chemical methods or indirectly by vectors. Genes can be introduced into plants in a number o dierent ways including microinj ection, electroporation, virus inection, ballistic incorporation and incubation with Agrobacterium tumefaciens. Incubating host cells at cold temperatures in calcium chloride solution and then heat shocking the solution is a chemical method that was one o the original methods or transorming cells. E lectroporation is a physical method that involves applying an external electric feld that leads to the ormation o temporary pores in the cell membrane allowing recombinant D NA to get into a cell. Microinj ection is another physical method o introducing genes. A pipette is used to aspirate and hold a cell in a fxed position while a needle is used to inj ect genes o interest. In biolistics, metal particles coated with the gene o interest are fred at an entire plant. A vector is a virus, a plasmid or some other biological agent that transers genetic material rom one cell to another. In the next section the use o the Ti plasmid vector is explained. O n page 5 70 the use o a virus as a vector is explained.

The use o Ti plasmid as a vector Use of tumour-inducing (Ti) plasmid of Agrobacterium tumefaciens to introduce glyphosate resistance into soybean crops. O ne way to introduce transgenes into plants is to use Agrobacterium tumefaciens. This is a species o bacteria that has a plasmid, called the Ti plasmid, that causes tumours in the plants it inects. The glyphosate resistance gene is inserted into the Ti plasmid along with an antibiotic resistance glyphosate resistance gene plasmid

bacterial cell

gene. The construct is then re- inserted into an A. tumefaciens bacterium. Plant cells are then exposed to the transgenic bacterium and cultured on a plate containing antibiotic. The only plant cells that grow are those that have taken up the plasmid. The others are killed by antibiotic.

plant cell gene transfer

DNA

bacterial antibiotic resistance gene suspension

dead cell

callus

antibiotic medium 1 Cut leaf

 Figure 5

2 Expose leaf to bacteria carrying an antigen gene and an antibiotic resistance gene. Allow bacteria to deliver the genes into leaf cells

3 Expose leaf to an antibiotic to kill cells that lack the new genes. Wait for surviving (gene-altered) cells to multiply and form a clump (callus)

4 Allow callus to 5 The plants sprout shoots and are transferred roots to soil where they can develop into fully dierentiated adult plants that are glyphosate resistant

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Edible viruses Genetic modifcation o tobacco mosaic virus to allow bulk production o Hepatitis B vaccine in tobacco plants. Vaccination programmes are oten impacted by lack o access to remote areas as well as the challenge o rerigerating vaccine preparations. O ne initiative has been to develop edible vaccines by incorporating antigens into plant matter. O ne attempt involved genetically modiying tobacco mosaic virus with antigens rom the Hepatitis B virus and then inecting tobacco plants.

Hepatitis B gene coding for antigen that will stimulate an immune response Capsid gene for tobacco mosaic virus (TMV)

+

Fusion of two genes and incorporation into virus Hepatitis B gene

Capsid gene

Infect plant

antibodies

Plant expresses the antigen

Feed to the animal whose immune system responds by creating antibodies to the Hepatitis B virus

 Figure 6

Potatoes modifed to produce starch containing only amylopectin Production o Amora potato (Solanum tuberosum) or paper and adhesive industries. Potatoes are used in industry as a source o starch. S tarch can be used or a number o purposes including use as an adhesive. Normally, potato starch consists o two dierent types

HO O HO

O HO

o starch polymers ( see fgure 7) . 80% o potato starch consists o the branched chain amylopectin and 2 0% is the straight chain orm, amylose.

OH O HO amylose

O

OH

HO

O

O HO

HO

OH O HO O

HO O HO

O

OH

HO

O HO O HO

O HO

O HO

amylopectin  Figure 7

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O O HO

OH O HO

O HO

O

B . 2 B i o t e c h n o l o g y i n a g r i c u lt u r e

When a starch mixture is heated and then cooled, it tends to orm a gel which is undesirable or some applications such as paper manuacturing and adhesive production. To prevent this, conventional methods use chemical treatment to remove the amylose. The company B ASF produced a genetically modied potato where one o the genes involved in the production o amylose was deactivated. The gene product was granule bound starch synthase. The method used was antisense technology. This involves inserting a version o the gene that is inverted such that it produces the antisense mRNA. The result would be that the normal sense strand would be produced as well as the antisense strand. The two would bind and the double stranded mRNA molecule gets degraded

rather than being translated to orm protein ( gure 8) . digestion

antisense strand

RNase

transcription translation DNA duplex formation mRNA  Figure 8

Assessing risks of transgenes entering wild populations Assessing risks and benefts associated with scientifc research: scientists need to evaluate the potential o herbicide resistant genes escaping into the wild population. Gene fow is the movement o genes or genetic material rom one population to another. In plant populations, it can occur through the transer o pollen between related species. Herbicide resistant genetically modied ( GM) crops are the most common type o genetically modied crop grown. The potential fow o transgenes rom the GM crop to non- GM crops and rom the GM crop to wild weed populations is an economic concern. I the transgene becomes expressed in the wild population, then controlling the eect o the weed population within a crop area would become

dicult. I the transgene is or insect resistance, then ecological balance could be disrupted. Assessing the risk requires estimating how requently gene fow occurs, determining whether the transgene becomes expressed and determining the changes to the phenotype o the plant. O ne strategy or reducing risk is to incorporate mitigator genes with the transgene which is designed to reduce the success o any hybrid plants that might be accidentally created. Another strategy is to transorm chloroplasts rather than nuclear D NA as the chloroplast D NA is not expressed in pollen.

Evaluating the environmental impact of a GM crop Evaluation o data on the environmental impact o glyphosate-tolerant soybeans. Weeds reduce crop yields by competing with crop plants or space, sunlight, water and nutrients. Glyphosate is a chemical that kills a very broad spectrum o plants. Soybeans as well as a number o other crop species have been genetically

modied to be glyphosate resistant allowing armers to use a single broad-spectrum herbicide. There are two potential environmental aspects to consider: the environmental risks o the genetic modication o a crop plant and the

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There has been broad academic consensus that there has been at least some environmental beneft o genetically modifed glyphosphate-tolerant crops in replacing previous widespread systems o weed reduction. The beneft to this modifcation is that crop weeds can be controlled by herbicide without the risk o reduced crop yields because the crop is resistant to the herbicide. Further the level o herbicide required to be applied is lower (table 1 ) than beore the GM crop was introduced. While the data is disputed, many researchers claim that glyphosate is nearly the least toxic pesticide used in agriculture.

% chane in herbicide use in comparison to non-gM crops in 1997 Heartland -23% Northern Crescent -15% Mississippi Portland -11% Southern Seaboard -51%

georaphic reion

 Table 1

Percentage reduction in the amount o herbicide applied in genetically modifed crops over traditional crops in various regions o the US

Tillage is the practice o turning over the soil and has been commonly practised as a component o weed management strategies. The loss o top soil and erosion is one o the consequences o tillage. Glyphosate and glyphosate- resistant crops have enabled signifcantly less tillage and thereore preserved soil ertility. This has reduced

the ossil uel use required or tillage and reduced the need or inputs required to supplement soil ertility. Figure 9 shows the growth in the area cultivated with GM soybeans in Argentina with a corresponding growth in no- till agriculture. Glyphosate resistance ( GR) is under intense selection pressure given the widespread use o the crop and the reduced use o other herbicides. The environmental consequences o resistant weeds will include reduced crop yields or the same inputs and the need to increase the use o tillage and alternative herbicide ormulations. A review conducted in 2 002 by the European Union reached the conclusion that there was little data to support claims o health impacts o glyphosate on humans. S ome studies suggest that other components o the herbicide mixture used in combination with glyphosate did have environmental impacts. The Australian government has banned the use o some ormulations o glyphosate near water. 18 16 14 12 10 8 6 4 2 0 1996

25

GM soybean no-till farming

20 15 10 5

no-till farming (million ha)

environmental risks o the widespread use o glyphosate as an herbicide that is encouraged by the prevalence o the GM crop.

GM soybeans (million ha)

B

0 1998

2000

2002

2004

 Figure 9

Open reading frames An open reading rame is a signifcant length o DNA rom a start codon to a stop codon. When the D NA o an organism has been sequenced, researchers will then look or the location o genes. The starting point or this search is to look or open reading rames. The search or open reading rames (ORF) depends on the ollowing concepts:

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

There are 64 triplets o bases that are called codons.



61 codons are used to code or an amino acid.



There are 3 three stop codons ( TAA, TAG and TGA) that signal the end o an open reading rame.

B . 2 B i o t e c h n o l o g y i n a g r i c u lt u r e There is one start codon ( ATG) that signals the start o an open reading rame and also codes or an amino acid.



O pen reading rames in D NA are identifed by searching or base sequences long enough to code or the amino acids in a polypeptide between a start codon and one o the three stop codons. In other words, they look or sequences where stop codons are absent. Researchers usually look or a base sequence long enough to code or one hundred amino acids. The average size o an O RF in E. coli is 3 1 7 amino acids long.

Identifying open reading frames Identifcation o an open reading rame (ORF) . 2

A short base sequence is shown below. AATTC ATGTTC GTC AATC AGC AC C TTTGTGGTTC TC AC C TC GTTGAAGC TTTGTAC C TTGTTTGC GGT GAAC GTGGTTTC TTC TAC AC TC C TAAGAC TTAA TAGC C TGGTG 1

a) C alculate the percentage chance o fnding a start codon in a piece o D NA with a random sequence o ten base pairs.

Find the frst start codon and the frst stop codon ater it in the sequence. a) S tate how many bases there are beore the start codon. [1 ] b) State how many codons there are in the open reading rame that you have ound.

Researchers need to distinguish between open reading rames that code or polypeptides and random base sequences in the genome that by chance have start codons ollowed by an extended sequence without a stop codon.

[2 ]

b) I the start codon is ound in a random base sequence, calculate the percentage chance that the next triplet o bases codes or an amino acid. [1 ]

[1 ]

c) Calculate how many amino acids are encoded in this open reading rame. Show how you worked out your answer. [3]

c) C alculate the percentage chance that the next 1 00 triplets all code or amino acids.

[2 ]

d-bs qss: Determining an open reading frame O nce the sequence o bases in a piece o D NA has been determined, a researcher may want to locate a gene. To do this, computers search through the sequences looking or open reading rames. An open reading rame is one that is uninterrupted by stop sequences and could thereore code or the production o a protein. The stop codons are UGA, UAA and UAG. 1

S tate the number o codons in the genetic code.

dna 3' A T T A mrna 5' rF1 U A A U rF2 A A U rF3 A U

2

D etermine the raction o codons that are stop codons in the genetic code. [2 ]

3

In table 2 , the codons could start with the frst, second or third base. These correspond to three dierent reading rames ( RF1 , RF2 or RF3 ) . D etermine which o the reading rames, 1 , 2 or 3 , might be an open reading rame. [2 ]

[1 ]

A C T A T A A A G A C T A C A G A G A G G G C T A G T A C U G A U A U U U C U G A U G U C U C U C C C G A U C A U G U G A U A U U U C U G A U G U C U C U C C C G A U C A U G U G A U A U U U C U G A U G U C U C U C C C G A U C A U G

 Table 2

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aciviy Alcanivorax borkumensis is a rod-shaped bacterium that utilizes oil as an energy source. It is relatively uncommon but quickly dominates the marine microbial ecosystem ater an oil spill. Scientists sequenced the genome o this bacterium in an efort to identiy the genetic aspects o its oil digesting ability. The entire genome can be accessed rom the database GenBank. Visit GenBank and search by genome to locate the genome o this organism. Click on FASTA to identiy the

toK Wh knwledge issues re creed by he rpid grwh in he mun f vilble d nd infrmin? The technology o DNA sequencing and bioinormatics has evolved at a rapid pace. In 2009, the biggest problem or researchers was developing solutions to improve the sequencing o DNA. Time and cost limited the production o DNA sequence inormation. By 2013, researchers can sequence a whole human genome within a single day. The challenge has now shited rom sequencing DNA to managing and analysing the extraordinary volume o sequence data that is being produced. It has been estimated that ve months o analysis are needed or every month's worth o data generated.

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organisms GI number. It is listed in the title. (GI number #110832861) . View the genome. Go to the open reading rame nder (http://www.ncbi.nlm. nih.gov/projects/gor/) . Enter the GI number and speciy the range o bases that you are going to search. Perhaps as a class, the genome can be divided up into 2000 bp pieces. Share inormation with one another about the open-reading rames identied.

Identifying target genes Bioinormatics plays a role in identiying target genes. B ioinormatics is the use o computers to investigate biological phenomenon. O pen reading rames are identifed by subj ecting genomic inormation held in a database to searches to fnd extended sequences without stop codons. O nce an open reading rame is identifed, a B LAS T search can be conducted. The acronym reers to B asic Local Alignment S earch Tool. A B LAS Tn search would search through databases to determine i an open reading rame with a similar nucleotide sequence existed in another species. A B LAS Tx search would search a protein database based on the translated sequence o the open reading rame. Alternatively, i a researcher has ound a protein and wants to determine the location o a gene, they can conduct a tB LAS Tn search using a computer search o multiple genomes using the translated sequence to search or potential genes that could have been transcribed to produce the protein. All three methods play a role in identiying target genes.

B . 3 e n vi r o n M e n tal pr o te cti o n

B.3 em  Understanding  Responses to pollution incidents can involve

      

bioremediation combined with physical and chemical procedures. Microorganisms are used in bioremediation. Some pollutants are metabolized by microorganisms. Cooperative aggregates o microorganisms can orm bioflms. Bioflms possess emergent properties. Microorganisms growing in a bioflm are highly resistant to antimicrobial agents. Microorganisms in bioflms cooperate through quorum sensing. Bacteriophages are used in the disinection o water systems.

Applications  Degradation o benzene by halophilic bacteria

such as Marinobacter.  Degradation o oil by Pseudomonas.  Conversion by Pseudomonas o methyl mercury into elemental mercury.  Use o bioflms in trickle flter beds or sewage treatment.

Skills  Evaluation o data or media reports on

environmental problems caused by bioflms.

Nature of science  Developments in scientifc research ollow

improvements in apparatus: using tools such as the laser scanning microscope has led researchers to deeper understanding o the structure o bioflms.

Methods used to address pollution incidents Responses to pollution incidents can involve bioremediation combined with physical and chemical procedures. When chemicals are released to the environment by accident or through carelessness, the result can be signifcant in terms o ecological disruption. B ioremediation is the use o microbes to remove environmental contaminants rom water or soil. In this section, we consider bioremediation strategies or addressing pollutants such as benzene, petroleum oil, heavy metals and sewage. Not all pollution incidents can be addressed solely through bioremediation. B ioremediation may be undesirable in the case o heavy metals because these need to be removed rom the ood chain. In such cases phytoremediation, which relies on plants, might be employed. The heavy metals can bioaccumulate in the biomass o the crop. The crop can then be incinerated to

575

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B I O T E C H N O LO G Y AN D B I O I N FO R M AT I CS concentrate the metal and then the metal can either be recycled or properly contained. There are a number o physical and chemical procedures that can be combined with bioremediation to respond to pollution incidents.

 Figure 1

Soil undergoing bioremediation at Fawley Refnery, an oil refnery and chemical plant located in Fawley, Hampshire, UK



Physical methods or oil spills include the use o scrubbers, detergents and dispersants.



C hemical- contaminated soil can be removed and incinerated to degrade volatile organic chemicals.



S oil can be removed, crushed, sited and then suspended in water that includes chemicals that will aid in dissolving the chemicals into the water. The chemical- contaminated water can then be puried separately.



O xidizing chemicals such as ozone and peroxide are sometimes inj ected into soils to accelerate the destruction o toxic organic compounds.

Microorganisms have properties that make them useful for bioremediation Microorganisms are used in bioremediation. B acteria and archaeans are useul in bioremediation because they can multiply very quickly by binary ssion and they are varied in their metabolism. They carry out a wider range o chemical reactions, especially inorganic reactions, than any other group o organisms. There is oten a species o prokaryote that will perorm the necessary reaction in a bioremediation process. Figure 1 shows a biopile. This is a method or addressing pollution in soil. A bulking agent such as compost, hay or other nutrient source is dug into the piles and the piles are constantly watered. The microbial community which fourishes digests the contaminants.

Bioremediation relies on microorganism metabolism  Figure 2

Some pollutants are metabolized by microorganisms. Microorganisms can use pollutants as energy sources, carbon sources and electron acceptors in cellular respiration. The bacterium Dehalococcoides ethenogenes ( shown in red in gure 2 ) has been used to break down chlorinated solvents in soil. It uses the chlorine compounds as electron acceptors in cellular respiration. The bacterium Geobacter sulfurreducens uses uranium as an electron acceptor converting it rom a soluble to an insoluble orm, which allows the uranium to settle out and be collected.

 Figure 3

576

Figure 3 shows the bacterium Acidovorax sp. ( yellow) partially coated with iron ( orange) . This bacterium is able to precipitate iron and arsenic out o the soil and bind it. D ue to this, it is being investigated as a means o reducing the amount o arsenic present in rice elds.

B . 3 e n vi r o n M e n tal pr o te cti o n

Microorganisms can orm bioflms Cooperative aggregates o microorganisms can orm bioflms. A biolm is a colony that coats a surace as a consequence o cooperation between individual cells. Members o a biolm colony secrete signalling molecules that recruit independent, or planktonic, cells into the colony. They also secrete molecules that acilitate the aggregate adhering to the surace and acilitate individual cells sticking together. O n their cell membranes, cells produce protein channels that acilitate the exchange o materials with other members o the colony. While biolms normally orm on solid suraces, they can orm on the surace o fuids. Sometimes, they can be composed o a community o organisms including bacteria, archaea, protozoa, algae and ungi. D ental plaque is a biolm that can contain up to 5 00 taxa o microorganisms while the biolm that orms in the lungs o patients aficted with cystic brosis is oten composed o a single species: Pseudomonas aeruginosa. Figure 4 shows a magnied view o a bristle rom a used toothbrush. The surace o the bristle is covered in a biolm o cooperating bacteria. Figure 5 shows a biolm inside a catheter. A catheter is a tube used in medical treatment to drain urine or maintain a connection to the bloodstream. The centre part is meant to be hollow but is covered in a white-coloured biolm.

 Figure 4 Bioflm

on the bristle o a used toothbrush

Emergent properties Bioflms possess emergent properties. Properties that emerge rom the interaction o the members o a collective that are not present in the single cell orm are reerred to as emergent properties. In biolms, the ability o the cells to sel-organize into a complex structure is an emergent property. Members o the colony secrete a chemical known as exopolysaccharide ( EPS) that orms into a matrix that holds the colony together and protects it. This matrix is an emergent property. Increased resistance to antibiotics; signalling between members o the colony; increased virulence; the ormation o channels or water fow inside the colony; and the ability o cells to use the matrix to move leading to the colony itsel moving are all considered emergent properties.

 Figure 5 Bioflm

ormed on the inside o a catheter

Bioflms resist antimicrobial agents Microorganisms growing in a bioflm are highly resistant to antimicrobial agents. Hospital acquired inections, or nosocomial inections, are commonly caused by biolms. Increased resistance to antibiotics sometimes occurs in biolms and is o concern to inection control ocers within hospitals. There are a number o proposed mechanisms or biolm antibiotic resistance. In part, the resistance is due to the exopolysaccharide (EPS) matrix providing a physical barrier to the entry o the antibiotic into the colony.

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B I O T E C H N O LO G Y AN D B I O I N FO R M AT I CS Antibiotics oten act on mechanisms that inhibit cell division. In some bioflms, limited supplies o nutrients leads to a suppression o the collective division rate which minimizes the eect antibiotics can have. This can especially be true o individuals deeper into the colony.

Quorum sensing Microorganisms in bioflms cooperate through quorum sensing. Quorum sensing is a system o behaviours that are triggered as a unction o population density. It is observed in a diverse range o organisms. In bacteria that orm bioflms, gene expression can be aected by population density. S ignalling molecules released by one cell bind to receptor molecules on another cell and lead to the expression o genes that are likely to acilitate the development o the bioflm. When population density is low, the concentration o the signalling molecule is low and is insufcient to trigger coordinated behaviour. When the population passes a threshold level; i.e., when the quorum is achieved, the concentration o the signalling molecule reaches a critical concentration and the behaviour becomes coordinated. The pathogen Pseudomonas aeruginosa uses quorum sensing to coordinate movement, EPS production, cell aggregation and the ormation o bioflms. locally high signal molecule concentration EPS matrix signal molecule secreted modied metabolism

relatively low concentration of signal molecule from other cells

signal molecule receptors

Free form

signal molecule secreted

Biolm

 Figure 6

Using viruses to kill bacteria in water systems Bacteriophages are used in the disinection o water systems. When bacteria produce a bioflm, they can be difcult to eradicate. The control o bioflms within water systems is essential. S ome o the damage that can be done includes:

 Figure 7

Bacteriophages (pink) shown infecting a population of bacteria shown as green

578



Anaerobic sulphate reducing bacteria produce sulphuric acid which can corrode pipes.



B ioflms can aect heat exchange in systems where the release o waste heat to the environment is important.

B . 3 e n vi r o n M e n tal pr o te cti o n



A prolierating bioflm can reduce the diameter o a pipe. This results in rictional drag, which lowers water pressure which leads to a need or increased pumping power.

B acteria can be difcult to kill when they orm a bioflm. The outer layer o bacteria in these bioflms can be killed by disinectant, but the inner bacteria are sheltered. Viruses solve this problem because they spread through the entire bioflm community. Viruses which attack bacteria are known as bacteriophages and they are specifc to certain bacteria. O ne study achieved the greatest success in killing bioflms by using a combination o bacteriophages and chlorine. An initial treatment with viruses ollowed by chlorine killed 97 percent o bioflms within fve days o exposure while chlorine alone removed only 40 percent. In addition, there may be specifc pathogenic bacteria that are living in the bacterial community, such as coliorm bacteria. B acteriophages that are specifc to the pathogen can be added to ensure reduction o the particular pathogen. The T4 bacteriophage is specifc to E. coli.

Bioremediation in saline conditions Degradation of benzene by halophilic bacteria such as Marinobacter. The production o oil in marine environments generates volumes o saline ( salty) wastewater that is contaminated with hydrocarbons such as benzene and toluene. B enzene ( fgure 8) is o particular concern as it can persist in the environment or a long time, is moderately soluble in water and is carcinogenic; i. e., it can lead to cancer. B ioremediation becomes difcult in this case as the salt content may be so high in the waste water that it kills most populations o bacteria.

H

H C

H

C

C

C

 Figure 8

H

Benzene molecule

H

C

C H = hydrogen C = carbon

S ome archaea are adapted to living in extreme environments such as highly saline water ( fgure 9) . They are reerred to as halophiles. This adaptation has been useul in the bioremediation o saline wastewater. O ne species o halophilic archaea, Marinobacter hydrocarbonoclasticus has been shown to be able to ully degrade benzene.

H benzene

 Figure 9

The colour in this salt pan pool is a indicator of the presence of a population of halophilic bacteria

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Bioremediation o oil spills Degradation o oil by Pseudomonas. In natural environments, some petroleum seeps through cracks and vents in the ocean foor. Some members o the genus Pseudomonas thrive in these communities as they can use crude oil as an energy and carbon source. C lean-up at oil spills will oten involve seeding the spill with Pseudomonas. These microbes also require substances such as potassium and urea as nutrients to metabolize the oil at a aster rate. These nutrients are oten sprayed on to an oil spill to aid the bacteria in their work. Figure 1 0 shows a population o bacteria degrading a droplet o oil suspended in water.

 Figure 10

Bioremediation o methyl mercury Conversion by Pseudomonas o methyl mercury into elemental mercury. Mercury ends up in garbage dumps as a component o some paints and some types o light bulbs. Elemental mercury is converted in this environment to the highly toxic organic methyl mercury by the bacterium Desulfovibrio desulfuricans. This orm o mercury more easily enters ood chains because it adheres to cell membranes and can dissolve in the cell membrane. It can bioaccumulate within the biomass o organisms and it can biomagniy up the ood chain.

The bacterium Pseudomonas putida can convert the methyl mercury to methane and the mercury ion. O ther bacteria then use the soluble mercury ion as an electron acceptor resulting in insoluble elemental mercury being reormed. In a bioreactor, such elemental mercury can be separated rom waste water as it is insoluble and will sink due to its density.

Bioflms used in trickle flter beds Use o bioflms in trickle flter beds or sewage treatment. The consequence o not treating sewage and allowing it to fow into watercourses is nutrient enrichment, or eutrophication o bodies o water. This avours algal blooms. When the mats o algae die, it leads to a loss o oxygen, because o bacterial activity on the dead organic matter. This is called biological oxygen demand.

adds oxygen to the sewage, which is necessary or the aerobic bacteria to digest the sewage content.

Many sewage treatment plants make use o biolms to address eutrophication. A trickling lter system has a rock bed that can be up to 2  metres deep. The rocks are colonized by a biolm o aerobic bacteria. S ewage water is sprayed onto the rocks. The process o spraying  Figure 11

580

B . 3 e n vi r o n M e n tal pr o te cti o n

Media reports on bioflms Evaluation o data or media reports on environmental problems caused by bioflms.  Most people expect to nd Salmonella on raw meats but dont consider that it can survive on ruits, vegetables or dry products, which are not always cooked,  said Ponder.

B iolms are commonly eatured in the media as they have a number o novel and interesting properties. They are employed as innovative solutions to problems. At the same time they have been implicated in a number o environmental issues:

In moist conditions, Salmonella thrive and reproduce abundantly. I thrust into a dry environment, they cease to reproduce, but turn on genes which produce a biolm, protecting them rom the detrimental environment.

Virginia Tech scientists have provided new evidence that biolms  bacteria that adhere to suraces and build protective coatings  are at work in the survival o the human pathogen Salmonella.

Researchers tested the resilience o the Salmonella biolm by drying it and storing it in dry milk powder or up to 3 0 days. At various points it was tested in a simulated gastrointestinal system. Salmonella survived this long- term storage in large numbers but the biolm Salmonella were more resilient than the ree-foating cells treated to the same conditions.

One out o every six Americans becomes ill rom eating contaminated ood each year, with over a million illnesses caused by Salmonella bacteria, according to the C enters or Disease C ontrol and Prevention. Finding out what makes Salmonella resistant to antibacterial measures could help curb outbreaks. Researchers aliated with the Fralin Lie Science Institute discovered that in addition to protecting Salmonella rom heat- processing and sanitizers such as bleach,  biolms preserve the bacteria in extremely dry conditions, and again when the bacteria are subj ected to normal digestive processes.

The bacterias stress response to the dry conditions also made them more likely to cause disease. B iolms allowed the Salmonella to survive the harsh, acidic environment o the stomach, increasing its chances o reaching the intestines, where inection results in the symptoms associated with ood poisoning. This research may help shape Food and D rug Administrations regulations by highlighting the need or better sanitation and new strategies to reduce biolm ormation on equipment, thus hopeully decreasing the likelihood o another outbreak.

O utbreaks o Salmonella associated with dried oods such as nuts, cereals, spices, powdered milk and pet oods have been associated with over 900 illnesses in the last ve years. These oods were previously thought to be sae because the dry nature o the product stops microbial growth.

Source: http://www.sciencedaily.com/releases/2013/04/ 130410154918.htm

ay



Choose one or more o the ollowing environmental issues related to bioflms. Create a brie research report outlining the scope o the problem. Ensure that you include the role o bioflms. Evaluate possible solutions to the problems caused by the bioflm.

The development o bioflms on equipment and piping systems in industry such as paper making acilities.



The development o bioflms in clean water pipes at water treatment acilities.



The binding o positively charged heavy metals to negatively charged bioflms.



The sequestering o toxins within the bioflm.



The role o bioflms in increasing biological oxygen demand in eutrophic bodies o water.

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Laser microscopes have enhanced our knowledge o bioflms Developments in scientic research ollow improvements in apparatus: using tools such as the laser scanning microscope has led researchers to deeper understanding o the structure o biolms. B iolms have a complex structure. The position o individual cells in relation to one another and the E PS matrix infuences roles and unctions. Three- dimensional visualization o living cells serving dierent unctions can be carried out using a laser- scanning microscope in combination with dyes. This technique allows direct observation o the biolm without disrupting its structure. Figure 1 2 shows an image o a ragment o biolm extracted rom amniotic fuid. The image was

 Figure 12

generated using a laser scanning microscope. Red dots indicate EPS , green dots indicate bacteria and grey dots represent host cells.

B.4 Medicine (ahl) Understanding  Inection by a pathogen can be detected by

   





the presence o its genetic material or by its antigens. Predisposition to a genetic disease can be detected through the presence o markers. DNA microarrays can be used to test or genetic predisposition or to diagnose the disease. Metabolites that indicate disease can be detected in blood and urine. Tracking experiments are used to gain inormation about the localization and interaction o a desired protein. Biopharming uses genetically modied animals and plants to produce proteins or therapeutic use. Viral vectors can be used in gene therapy.

Applications  Use o PCR to detect diferent strains o

inuenza virus.  Tracking tumour cells using transerrin linked to luminescent probes.  Biopharming o antithrombin.  Use o viral vectors in the treatment o Severe Combined Immunodeciency (SCID) .

Skills  Analysis o a simple microarray.  Interpretation o the results o an o ELISA

diagnostic test.

Nature o science  Developments in scientic research ollow

582

improvements in technology: innovation in technology has allowed scientists to diagnose and treat diseases.

B . 4 M e d i ci n e ( ah l)

Innovations in diagnostic techniques Developments in scientifc research ollow improvements in technology: innovation in technology has allowed scientists to diagnose and treat diseases. To be useul, new methods used to diagnose a disease must be accurate and preerably simple to use. They should provide a result that is timely and increases the time to carry out treatment in such a way that long- term complications do not result. In the case o inectious diseases, aster and more accurate diagnosis can lead to treatment which prevents the spread o the pathogen.

bacterial inection exists, the sample can be plated on culture media to look or the growth o the kind o bacterial colonies which characterize a certain disease. The limitation o this procedure is that sometimes dierent microorganisms present in the same way. Further, some pathogens are difcult or slow to culture.

Inection by parasites has oten been diagnosed by microscopic analysis to look or the presence o the organism or evidence o its activity.

Diagnosis o genetic diseases has traditionally been carried out by reviewing a combination o clinical observation and searching or the presence o high levels o unusual metabolites in the urine or blood.

D iagnosis by bacterial inection has traditionally been done by collecting samples o urine or stool, or swabs can be taken rom an inected site. I

Improvements in methods o diagnosis have increased the specifcity, the speed and the reliability o diagnosis.

High levels of metabolites can indicate disease Metabolites that indicate disease can be detected in blood and urine. Inborn errors o metabolism is a term applied to a broad group o genetically inherited disorders that aect metabolism. The maj ority o these diseases are due to mutations in single genes that code or enzymes oten resulting in a non- unctional enzyme. This results in a build- up o substances which are toxic or a shortage o important molecules necessary or normal unction leading to secondary symptoms. Table 1 shows three such diseases and the metabolites that are detected in blood and urine when an individual is aected. Newborn inants are subjected to a heel prick test to detect phenylketonuria (PKU) , in which a blood sample is taken rom the heel o the oot. I the child is aected, there will be elevated levels o phenylpyruvate in the blood indicating the child lacks an enzyme or converting the amino acid phenylalanine to tyrosine. I diagnosed quickly enough, diet modifcation can prevent severe consequences or the child.

dss

Mtbo ptwy tt s ot futog

Mtbot tt s tt

LeschNyhan syndrome

Production o purines

Uric acid crystals in the urine

Alkaptonuria

Breakdown o the amino acid tyrosine

High levels o homogentisic acid detected in both the urine and the blood by thin layer chromatography and paper chromatography

Zellweger syndrome

Assembly o peroxisomes (organelles essential or the degradation o long chain atty acids)

Elevated very long chain atty acids in the blood

 Table 1

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Indicators of infection by a pathogen Infection by a pathogen can be detected by the presence of its genetic material or by its antigens. Modern molecular methods have the advantage o being much better at discriminating between pathogens. They can be automated to speed up the process and they dont present the challenge o having to culture the pathogen separately. The E nzyme- Linked Immunosorbent Assay ( E LIS A) detects the presence o antibodies to pathogens. The challenge with this diagnostic test is that it is usually only eective once the patient has developed an immune response to the pathogen resulting in the production o antibodies. Recent versions o the E LIS A test or the antigen directly such as the p2 4 antigen rom the HIV virus. PC R can be used to detect the genetic material o a pathogen. I primers that have the same nucleotide sequence as the genetic material o the

The ELISA test Interpretation of the results of an ELISA diagnostic test. An ELISA test can be used to detect the presence o inection by a pathogen. The test works by testing or the presence o antibodies to the antigens o the pathogen. Alternatively, it can test or the antigen directly. Figure 1 shows the basis o a positive test or HIV. A capture molecule is fxed to a surace. In the fgure, these capture molecules are antibodies to the HIV p2 4 capsid protein. The sample to be tested is exposed to the capture surace. B ecause the target molecules are present in a positive test, they bind to the capture molecules. Next a ree version o the capture molecule is added. This version o the capture molecule is

+

 Figure 1

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Figure 2 shows a tray o wells containing human blood serum rom dierent individuals being tested or antibodies to the hepatitis C virus. Wells which remain uncoloured are negative. Those that change colour to yellow/orange are positive and confrm that the patient has antibodies or hepatitis C virus.

+

substrate +

capture antibody antigen

linked to an enzyme. The solution is rinsed. In a negative test, this would wash away the ree version o the capture molecule. In a positive test, they bind to the target molecule and they are not washed away. The last step is to add the substrate o the enzyme which changes colour when acted upon by the enzyme. A positive test is thereore indicated by a coloured solution (see fgure 2) .

colour change by activity of conjugated enzyme

detection antibody enzyme attached to detection antibody converts substrate to coloured product

Steps in a positive ELISA test

 Figure 2 Results of multiple ELISA tests for the Hepatitis C virus

B . 4 M e d i ci n e ( ah l) pathogen are added to a sample rom the patient, then amplication will only occur i the genetic material o the pathogen is present. Another way to detect the presence o a pathogen is to use D NA probes in a microarray. These can be used to detect mRNA sequences complementary to the pathogen in samples rom a patient.

atvty

2

1 Explain how the standard curve could be used. 2 Determine the concentration o antigen present at an optical density o 1.0.

O.D.

Figure 3 shows a standard curve that relates quantity o antigen present in the test serum to optical density, a measure o the colour o solution. The darker the colour, the higher the optical density.

1

[2 ] 0 0

[1]

100

200

300

400

500

antigen concentration /pg mL- 1  Figure 3

PCR as a diagnostic tool Use o PCR to detect diferent strains o inuenza virus. There are a number o clinical signs and tests that can indicate inection by an infuenza virus. For some people, inection with more serious strains such as swine fu needs to be diagnosed quickly. This includes such patients as pregnant women, elderly patients or patients whose immune system is compromised, as the inection can result in death. Further, some strains can produce more serious side eects. In addition, rapid detection can prevent a serious epidemic. The PC R test is most likely to be able to identiy the specic strain o the virus that inects a person.

mRNA being sought was present in the original sample and the cD NA will be amplied. A recent modication is to include fuorescent dyes into the sample that bind specically to double-stranded D NA. As the quantity o double- stranded D NA increases, fuorescence will be detected indicating a positive test. mRNA reverse transcriptase mRNA cDNA

B ecause the infuenza virus is an RNA virus, a variation o PC R called reverse transcription polymerase chain reaction ( RT- PC R) is used. Reverse transcriptase will produce a D NA molecule rom an RNA template called cD NA. The rst step involves puriying mRNA rom cells o an inected patient. The mRNA extract is converted into cD NA. Then primer sequences specic to the strain o infuenza virus being tested or are added. I the infuenza primers bind to sequences in the cD NA, this means that the

RNase cDNA primer 3 + Taq polymerase Double-stranded cDNA (target) Amplication  Figure 4

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chromosome 13

Genetic markers Predisposition to a genetic disease can be detected through the presence of markers.

BRCA 2 BRCA 1

 Figure 5 Chromosomal

location of the BRCA 1 and BRCA 2 genes

Genetic markers are particular alleles which are associated with a predisposition to having a genetic disease. They can be single nucleotide polymorphisms or tandem repeats. D etection o the marker can be achieved through such methods as PC R, and D NA proling. Markers may be part o a coding or non-coding sequence; i.e., they may contribute to the disease or they may be genetically linked to the gene that infuences the condition. To be useul, non- coding markers need to lie near to the deective gene to avoid being separated by crossing over. The marker should be an allele or which the population is polymorphic; that is, there should be a number o possible genotypes at the locus. Researchers look or alleles which are ound more requently than expected by chance in those people aected by the disease. For example, mutations in the B RC A 1 and B RC A 2 genes indicate an increased risk o breast cancer and ovarian cancer in women and the gene itsel contributes to the onset o the cancer. The genes are ound on chromosome 1 7 and chromosome 1 3 respectively.

 Figure 6

There are dierent alleles o B RC A mutations. Figure 6 shows the separation o proteins by electrophoresis. In this case, radioactively labelled amino acids were supplied during protein synthesis and the products were separated by electrophoresis and photographed using lm that detects radioactivity. The arrows indicate the various types o marker proteins produced by dierent mutations o the B RC A 1 gene. The presence o such marker proteins in a blot rom an individual would indicate a predisposition to cancer. For diseases which are linked to a single gene, the marker has more predictive power. Where diseases are strongly infuenced by the environment or are polygenic, particular markers have less predictive power, though considerable progress has been made recently in establishing statistical probabilities rom more complex inheritance patterns.

DNA microarrays DNA microarrays can be used to test for genetic predisposition or to diagnose the disease. A microarray is a small surace that has a large range o D NA probe sequences adhering to its surace. Microarrays can be used to test or expression o a very large number o D NA sequences simultaneously.

 Figure 7 A DNA microarray cartridge being loaded into a machine that will be used to analyse the results from this test

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The sample to be tested is the mRNA being expressed by a cell. cD NA is ormed rom the mRNA using reverse transcriptase. At the same time as synthesis, fuorescent dyes are linked to the cD NA. The microarray is exposed to the cD NA sample long enough or any complementary sequences to bind to the xed probes and then the chip is rinsed. The chip is then exposed to laser light which will cause the fuorescent

B . 4 M e d i ci n e ( ah l) probes to give o light where there has been hybridization between the cD NA and the D NA probes within the chip. The brighter the light, the higher the level o gene expression in that region. 1.28 cm 1.28 cm actual size of GeneChip array

A

A T C A T G

A T T C C A A A T T G T C G

A T G

A T C A T G

A T C A T G

A T C A T G

A T C A T G

A T C

A A T T G CA AT TC GA

A T C A T G

T G

A T C A T G

A T C A T G

A T C A T G

A T C A T G

non-hybridized DNA

millions of DNA strands built in each location 6.5 million locations on each GeneChip array

actual strand = 25 base pairs

hybridized DNA

 Figure 8

Interpreting a microarray Analysis of a simple microarray. As an example o the use o a microarray, an experimenter may want to assess the range and level o gene expression in a cancerous cell. They would extract mRNA rom control cells and produce labelled cD NA rom this sample. They would modiy this cD NA with a green fuorescent dye. They would then extract mRNA rom cancerous cells, produce cD NA and label it with red dye. They would then expose the microarray chip to both samples, allow time or hybridization 1 Spot DNA fragments on glass slide to make microarray

and then wash the chip to remove unhybridized cD NA. The chip would then be exposed to fuorescent light. The part o the chip where green light is observed indicates sequences being expressed in the control only. The part o the chip where there is red light is where the sequences are being expressed by the cancerous cells only. Yellow light, which is a combination o green and red light, corresponds to regions where both types o cells are expressing the sequence. 2 Isolate mRNA from cells

normal

cancerous

3 Use mRNA to produce cDNA for stability and label with dyes

Yellow: equal activity for both cell types Green: higher gene activity for normal cells

4 Mix and wash over microarray. Scan with laser and detect levels of binding/expression using uorescent detection

Red: higher gene activity for cancer cells  Figure 9

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Protein tracking experiments Tracking experiments are used to gain inormation about the localization and interaction o a desired protein. Proteins circulating in the blood can be traced i radioactive probes are attached to them. S uch tracking experiments can allow researchers to ollow distribution and localization patterns. They can also allow researchers to determine how the proteins interact with the target tissue. Radioactive atoms or molecules can be attached to the proteins and their distribution can be tracked with PE T scans.

Tracking experiments involving transferrin Tracking tumour cells using transerrin linked to luminescent probes. Transerrin is a molecule which binds iron. It is taken up by more by tumour cells than by surrounding cells. Figure 1 0 shows a sequence o photos taken using luminescent dyes linked to transerrin molecules. The experiment is being used to study receptor- mediated endocytosis in lymphoma cells. At zero minutes, the transerrin is shown bound to receptors on the surace o cells. The dots represent the fuorescent dye. The bottom image shows some o the receptortranserrin complexes having entered the cell. O nce they have delivered their load o iron, the receptor transerrin complex is recycled to the cell- surace membrane ( top right) .

 Figure 10

Biopharming Biopharming uses genetically modifed animals and plants to produce proteins or therapeutic use. There are thre e main categorie s o prote ins used in the rapy: antibodie s, human proteins and viral or b acterial proteins ( used in vaccines) . The production o simple human recombinant proteins or therapy, such as insulin and growth hormone, has been most successully carried out in genetically modied bacteria. However, the production o more complex therapeutic proteins is more dicult to produce

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B . 4 M e d i ci n e ( ah l) in these living systems. Prokaryotic systems do not carry out the required post- translational modifcation such as the addition o sugars. S ometimes, only mammal cells are capable o perorming these modifcations. Producing these proteins in transgenic arm animals addresses the post-translational modifcation problem. Some domestic varieties o cows, sheep and goats have been selectively bred to produce high yields o milk. Lactating emale animals have been engineered to secrete recombinant proteins into their milk. The combination o these two actors means a small herd o animals can yield a relatively large mass o therapeutic protein. Plant- made therapeutic proteins have been made using whole plants and plant cell cultures. In May 2 01 2 , the frst plant- made human therapeutic protein was approved or use in humans by the US Food and D rug Administration ( FD A) as enzyme- replacement therapy to treat the symptoms o Gauchers disease.

Biopharming to produce ATryn Biopharming of antithrombin. Antithrombin defciency is a condition that puts patients at risk o blood clots during childbirth and surgery. ATryn is the commercial name o antithrombin that has been produced in the mammary glands o genetically modifed goats. To achieve this genetic modifcation, the gene o interest and specifc additional sequences have to be added. A specifc promoter sequence that will ensure that the gene is expressed in milk is necessary in creating the gene construct. In addition, a signal sequence has to be added to ensure that the protein is produced by ribosomes on the endoplasmic reticulum rather than by ribosomes that are ree in the cytoplasm. This is to ensure that the antithrombin protein is secreted by the mammary cells rather than released intracellularly.

mammary gland-specic regulatory gene of sequences interest

isolate oocytes & enucleate

+ transfer reconstructed embryo into recipient female

target protein expression vector transfect cells

select cell

fuse transgenic cell to enucleated oocyte

verify presence of transgene

 Figure 11

Gene therapy Use of viral vectors in gene therapy. S ome inherited diseases are caused by a deective gene, that results in the lack o a particular enzyme or protein. C ystic fbrosis is one such disease. It is caused by the lack o cystic fbrosis transmembrane protein ( C FTP) . This protein normally transports chloride ions out o cells and into mucus. The chloride ions draw

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B I O T E C H N O LO G Y AN D B I O I N FO R M AT I CS retroviral vector capsid envelope

adenoviral vector

reverse transcriptase DNA genome RNA genome

cell membrane

ribosome

nuclear membrane DNA therapeutic gene

Gene therapy may oer a cure or inherited diseases like cystic fbrosis. In gene therapy, working copies o the deective gene are inserted into a persons genome. To do this, a gene delivery system, or vector, is needed. Figure 1 2 shows two dierent ways o using viruses as vectors. The viral genome is altered so that the particles are not virulent. The therapeutic gene is then inserted into the virus. Viruses that contain doub le - strande d ( ds) D NA, such as adenovirus, cannot cause the prob lems ound with re troviruses b ecause the viral D NA is not inserted into the ge nome. However, the therapeutic gene is not passed on to the next generation o cells, so tre atment has to be repeate d more re quently. A challenge o using viruses as ve ctors is that the host may de velop immunity to the virus.

therapeutic protein

RNA/DNA

water out o the cells and make mucus watery. C ystic fbrosis patients suer rom thick mucus, which builds up in the airways.

nuclear pore

therapeutic gene

The treatments described above are called somatic therapy, because the cells being altered are somatic ( body) cells. An alternative method would be to inj ect therapeutic genes into egg cells. The missing gene would be expressed in all cells o the organism. This is called germ line therapy.

 Figure 12

Two diferent gene therapy techniques involving viral vectors

Gene therapy to treat SCID Use o viral vectors in the treatment o Severe Combined Immunodefciency (SCID) . Defciency o the enzyme adenosine deaminase (ADA) leads to the accumulation o deoxyadenosine within cells. This is particularly toxic to T and B lymphocytes. The lack o unctional immune cells leads to severe combined immunodefciency syndrome (SCID) which is characterized by an inability to fght o the simplest o inections. ADA defciency was the frst condition successully treated by gene therapy. The steps involved in the successul therapy included:

590



Removing AD A defcient lymphocytes rom the patient with SC ID .



C ulturing the cells in vitro.



Inecting the cultured cells with genetically modifed retrovirus containing the gene that can produce unctional AD A.



D elivering the modifed lymphocytes by transusion back into the patient.

The eect lasted or our years ater the start o gene therapy in one patient.

B . 5 B i o i n F o r M at i c s ( a h l )

B.5 Bfm (ahl) Understanding  Databases allow scientists easy access to      

 

inormation. The body o data stored in databases is increasing exponentially. BLAST searches can identiy similar sequences in diferent organisms. Gene unction can be studied using model organisms with similar sequences. Sequence alignment sotware allows comparison o sequences rom diferent organisms. BLASTn allows nucleotide sequence alignment while BLASTp allows protein alignment. Databases can be searched to compare newly identied sequences with sequences o known unction in other organisms. Multiple sequence alignment is used in the study o phylogenetics. EST is an expressed sequence tag which can be used to identiy potential genes.

Applications  Use o knockout technology in mice to

determine gene unction.  Discovery o genes by EST data mining.

Skills  Explore the chromosome 21 in databases (or

example in Ensembl) .  Use o sotware to align two proteins.  Use o sotware to construct simple cladograms and phylograms o related organisms using DNA sequences.

Nature of science  Cooperation and collaboration between groups

o scientists: databases on the internet allow scientists ree access to inormation.

The role of databases in genetic research Databases allow scientists easy access to inormation. A database is a structured collection of information stored on a computer. It can include data in a range of formats including qualitative information, articles, images or quantitative information. Types of databases used in bioinformatics include: 

Nucleotide sequence databases such as E MB L ( The European Molecular B iology Laboratory) .



Protein sequence databases such as SwissProt.



Three-dimensional structure databases such as PDB (Protein Data Bank) .



Microarray databases such as ArrayE xpress which contain information about the level and types of mRNA expressed in different cells.



Pathway databases which contain information about enzymes and reactions and can be used to model metabolic pathways. An example of such a database is KEGG (Kyoto Encyclopedia of Gene and Genomes) .

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toK

Hypothesis testing is increasingly possible by extracting data rom a database rather than the researcher collecting the data directly or themselves.

t wh exen des scenfc reserch requre reguln? i  des requre reguln wh shuld dmnser he regulns?

A researcher can employ a database to do a number o tasks:

In 1999 a patient died as a result o participation in clinical trials or gene therapy. He sufered rom ornithine transcarbamylase deciency, or OTC, a liver disease marked by an inability to metabolize ammonia. Ammonia is a waste product o amino acid metabolism. He had been able to survive up to that point because o dietary modication and medication. The trial he participated in involved being injected with adenoviruses carrying the gene or transcarbamylase. He died within days due to a strong immune response to the viral vector. An investigation concluded that the scientists involved in the trial violated several rules o conduct. 

Four other patients who had received the treatment had reactions that were deemed so severe that the trial should have ended.



The inormed consent orms did not include inormation about primates that had died in similar trials.



The patient had levels o ammonia that were so high he should have been excluded rom the study.



A principal investigator o the study had a major interest in the outcome o the trial as he held patents on the OTC treatment.

From Welcome to the Genome by Bob De Salle and Michael Yudell 1

Explain what is meant by inormed consent.

2

) Suggest what policy instruments might be put in to place to prevent such occurrences. b) Who should administer these policies  governments, other scientists or research institutions?



add the results o their research or others to access



extract subsets o data



query the database by searching or a particular piece o data.

Growth in information housed in databases The body o data stored in databases is increasing exponentially. Advances in technology have meant that the rate o creation and publication o data is increasing. Advances in genome sequencing technology, microarrays, 3 -D modelling programmes and computing power have resulted in a number o large- scale collaborative research proj ects which have generated an exponential growth in data housed in databases. O ne research report tracked the growth in inormation in bioinormatics databases and concluded that it has a doubling time o between 1 2 and 2 4 months.

access  nrmn ssues n bnrmcs Cooperation and collaboration between groups o scientists: databases on the internet allow scientists ree access to inormation. Most people presume that collaboration and cooperation between researchers characterizes the scientifc endeavour. Most o the important bioinormatics databases are public and reely accessible to all researchers. O ten, once data is added to one database, it is immediately synchronized with data in other databases. S uch open access and synchronization acilitates collaboration and a spirit o cooperation. O ne view is that the commercialization o bioinormatics databases is a threat to this spirit. S ome researchers working in private companies do not post their sequence inormation because o the need to make a proft. Some databases that have been public in the past have been taken over by or- proft companies who have started to charge or access to sequence inormation. Two examples are the Saccharomyces cerevisiae ( yeast) and Caenorhabditis elegans ( soil roundworm) databases, two o the most widely studied eukaryote model organisms. This was controversial as some o the inormation in the databases was derived rom published studies and personal communications. The academic j ournal Science twice created controversy due to the competing imperatives o public and private science. In 2 001 , the j ournal published the company C eleras version o the sequence o

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the human genome sequence while allowing the company to house the sequence on their own database. In 2 002 , Science published a version o the rice genome while allowing the company S yngenta to keep the data on their own private database. These two papers broke the industry standard o the previous 2 0 years that had seen data being published on the public database GenB ank. It also did not comply with a second tradition o publication that was much more longstanding. Traditionally data supporting published reports has always been assumed to be published and thereore reely available to the scientifc community so that, at a minimum, verifcation was possible.

Bioinformatics BLAST searches can identiy similar sequences in diferent organisms. O nce a researcher frst identifes a sequence o interest by sequencing a protein, identiying an open reading rame or fnding high levels o a certain type o mRNA within a cell, their next step would be to conduct a B LAS T search. The acronym reers to B asic Local Alignment S earch Tool. The tool fnds regions o similarity between sequences. The computer program compares protein or nucleotide sequences housed in databases and carries out statistical calculations to determine matches with other sequences. There are three main nucleotide databases: GenB ank, E MB L and D D JB . Two o the most important protein sequence databases are PIR International and S wissProt.

BLASTn and BLASTp searches BLASTn allows nucleotide sequence alignment while BLASTp allows protein alignment. A researcher can identiy open reading rames in nucle otide se quences. O nce an open re ading rame is ide ntiied, a B LAS Tn search can be conducted which involves searching through nucleotide databases to determine i a similar o p e n reading rame exists in another spe cies. A B LAS Tp search uses a protein sequence to search a protein database. A B LAS Tx searches a protein database based on the translated sequence o an open reading rame. Alternatively, i a researcher has ound a protein and wants to determine the location o a gene, they can conduct a tB LAS Tn search using a computer

 Figure 1

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B

B I O T E C H N O LO G Y AN D B I O I N FO R M AT I CS search o multiple genomes using the translated sequence to search or potential genes that could have been transcribed to produce the protein. Figure 1 shows a B LAS Tn search that is about to be conducted. A sequence rom human mitochondrial D NA has been entered to search or similar sequences in mouse D NA.

Matching new sequences with those found in databases Databases can be searched to compare newly identifed sequences with sequences o known unction in other organisms. I a researcher has a sequence o unknown unction, they can search a database to determine i a similar sequence has been identifed in another organism. I the sequence were a protein sequence, they could conduct a B LAS Tp search. The outcome would allow the researcher to determine i a protein o similar sequence exists in another organism and what its unction might be. I the researched sequence were a nucleotide sequence, they might conduct a B LAS Tn search to determine i a similar sequence o known unction exists in another organism or a B LAS Tx search to see i a gene product o a similar sequence has been identifed in another organism.

Knockout mice Use o knockout technology in mice to determine gene unction. O ne method o determining gene unction is to genetically modiy mice by knocking out a gene. This involves replacing the unctional sequence with a non- unctional sequence within stem cells and then using the stem cells with an embryo. The resulting mouse is a chimera. The chimeras are mated with normal mice. Heterozygotes are interbred until a purebred knockout mouse is generated.

mutation. Figure 2 shows a wild type mouse on the right and an obese ( ob/ob) knockout mouse on the let. This is part o the evidence that leptin plays a role in regulating at deposition and energy metabolism.

The loss o the activity o the gene will oten lead to a detectable change in the phenotype o the mouse. This allows researchers to determine the likely unction o the gene. The gene or the production o the hormone leptin was knocked out by introducing a point

594

 Figure 2

B . 5 B i o i n F o r M at i c s ( a h l )

Model organisms Gene unction can be studied using model organisms with similar sequences. A model organism is a species that has been extensively studied b ased on the assumption that discoveries made in the mode l o rganism will have relevance to other organisms. S ome of the most e xtensive ly studied model organisms are Caenorhabditis elegans ( a soil roundworm) , Mus m usculus ( the common house mouse) , Drosophila m elanogaster ( the fruit fly) , Arabidopsis thaliana ( a plant with the common name Thale cress) , E. coli and Saccharom yces cerevisiae ( yeast) . The genome s of these organisms have been se quenced as has the genome of humans. Acro ss the diversity of life, there are some conserved metabolic pathways and some conserved ge netic sequence s. Model organisms can be used as living, or in vivo, models o f disease s relate d to these conserved pathways or dise ases related to mutations in seque nces. S uch studies might not be fe asible or might b e une thical in humans.

Computer-based sequence alignment Sequence alignment sotware allows comparison o sequences rom diferent organisms. S e que nces that are similar betwe en organisms suggest evolutionary re lationships. The greater the similarity, the closer the re lationship. Visual co mpariso n is possible when comparing two relatively short se quence s, but comparing longe r sequences or multip le sequence comp arison relies on the use of computer algorithms. There are a number o f software programmes used to carry out se quence alignme nt including C lustalW and MUS C LE . Incre asingly alignme nts can be carried out using we b- b ased interface s. For e xample the B LAS T search web page of the National C e ntre of B iotechnology Information ( NC B I) carries out the alignment of two se quence s and the C lustalO me ga web page will carry out multiple se quence alignme nt. Figure 3 shows a D NA sequence alignment of nine different o rganisms gene rated using the programme C lustalX.

 Figure 3

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B

B I O T E C H N O LO G Y AN D B I O I N FO R M AT I CS S equence alignment tools oten start with the deault o searching or global relationships over the e ntire length o the sequence. However, in terms o unctions, two prote ins might share a ew common are as that are close ly linked to a common unction with other regions having little or no homologous areas. For this re ason, alignment tools oer a choice b e twee n local or global alignme nt.

Using BLAST to align two proteins Use of software to align two proteins. There are a number o applications or aligning two protein sequences. The ollowing instructions are or using the B LAS T sequence alignment tool at the NC B I website ( http: //www. ncbi. nlm. nih. gov/protein/) . In this example, we will conduct a sequence alignment using the cytochrome oxidase ( cox1 ) protein or two species o primates called tarsiers. Horsfeld' s tarsier, variously classifed as Cephalopachus bancanus and Tarsius bancanus, is a threatened species that lives in B orneo and S umatra. The cox1 sequence or this tarsier will be compared to the sequence o the same gene or the Philippine tarsier ( Tarsius syrichta) . There is some uncertainty over the classifcation o Horsfeld' s tarsier and it is this type o sequence comparison which is oten used to resolve this kind o controversy.

 Figure 4 Horsfelds tarsier

596

 Figure 5 Philippine tarsier

B . 5 B i o i n F o r M at i c s ( a h l ) Choose the protein database rom the NCBI site and enter cox1 tarsius

From the next screen, choose FASTA and the sequence or the protein will be shown. Alternatively, copy the two accession numbers: NP_148740.1 and YP_002929466.1 Go to the BLAST home page, (http://blast.ncbi.nlm. nih.gov/Blast.cgi) , choose protein, blast.

On the Enter Query Sequence page, check the box or sequence alignment, paste in the accession numbers and click on the BLAST button.

Scroll down to review the diferences between the sequence or this protein in the two species.

 Figure 6 Using BLAST to align

two proteins

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B I O T E C H N O LO G Y AN D B I O I N FO R M AT I CS

Multiple sequence alignment Multiple sequence alignment is used in the study of phylogenetics. Phylogeny is the evolutionary history o a species or a group o species. A phylogenetic tree is a diagram that describes phylogeny. When multiple sequences are compared, a consensus sequence is oten identifed based on the amino acid or nucleotide that appears at a certain position in the aligned sequences. As an example, i you aligned six sequences and the nucleotides at position 1 0 are G, A, G, G, C and G, then the consensus sequence will have a G at position 1 0. S imilarities in sequences can be caused by actual evolutionary relationships in which case the sequence similarities are said to homologous. Alternatively sequences which are the same by chance are reerred to as analogous. Most homologous sequences have many positions where mutations have occurred several times. However, not all mutations have the same eect. A mutation in a coding region which results in a change in amino acid sequence is less likely to persist in a population. The probability o a match by chance is higher or D NA sequences than it is or protein sequences. Nonetheless, computer based algorithms have been developed that can use sequence alignments to suggest evolutionary relationships.

Constructing phylograms and cladograms using computer applications Use of software to construct simple cladograms and phylograms of related organisms using DNA sequences. A phylogenetic tree that is created using the cladistics methods discussed in sub-topic 5 .4 is a cladogram. This type o tree only shows a branching pattern and the length o its branch spans do not represent time or the relative amount o change that occurs along a branch. A phylogram is a phylogenetic tree that has branch lengths that are proportional to the amount o character change ( see fgure 8) . The ollowing activity requires the use o two types o sotware: C lustalX and PhyloWin. This activity is based on an activity developed by the American Museum o Natural History. In this activity, we will conduct multiple sequence alignment or the gene or cytochrome oxidase or a number o primate species.

598

1

Visit the NC B I website and choose gene.

2

S earch cox1 primate.

3

C hoose the species that you want to be included in the tree.

4

Under the Genomic regions, transcripts and products, choose FAS TA.

Highlight all o the D NA sequence including the title ( or example >gi|1 961 2 3 5 78: 5 667-7670 Homo sapiens neanderthalensis.) 5

O pen either Notepad rom your PC or TextEdit on a Mac.

6

Paste your sequence into the text editing document.

7

Repeat with several other sequences rom dierent organisms.

8

Edit the titles but remember to include the > symbol and to separate words in the title with an underscore. For example: >Homo_sapiens_ neanderthalensis.

B . 5 B i o i n F o r M at i c s ( a h l )

 Figure 7

9

A screen capture of an image generated using PhyloWin

When your document is complete, there is an extra step or Mac users. Under the Format menu choose make plain text.

1 0 S ave your fle as sequences.asta 1 1 O pen the C lustalX sotware. 1 2 Under the File menu, chose load sequences.

cytosine to a thymine back to a cytosine back to a thymine is a possible series o events, but maximum parsimony presumes that the change was simply cytosine to thymine. This means that you are choosing the tree that involves the least evolutionary change. Figure 8 is one possible phylogenetic tree showing the evolutionary relationship o the nine primates.

1 3 B rowse your fles and open the sequences. asta fle. 1 4 O nce the sequences are loaded, choose do complete alignment under the Alignment menu. Make a note o where the output fle sequences.asta.aln is saved. 1 5 O pen PhyloWin. 1 6 B rowse or the sequences.asta.aln sequence. 17 In the tree building window, choose max. parsimony. There are a number o possible ways the sequences could have ended up the way they have. For example, a change between a

 Figure 8

A phylogram

Expressed sequence tags EST is an expressed sequence tag which can be used to identify potential genes. I a gene is being expressed, then mRNA transcribed rom that gene will be ound within the cell. The mRNA can then be used to search or the gene that produced it using expressed sequence tags ( E STs) . Scientists use the mRNA along with the enzyme reverse transcriptase to produce cDNA. This cDNA will not have any introns in it. Scientists use this DNA to synthesize ESTs. These are short sequences o DNA about 200 to 500 nucleotides long that are generated rom both the 5 end and the 3 end. The 5 end tends to have a sequence that is conserved across species and within gene amilies. The 3 end is more likely to be unique to the gene.

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Using ESTs to locate genes Discovery o genes by EST data mining. B ecause o their useulness and the ease with which they are generated, a very large number o ESTs have been generated. The sequences have been deposited within their own database called dbEST. This database contains ESTs rom over 300 organisms. Scientists can conduct a B LAST search once they have an EST to determine i it matches a D NA sequence rom a known gene with an identifed unction.

The location o the gene within the genome then can be located through physical mapping techniques or by searching through database libraries o known ES Ts.

Exploring chromosome 21 Explore the chromosome 21 in databases (or example in Ensembl) . The Ensembl proj ect collates genome inormation or 75 organisms. It allows or detailed exploration o the coding and non- coding sequences o each o the chromosomes rom these species. C hromosome 2 1 is the shortest human chromosome and perhaps best known or D owns syndrome, or trisomy 2 1 .

activity To explore the inormation available about chromosome 21, visit the website o the web-based database Ensembl (www.ensembl.org) . The frst column shows the position o the centromere as being acrocentric which means to one side o the middle. 1 Click on a red bar to go to a detailed view o that coding region. 2 Click on three protein coding regions to determine the gene that they code or. The search can be refned by looking at protein coding regions to determine what genes have a locus on the chromosome. Visually, it can be seen that there the q arm (the longer arm) has ar more coding sequences per unit length that have been discovered.

600

Question s

Questions

hours after untreated sewage added 5 10 15 20 25 30

0 10 5

Day 1

2

[1 ]

Wastewater rom actories producing polyester fbres contains high concentrations o the chemical terephthalate. Removal o this compound can be achieved by certain bacteria. The graph below shows the relationship between breakdown o terephthalate and conversion into methane by these bacteria in an experimental reactor. 100

4 terephthalate concentration methane production

80

3 60 2 40 1

methane/ml

A two- day experiment was carried out with untreated sewage added to seawater. B oth days were sunny with no clouds. The fgure below shows the inactivation o the microbes in seawater as a unction o the cumulative amount o sunlight and time. The survival curves o the two microbes are plotted against sunlight exposure ( lower x axis) during daylight periods and against time during the overnight period ( upper x axis) . The y axis gives counts o bacteria and viruses per 1 00 ml.

c) For an accidental sewage spill, suggest, giving a reason, which o the two microbes may be most useul as a ecal indicator two days ater the spill.

concentration/mg dm -3

Release o sewage in marine waters is a common practice but it can cause water contamination with pathogens. A series o experiments were conducted to compare inactivation rates o two dierent groups o microbes with dierent sunlight exposures. O ne group were ecal coliorm bacteria and the other were coliphage viruses. E xperiments were conducted outdoors using 3 00-litre mixtures o sewage- seawater in open- top tanks.

counts of bacteria virises/100 ml -1

1

20

Day 2

10 4

0

0 4

10 3

8 12 time/days

16

20

Source: Jer-Horng Wu, Wen-Tso Liu, I-Cheng Tseng, and Sheng-Shung Cheng, Characterization of microbial consortia in a terephthalatedegrading anaerobic granular sludge system, Microbiology, Volume 147 (2001) , pp. 373382,  Society for General Microbiology. Reprinted with permission.

10 2 10 dark period 230

3

 Figure 9

a)

O utline the changes in soil properties that are seen. [1 2 ]

75 50 25

525 350 175

0

0 alder spruce

1.2 0.6 0

pioneer dryas

alder spruce

pioneer dryas

alder spruce

pioneer dryas

alder spruce

8

1.8

6

1.2 pH

bulk density (g/cm 3 )

[2 ]

1.8

700 nitrogen (mg/g)

100 moisture (mg/g)

organic content (mg/g)

b) D educe the stage where the greatest changes in soil properties are observed.

pioneer dryas

200

b) Outline the changes in the relative numbers o species types (species evenness) [2 ]

E4

0 40

20

10

E2 0

30

trees tall shrubs low shrubs and herbs mosses, liverworts and lichens

 Figure 10

E8

E3

[2 ]

0

E6

0.4

[2 ]

Figure 1 0 shows the number o species ound in Glacier B ay, Alaska as a unction o time since the glacier covered the area.

Figure 9 shows the mean stem diameter and range o diameter o plants as a unction o time since the tongue o the glacier covered the area at eight sites ( E1 -E 8) .

E5

O utline the changes in mean stem diameter with time.

b) E xplain the change in mean stem diameter.

The rst species to colonize the bare rock are bacteria, lichens and moss. Mountain avens ( Dryas drummondii) is a fowering shrub that dominates ater the moss stage. D eciduous alder trees ( Alnus sinuata) invade next ollowed by the most stable ecosystem which is a spruce and hemlock orest.

0.6

a)

0.6

4 2

0

0 pioneer dryas

alder spruce

 Figure 11

621

C

E C O LO G Y AN D C O N S E R VAT I O N

Respiration rates and biomass accumulation

gross production total biomass

total com mun ity resp irati on

0

100

time (years)

 Figure 12

The percentage of ingested energy converted to biomass is dependent on the respiration rate. Production in plants happens when organic matter is synthesized by photosynthesis. In animals it occurs when ood is absorbed ater digestion. Energy units are usually used or measuring production e.g. kiloj oules. The amounts o energy are given per unit area, usually per m 2 and per year. Gross and net production values can be calculated using this equation: net production = gross production - respiration Gross production is the total amount o organic matter produced per unit area per unit time by a trophic level in an ecosystem. Net production is the amount o gross production remaining ater subtraction o the amount used or respiration by the trophic level. In the early stages o primary production, the high availability o sunlight means that gross production is high and there is little total biomass in the community. As a result, the total amount o respiration to support the small biomass is low. As succession proceeds, the standing biomass increases and the total amount o respiration increases. Further, the amount o gross production begins to decline once all available spaces or stems become lled. An equilibrium is reached where the total community production to total community respiration (P/R) ratio equals 1 . When this occurs, the ecosystem has reached a relatively stable stage.

Data-based questions: Calculating productivity values The energy fow diagram in gure 1 3 is or a temperate ecosystem. It has been divided into two parts. O ne part shows autotrophic use o energy and the other shows heterotrophic use o energy. All values are kJ m 2 yr 1 . 1

C alculate the net production o the autotrophs.

[1 ]

2

3

C ompare the percentage o heat lost through respiration by the autotrophs with that lost by the heterotrophs.

[1 ]

Most o the heterotrophs are animals. S uggest one reason or the dierence in heat losses between the autotrophs and animal heterotrophs.

[1 ]

autotrophs

heterotrophs heat 23,930

heat 14,140 heterotrophic respiration

autotrophic respiration photosynthesis

gross production 43,510

net production X

storage 4,900

 Figure 13

622

An energy fow diagram or a temperate ecosystem

feeding 14,690

storage 540

C . 2 C O m m U n i T i e S a n D e C O S yS T e m S

Secondary succession Disturbance infuences the structure and rate o change within ecosystems. S econdary succession takes place in areas where there is already, or recently has been, an ecosystem. The succession is initiated by a change in conditions. C onstruction sites or roads might become disused and eventually plants grow up in the remains. O ld- feld succession occurs when an arable feld or meadow ( feld o grassland) is abandoned. The lack o tillage or grazing initiates the succession. Figure 1 4 shows the sequence o communities ollowing the abandonment o an arable feld, with approximate timings. Examining the time scale in fgure 1 4 indicates that the pace o change slows as succession proceeds. C lose to the time o the disturbance, rates o system respiration and productivity increase rapidly and there is an accumulation o biomass. Species diversity increases close to the time o the disturbance. At the climax stage shown in the diagram, changes are still occurring, but they are slower and the ecosystem is viewed as being more stable and resistant to change at the climax stage.

open closed herb scrub pioneer community community (shrubs, (annuals) (perennials) small trees) 12

young broad- climax, leaved old woodland woodland

35 1630 3150