Biotechnology 9788184883596

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BIOTECHNOLOGY MOHAN P. ARORA M.Sc., M.Phil., Ph.D., F.E.S.I., F.A.Z., F.A.S.E.A.; A.l.e.c.E.

Reader, Department of Zoology, M.M.H. Post-graduate College, Ghaziabad.

Edited by

CHANDER KANTA

Hal Gflimalaya GJlublishingGJIouse MUMBAI • DELHI • NAGPUR • BANGALORE • HYDERABAD

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ISBN: 978-81-84883-59-6 REVISED EDITION : 2007

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CONTENTS 1.

INTRODUCTION

1-10

Emergence of Molecular Biotechnology, What is Gene Cloning?, Gene Cloning Requires Specialized Tools and Techniques, Vehicles, Techniques for Handling DNA, The Diversity of Cloning Vectors, Scope and Importance of Biotechnology, Tissue Culture Techniques in Biotechnology, Gene Technology as a Tool for Biotechnology, Hybridoma and Monoclonal Antibodies in Biotechnology, Biotechnology in Medicine, Biotechnology and Protein (or Enzyme) Engineering, Biotechnology and Metabolic Engineering, Biotechnology in Agriculture, Biotechnology and Industrial Microbiology, Biotechnology and Environment, Biotechnology and Intellectual Property Rights, Commercialization of Molecular Biotechnology, Concerns and Consequences.

2.

CELL ORGANISATION

11-27

Viruses, Prokaryotic Cells, Non-photosynthetic Eubacteria, Photosynthetic Bacteria, Eukaryotic Cells, Shape, Numbers, Size, A Typical Cell, Plasma Membrane, Cell Walls, Golgi Bodies, Lysosomes, Mitochondria, Endoplasmic Reticulum, Ribosomes, Peroxisomes, Crystals and Oil Droplets, Plastids, Origin, Types of Plastids, Vacuoles, Microtubules, Centrioles, Cilia and Aagella, The Nucleus, Cytoplasm.

3.

CHROMOSOMES

28-52

Viral Chromosomes, Prokaryotic Chromosomes, Eukaryotic Chromosomes, Morphology, Kinds of Chromosomes, Structure, Pellicle, Chromatids and Chromonemata, Heterochromatin and Euchromatin, Constitutive heterochromatin, Facultative Heterochromatin, Chromosome Banding, Ultra Structure of Chromosomes, Isochromosomes, Ring Chromosomes, Giant Chromosomes, Human Karyotype, Chromosomes in Fishes, Artificial Chromosomes.

4.

NUCLEIC ACID

53-73

Location of Nucleic Acid, Chemical Background of Nucleic Acids, DeOXyribonucleic Acid (DNA), DNA Contents, Structure of DNA, Molecular Weight of DNA, Molecular Structure of DNA, Nitrogenous Bases, Chargaffs Rules, Polarity of DNA, Unusual Bases in DNA, Sugar, Phosphoric Acid, Nucleosides, Nucleotides, The Primary Structure of DNA, The Secondary Structure of DNA, The Tertiary Structure of DNA, Double Stranded Linear DNA, Double Stranded Cyclic DNA, The Structure of the DNA in Eukaryotic Chromosome, Forms of DNA, Repetitive DNA or Satellite DNA, Structural Variation in DNA, Non-Chromosomal DNA, Catalytic Function, Watson & Crick's Model for Replication of DNA (Autocatalytic nature), Replication of DNA in Bacteria or Prokaryote Cells, Enzymes taking part in Replication, Biological Significance of DNA, The Meselson and Stahl Experiment, Cairn's Autoradiography Experiment, Taylor's Experiment on Vicia faba Root Tips, Identification of the Genetic Material, DNA as the Genetic Material.

5.

BIOLOGICAL REPLICATION

74-82

Mechanism of DNA Replication, Activation of Nucleotides, Recognition of the Initiation Point, Unwinding of DNA, Single Strandded DNA-binding Proteins, Template DNA, RNA Priming, Formation of DNA on RNA Primer, Excision of RNA Primers, Editing (Proof-reading) and DNA Repairs, Rate of Replication, Discontinuous Replication, Okazaki Fragments, Replicating the Ends of Chromosomes, DNA Ligase or Polynucleotide Ligase, DNA Repair, Three Types of DNA Repair, Dealing with DNA Damage, DNA Repair in Human Cells.

6.

ENZVMES OF DNA SYNTHESIS

83-93

DNA Polymerases, Mechanisms of Action, DNA Polymerase II, DNA Polymerase III, Reverse Transcriptase or RNA Dependent DNA Polymerase, DNA Ligases, Assay of DNA Ligase, Role of DNA Ligase, Topoisomerases.

7.

RIBONUCLEIC ACID (RNA)

94-108

Structure of RNA, Primary Structure of RNA, Secondary and Tertiary Structures of RNA, Types of Ribonucleic Acids, Transfer Ribonucleic Acid (t-RNA), Messenger Ribonucleic Add (m-RNA), Ribosomal Ribonucleic acid (r-RNA), Viral RNA (v-RNA), Mitochondrial RNAs, Structural variation of RNA, Replication of Ribonucleic Acid, Biosynthesis of Viral RNA, Biosynthesis of m-RNA, The notion of a Messenger, Biosynthesis of Transfer RNA, t-RNA Synthesis, Virus Nucleic Acids, Viroids, Prions.

8.

RNA BIOSYNTHESIS

109-120

Synthesis of RNA Chains Occurs in a Fixed Direction, DNA-Dependent RNA Polymerase, Bacterial DNA-Dependent RNA Polymerase, Eukaryotic DNA Dependent RNA Polymerases, Prokaryotic RNA Synthesis, Elongation of RNA Transcripts, Termination of Transcription in Prokaryotes, Eukaryotic RNA Synthesis, Initiation by RNA Polymerase II, Initiation by RNA Polymerase III, Initiation by RNA Polymerase I, Eukaryotic Factors, Transcription of Mitochondrial and Chloroplast Genes, RNA Polymerases and RNA Synthesis in DNA Viruses, RNA Bacteriophage, Eukaryotic RNA Viruses, Pi Carnatviruses (Class IV), Rhabdovirus (Class V), Myxovirus (Class V), Reoviruses (Class III), Reteroviruses (Class VI).

9.

PROTEIN SYNTHESIS

121-141

Row of Information, Central Dogma Reverse, Restatement of Central Dogma, Open Reading Frames (ORFs), Transcription, Intron and Exons, Post Transcriptional Control in Eukaryotes, Processing of the Primary Transcript, Transport of the Processed Transcript Out of the Nucleus, Selecting Which mRNAs are Translated, Selectively Degrading mRNA Transcripts, Post Transcriptional Control in Prokaryotes, Translation, Amino Acids have no Specific Affinity for RNA, Amino Acids are Aligned on RNA Templates by Means of Adaptors, Specific Enzymes Recognize Specific Amino Acids, The Adaptor Molecules are themselves RNA Molecules, Charging tRNA, Activation of Amino Acids, Isoacceptors, Peptide Bond Formation Occurs on Ribosomes, tRNA Binding Sites, Heterogeneity of Messenger RNA, Attachment of tRNA to mRNA, Polyribosomes, Formation of Polypeptide Chain, Initiation of Protein Synthesis, The Direction of mRNA Reading is 5' ~3' , Elongation of Polypeptide, Chain Elongation Requires GTP, Movement of mRNA Across the Ribosomal Surface, Termination of Polypeptide Chain, Gene Mutation, The Role of ER, Protein Folding and Processing, Chaperones and Protein Folding, Protein Cleavage, Glycosylation.

10.

GENETIC CODE

142-150

Properties of Genetic Code, The Code is Triplet, The Code is Degenerate, The Code is Non-overlapping, The Code is Comma less, The Code is Non-ambiguous, The Code is Universal, Colinearity, The Code has Polarity, Initiator Codons, Terminator Codons, Assignment of Codons, Assignment of Codons with Unknown Sequence, Co-polymers, Assignment of Codons with Known Base Sequence, Co-

polymers of Replicative Sequence, The Wobble Hypothesis, Mutations and the Triplet Code, Reverse Mutations and Suppressor Mutations, Intragenic Suppression, Intergenic Suppression.

11.

NATURE OF THE GENETIC MATERIAL

151-160

Requirements for the Genetic Material, Identifications of DNA as the Genetic Material, The Transformation Experiments, The Blendor Experiment, Properties of Genetic Material, How DNA Stores Information, Transmission of Genetic Information by DNA, Transmission of Information from Parent to Progeny, Chemical stability of DNA and of its Information Content, How DNA Generates Diversity, Mutation, RNA as Genetic Material in Small Viruses.

12.

CONCEPT OF GENE

161-171

About Gene and its Definitions, Classic Concept of Gene, Location of Gene, Genes and Genome, Gene Size, Shape of Gene, Number of Gene, Gene Families and Pseudogenes, Life of Gene or Gene Stability, Role of Gene, Chemical Composition of Gene, Fine Structure of Gene, Classification of Gene, Position Effect of the Gene, One Gene-One Enzyme Hypothesis, One Gene and Two Genes Theories, Overlapping and Included Genes, Broken or Split Gene, Jumping Genes, Longevity Genes, Cryptic Genes, Ultra Modern Concept of Gene, Cheating Genes, Gene Expression and Regulation, Other Types of Genes, Constitutive Genes (House Keeping Genes), Non-constitutive Genes (Luxary Genes), Inducible Genes, Repressible Genes, Repeated Genes, Pseudogenes, Processed Genes, Transposons, Tandem Clusters, Multigene Families, Dispersed Pseudogenes, Single-Copy Genes.

13.

GENE EXPRESSION

172-188

Regulation of Protein Synthesis, Modulation of Gene Activity, Operon Hypothesis, The Structural Gene, The Operator Gene, The Promotor Gene, The Regulator Gene, Elucidation of the Regulation of the Lactose Operon by Studies of Mutants, Regulation of the Tryptophan Operon, Arrangement of Sequences in the Tryptophan Operon, Repressor Control of the trp Operon, Attenuation of Transcription, Regulation in Eukaryotes, Modulation in Eukaryotes, Response to Steroid Hormones, Other Transcription Factors, Enhancers, Feedback Mechanism, Negative Control of Transcrption, Positive Control of the Operon (Catabolite Repression), Footprinting, Isolating Transcription Factors, The Arabinose Operon of E.coli.

14.

GENE ACTION AND RELATED DISEASES

189-200

Phenotype and Environment, Levels of Gene Action, Gene and Development, Gene Action and Related Diseases, Gene Amplification, Gene and Metabolism, Gene Effects, A Gene may Produce Manifold Effects, A Gene may not Always Produce an Effect, Mutation and the Structure of Individual Genes, Regulation of Gene Action, Gene Regulation in Developing Organisms, Genes in Action and Related Diseases, Detection of Genetic Diseases, Treatment of Genetic Diseases, Variations in Gene Effect, Environmental Influence on Genes.

15.

GENETIC ENGINEERING

201-218

Genetically Engineered Bacteria, Beneficial Bacteria, Harmful Bacteria, Applying Genetically Engineered Bacteria, Genetic Engineering of Plants, Protoplasts, Desirable Plant Traits, Genetic Engineering of Animals, Reproductive Technologies, Transgenic Animals, Introducing Genes into Animals, Chromosomal Integration of Foreign DNA, Gene Expression in Transgenic Animals, Applications of Genetic Technology, Cloning of Whole Organisms, Release of Genetically Engineered Organisms, Applications of Genetic Engineering, Commercial Possibilities, Uses in Research, Production of Useful Proteins, Genetic Engineering with Animal Viruses, Diagnosis of Hereditary Diseases, Genetic Engineering in Medicine, Somatostatin, Insulin, Human Growth Hormone, Gene Expressed in Mice, Interferons, Blood Factor VIII, Hepatitis B Vaccine, Genetic Engineering in Agriculture, Plants with a Pesticide Gene, Frost-Reducing Bacteria.

16.

TRANSGENIC ORGANISMS

219-250

Delivering DNA, Transgenic Pioneers-Nancy, Ethel, and Herman, Gene Targeting, Knocking Out Gene Function, Knock out Mice as Models, Transgenic Technology in Plants, Gene Transfer in Dicots and Monocots, Applications of Transgenic Plants, Altering Plants at the Cellular Level, Protoplast Fusion, Callus Culture, Transgenic Animal Modeling, Applications of Transgenic Animals, Production of Transgenic Laboratory Animals, Choice of Animals, DNA Microinjection, Retroviral Introduction of Transgenes, Embryonic Stem Cell Technology, Other Laboratory Animal Models, Production of Transgenic Domestic Animals, Traits Affecting Domestic Animal Productivity, Domestic Animals as Bioreactors, Examples From Domestic And Miniature Pigs, Analysis of Transgene Integration and Expression, Transgene Integration, Transgene-Encoded mRNA Expression, Protein Expression, Conclusions and Future Directions, Gene Transfer Today, Transgenic Animal Patents, Legal Considerations, Legal Status, Patentability ReqUirements, Scientific Developments, Economic Factors, Economic Incentives, Impact on Prices and Industry Structures, Royalty Payments, Implication for Farm Structure, Genetic Diversity, Ethical Issues, Public Attitudes Toward Animal Biotechnology and Patents, Animal Welfare, Animal Rights, Devalue Humanness, Transgenic Fish, Selection of Model Fish Species, Preparation of Gene Constructs, Methodology of Gene Transfer, Microinjection, Electroporation, Fate of the Transgenes, Identification of Transgenic Fish, Pattern of Transgene Integration, Examples of Transgenic Fish, Other Transgenic Fish, Applications and Perspectives, The Making of a Transgenic Mouse, Transgenic Cattle, Transgenic Birds, Transgenic Pigs.

17. DNA CLONING AND FINGERPRINTS

251-269

DNA Cloning and Sequencing, Obtaining DNA to be Cloned, Genomic DNA Isolation and Restriction Enzymes, Complimentary DNA (cDNA), Cloning Vectors, Plasmids, Phages, The Role of Bacteria, Creating DNA to Clone, Direct Formation of Clonable DNA, Probing For a Specific Gene, Southern Blotting, Probing for a Cloned Gene, Dot Blotting, Western Blotting, Chromosome Walking, Heteroduplex Analysis, Eukaryotic Vectors, Yeast Vectors, Animal Vectors, Plant Vectors, Expression of Foreign DNA in Eukaryotic Cells, Human DNA Ubraries, DNA Probes, DNA Fingerprint Analysis, Principles, Specimen Processing, Specimen Analysis, Data ProceSSing, Quality Assurance, Applications, Microorganisms, Plants, Perspectives, DNA Fingerprinting Versus Other Identification Techniques, Future Directions.

18.

GENETIC SCREENING

270-280

Methods of Testing in Prenatal Screening, Non-invasive Testing Methods, Invasive Testi~g Methods, Sharing Diagnostic Information, Anencephaly and Spina Bifida, Maternal Serum Alpha-Fetoprotein, Newborn Screening, Carrier Screening, Detection of Autosomal Recessive Carriers, Restriction Fragment Length Polymorphisms and DNA Probes, Molecular Analysis of Huntington's Disease, Cystic Fibrosis and Sickle-Cell Anemia, Restriction Fragment Length Analysis of the Human Growth Hormone Gene, Gene Probes for Haemophilia A, Gene Probes for Duchenne's Muscular Dystrophy.

19.

GENETIC COUNSEUNG

281-291

History of Genetic Counseling, Who Are Genetic Counselors?, Methods of Genetic Counseling, Nondirective Counseling, Directive Counseling, Reasons for Seeking Genetic Counseling, Counseling for Dominant Defective Phenotypes, Counseling for Recessive Defective Phenotypes, Counseling for X-Unked Defective Phenotypes, Counseling for Multifactorial Defective Phenotypes, Counselling for Consanguinity, Counseling for Chromosome-Related Defects, Psychodynamics of Genetic Counseling, Poor Prognosis and Counselee Adjustment, Parental Reaction to a Severely Defective Newborn, Sibling Reaction to a Severely Defective Newborn.

20.

GENE THERAPY

292-309

The Evolution of a Cure, Types of Gene Therapy, Treating the Phenotype-A short-term Solution, Types of Gene Therapy, The Mechanics of Gene Therapy, Gene Vectors, Introducing Vectors, Ex Vivo Gene Therapy, In Vivo Gene Therapy, Sites of Gene Therapy, Endothelium-{3ateways to the Bloodstream, Gene Therapy Against Cancer, Molecular Surgery-Targeting Brain Tumors, Encouraging an Immune Response, Perspective: A Slow Start, But Great Promise, The Future of Gene Therapy.

21.

REPRODUCTIVE TECHNOLOGY

310-319

New Ways to Make Babies, A Grandmother and Mother at the Same Time, Midlife Motherhood, A Five-Year Wait, Infertility, Male Infertility, Female Infertility, Infertility Tests, Assisted Reproductive Technologies, Donated Sperm-Artificial Insemination, A Donated Uterus-Surrogate Motherhood, In vitro Fertilization, Gamete Intrafallopian Transfer, Oocyte Banking and Donation, Embryo Adoption, Preimplantation Genetic Screening.

22.

GENETICS OF IMMUNITY

320-332

Antigens, Antibodies, Lymphocytes, Cells of the Immune System, Primary Immune Responses, T Cells, B Cells, Memory Cells, Monoclonal Antibodies, Secondary Immune Responses, Immlmological Inherited Diseases, Autosomal-Recessive Immunological Diseases, Inherited Immune Deficiencies, Autoimmune Diseases, Acquired Immune Deficiency Syndrome (AIDS), Genetic Structure of the AIDS Virus, AIDS Transmission, Hypersensitivites: The Allergies, Allergens.

23.

PROTEIN ENGINEERING

333-351

Objectives of Protein Engineering, Directed Mutagenesis Procedures, Oligonucleotide-Directed Mutagenesis with M13 DNA, Oligonucleotide-Directed Mutagenesis with Plasmid DNA, PCR-Amplified Oligonucleotide-Directed Mutagenesis, General Problems with the Production of Recombinant Protein in E.coli, Problems Resulting from the Sequence of the Foreign Gene, Problems caused by E.coli, Production of Recombinant Protein by Eukaryotic Cells, Recombinant Protein from Yeast and Filamentous Fungi, Using Animal Cells for Recombinant Protein Production, Protein Engineering, Adding Disulfide Bonds, Changing Asparagine to other Amino acids, Reducing the Number of Free Sulfh~ryl Residues, Increasing Enzymatic Activity, Modifying Metal Co-factor Requirements, Modifying Enzyme Specificity, Increasing Enzyme Stability and Specificity, Application of Protein Engineering to Understand Structure Activity Relationship, Purification of Enzyme Made Easier by Protein Engineering, Protein Engineering Applications to Hormones, Protein Engineering Applications for Biopharmaceuticals.

24.

VACCINE BIOTECHNOLOOGY

352-364

Nature of Vaccines, living Organisms (with attenuated virulence) as Vaccines, Genetically Engineered Viruses as Vaccines, Dead Organisms as Vaccines, Bacterial Toxins and Toxoids as Vaccines, Immunizing Sera as Vaccines, Vaccines of Defined Chemical Nature, Preparations of Active Immunization Products, Bacterial Vaccine, Killed Bacterial Vaccine, living Bacterial Vaccine, Toxoid or Toxin Preparation, Diphtheria Vaccine, Staphylococcus Toxoid, Tetanus Toxoid, Viral Vaccine, Influenza Vaccine, Poliomyelitis Vaccine, Rabies Vaccine, Smallpox Vaccine, Rickettssial Vaccine, Yellow Fever Vaccine, Typhus Vaccine, Subunit Vaccines, Herpes Simplex Virus, Foot-and-Mouth Disease, TuberculosiS, Peptide Vaccines, Attenuated Vaccines, Cholera, Salmonella Species, Leishmania Species.

25.

PlANT BIOTECHNOLOGY

365-395

Principles, Physiology of the Recipient Cells, Characteristics of Transferable Nucleic Acids, Characteristics of the Delivery Systems, DNA Delivery Methods, Agrobacterium-mediated Gene

Transfer, Chemical Methods and Electroporation, Microinjection, Ballistic Methods, Analysis of Transgenic Plant Material, Analysis of Foreign Gene Products, Physical Analysis of Foreign DNA, Clonal Propagation or Micropropagation, Plant Cell Culture Methods, Callus Culture, Cell Culture, Embryo Rescue, Anther and Pollen Culture, Endosperm Culture, The Gene Addition Approach to Plant Genetic Engineering, Plants that Make their Own Insecticides, Other Gene Addition Projects, Gene Subtraction, The Principle behind Antisense Technology, Antisense RNA and the Engineering of Fruit Ripening in Tomato, Other Examples of the Use of Antisense RNA in Plant Genetic Engineering, Insect-Resistant Plants, Virus-Resitant Plants, Herbicide-Resistant Plants, Fungus -and BacteriumResistant Plants, Nitrogen Fixation, Nitrogenase, Genetic Engineering of the Nitrogenase Gene Cluster, Hydrogenase, Genetic Engineering of Hydrogenase Genes, Nodulation, Competition among Nodulating Organisms, Genetic Engineering of Nodulation Genes, Biofertilizers, Green ManUring, Bacterization, Microbial Culture for Biofertilizer Production, Bioinsecticides, Bioherbicides. GLOSSARY

INDEX

396-424 425-442

1 INTRODUCTION Biotechnology is an exciting, revolutionary scientific discipline that is based on the ability of researchers to transfer specific units of genetic information from one organism to another. This conveyance of a genets) relies on the techniques of genetic engineering (recombinant DNA technology). The objective of recombinant DNA technology is often to produce a useful product or a commerical process. In molecular biotechnology, the organisms that are commonly used by molecular biotechnologists, some fundamentals of molecular biology and recombinant DNA procedures are presented. Essential molecular biotechnology laboratory techniques, including chemical synthesis of genes, the polymerase chain reaction (peR) and DNA sequencing are discussed. In addition to isolating (cloning) genes, it is important that these genes function properly in a host organism. To this end, strategies for optimizing the expression of a cloned gene, either prokaryotic or eukaryotic cells are reviewed. Finally, procedures for modifying cloned genes by the introduction of specific nucleotide changes (in vitro mutagenesis) to enhance the properties of the target proteins are examined. EMERGENCE OF MOLECUlAR BIOTECHNOLOGY

In the early 1970s, traditional biotechnology was not a well-known scientific discipline; research in this area was centered in departments of chemical engineering and occasionally in specialized microbiology programs. In a broad sense, biotechnology is concerned with the production of commerical products generated by the metabolic action of microorganisms. More formally, biotechnology may be defined as "the application of scientific and engineering principles to the processing of material by biological agents to proVide goods and services". Thus, from a historical perspective, biotechnology dates back to the time when yeast was first deliberately used to ferment beer and bacteria were first used to make yogurt. The term "biotechnology" was created in 1917 by a Hungarian engineer, Karl Ereky, to describe an integrated process for the large-scale production of pigs by using sugar beets as the source of food. According to Ereky, biotechnology was "all lines of work by which products are produced from raw materials with the aid of living things". This fairly precise definition was more or less ignored. For a number of years, biotechnology was used to describe two very different engineering disciplines. On one hand, it referred to industrial fermentation. On the other, it was used for the study of efficiency in the workplacewhat is now called ergonomics. This ambiguity ended in 1961 when the Swedish microbiologist Carl Goran Heden recommended that the title of a scientific journal dedicated to publishing research in the fields of applied microbiology and industrial fermentation be changed from the "Journal of Microbiological and Biochemical Engineering and Technology" to "Biotechnology and Bioengineering". From that time on, biotecnhology has clearly and irrevocably been associated with the study of "the industrial production of goods and services by processes using biological organisms, systems, and processes." And it was firmly grounded on expertise in microbiology, biochemistry and chemical engineering. 1

2

Biotechnology

An industrial biotechnology process that uses microorganisms for producing a commerical product typically has three key stages: 1. Upstream processing: preparation of a raw material so that it can be used as a food source for the target microorganism. 2. Fermentation and transformation: growth (fermentation) of the target microorganism in a large bioreactor (usually> 100 liters) with the consequent production (biotransformation) of a desired Upstream processing compOlmd, which can be, for example, an antibiotic, an amino acid, or a protein. 3. Downstream processing: purification of the desired compound from either the cell medium or the cell mass. Fermentation and Biotechnology research is dedicated to maximizing the overall biotransformation efficiency of each of the..;e steps and to finding microorganisms that make products that are useful as foods, food supplements and drugs. During the 1960s and 1970s, this research focussed on upstream processing, Downstream bioreactor design and downstream processing. These studies led to processing enhanced bioinstrumentation for monitoring and controlling the fermentation process and to efficient large-scale growth facilities that increased the yields of various products. The biotransformation components of the overall process was the most difficult phase to optimize. Commodity production by naturally occurring microbial strains on a large scale was often considerably less than optimum. Consequently, mutations induced by chemical mutagens Fig. 1.1. Principal steps of a bioor ultraviolet radiation were used to change the genetic constitution of ' engineered biotechnology existing strains, in an effort to create variants (mutants) with enhanced process. Parenthetically, product yields. However the level of improvement that could be achieved Karl. Erek(s .scheme . . .:. . . entaIled usrng rnexpenm thiS way was often limited biologically. If a mutated stram, for example, siue sugar beets (raw synthesized too much of a compound, other metabolic functions often material) to feed pigs (biowere impaired, thereby causing the strain's growth during large-scale transfo~mation) for the fermentation to be less than desired. Despite this constraint, the traditional productIon of pork. 'induced mutagenesis and selection' strategies of strain improvement were extremely successful for a number of processes such as the production of antibiotics. The traditional genetic improvement regimens were tedious, time-consuming and costly because of the large numbers of colonies that had to be selected, screened and tested_ Moreover, the best result that could be expected with this approach was the improvement of an existing inherited property of a strain rather than the expansion of the genetic capabilities of a strain. Despite these limitations, by the late 1970s, effective processes for the mass production of a wide range of commerical products had been perfected. However, the nature of biotechnology was changed forever by the. development of recombinant DNA technology. With these techniques, the optimization of the biotransformation phase of a biotechnology process was achieved more directly. Genetic engineering prOvided the means to create rather than merely isolate highly productive strains. Microorganisms and eukaryotic cells could be used as 'biological factories' for the production of insulin, interferon, growth hormone, viral antigens and a variety of other proteins. Recombinant DNA technology coulq also be used to facilitate the biological production of large amounts of useful low-molecular-weight compounds and macromolecules that occur naturally in minuscule quantities_ Plants and animals became natural bioreactors producing new or altered gene products that could never have been created either by mutagenesis and selection or by crossbreeding. Finally, this new technology facilitated the development of radically new medical therapies and diagnostic systems.

Introduction

3

The union of recombinant DNA technology with biotechnology created a vibrant, highly competitive field of study that has been called molecular biotechnology. This new research area, like molecular biology in its early stages in the 1960s, is a brash discipline in which claims and ey.pectations sometimes exceed the researchers' abilities to produce the desired results. As well, the strategies and experimental foundations of .molecular biotechnology undergo rapid changes. It is not unusual for various approaches to be superseded by different strategies within a short time. What is inevitable is that in the future, molecular biotechnology will be the standard method for developing living systems with novel functions and capabilities for the synthesis of important commerical products. Table 1.1. Historical development of molecular biotechnology. Date

Event

1917 1943 ·1944 1953 1961 1961-1966 1970 1972 1973 1975 1976 1976 1978 1980

Karl Ereky coined the term biotechnology. Penicillin produced on an industrial scale. Avery, MacLeod and McCarty demonstrated that DNA is the genetic material. Watson and Crick determined the structure of DNA Journal "Biotechnology and Bioengineering" established. Entire genetic code deciphered. First restriction endonuclease isolated. Khorana and coworkers synthesized an entire tRNA gene. Boyer and Cohen established recombinant DNA technology. Kohler and Milstein described the production of monoclonal antibodies. First guidelines for the conduct of recombinant DNA research issued. Techniques developed to determine the sequence of DNA. Genentech produced human insulin in E.coli. U.S. Supreme Court ruled in the case of Diamond u. Chakrabarty that genetically manipulated microorganisms can be patented. First commercial automated DNA synthesizer sold. 1981 1981 First monoclonal antibody-based diagnostic kit approved for use in the United States. 1982 First animal vaccine produced by recombinant DNA technology approved for use in Europe. 1983 Engineered Ti plasmids used to transform plants. 1988 U.S. patent granted for a genetically engineered mouse susceptible to cancer. 1988 Polymerase chain reaction (PCR) method published. 1990 Approval granted in the United States for a trial of human somatic cell gene therapy. 1990 Human Genome.Project officially initiated. 1994-1995 Detailed genetic and physical maps of human chromosomes published. 1996 First recombinant protein, erythropoietin, exceeds 1 billion dollars (U.S.) in annual sales. 1996 Complete DNA sequence of all the chromosomes of a eukaryotic organism, the yeast Saccharomyces cereuisiae determined. 1997 Nuclear cloning of a mammal, a sheep, with a differentiated cell nucleus.

Most new scientific disciplines do not arise solely on their own. They are often formed by the amalgamation of knowledge from different areas of research. For molecular biotechnology, the biotechnology component was perfected by industrial microbiologists and chemical engineers, whereas the recombinant DNA technology portion owes much to discoveries in molecular biology, bacterial genetics and nucleic acid enzymology. In a broad sense, molecular biotechnology draws on knowledge from a diverse set of scientific disciplines to create a ,wide range of commerical products.

Biotechnology

4 \ Molecular biology

Microbiology

Biochemistry

\

Genetics

Chemical engineering

Celt biology

Molecular biotechnology

Fig. 1.2. Many scientific disciplines contribute to molecular biotechnology, which generates a wide range of commercial products.

What is Gene Cloning? The basic steps in a gene cloning experiment are as follows: 1. A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a oector, to produce a chimera or recombinant DNA molecule. 2. The vector acts as a vehicle that transports the gene into a host cell, which is usually a bacterium, although other types of living cells can be used. 3. Within the host cell, the vector multiplies, producing numerous identical copies not only of itself but also of the gene that it carries. 4. When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place and 5. After a large number of cell divisions, a colony or clone of identical host cells is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule; and the gene carried by the recombinant molecule is now said to be cloned. GENE CLONING REQUIRES SPECIAUZED TOOLS AND TECHNIQUES

Vehicles The central component of a gene cloning experiment is the vehicle, which transports the gene into the host cell and is responsible for its replication. To act as a cloning vehicle, a DNA molecule must be capable of entering a host cell and, once inside, replicating to produce multiple copies of itself. Two naturally occurring types of DNA molecule satisfy these requirements: 1. Plasm ids, which are small circles of DNA found in bacteria and some other organisms. Plasmids can replicate independently of the host cell chromosome and 2. Virus chromosomes, in particular the chromosomes of bacteriophages, which are viruses that specifically infect bacteria. During infection, the bacteriophage DNA molecule is injected into the host cell where it undergoes replication.

5

Introduction

Techniques for Handling DNA Plasmids and bacteriophage DNA molecules display the basic properties required of potential cloning vehicles. But this potential would be wasted without experimental techniq· !es for handling DNA molecules in the laboratory. The fundamental steps in gene cloning require several manipulative skills. First, pure samples of DNA must be available, both of the cloning vehicle and of the gene to be cloned. Table 1.2. Basic skills needed to carry out a simple gene cloning experiment. 1. 2. 3. 4. 5. 6.

Preparation of pure samples of DNA Cutting DNA molecules Analysis of DNA fragment sizes Joining DNA molecules together Introduction of DNA into host cells and Identification of cells that contain recombinant DNA molecules.

Having prepared samples of DNA, construction of a recombinant DNA molecule requires that the vector be cut at a specific point and then repaired in such a way that the gene is inserted into the vehicle. The ability to manipulate DNA in this way is an offshoot of basic research into DNA synthesis and modification within living cells. The discovery of enzymes that Ciln cut or join DNA molecules in the cell has led to the purification of restriction endonucleases and ligases, which are now used to construct recombinant DNA molecule in the test-tube. The properties of these enzymes and the way they are used in gene cloning experiments are paramount in this research. Once a recombinant DNA molecule has been constructed, it must be introduced into the host cell so that replication can take place. Transport into the host cell makes use of natural processes for uptake of plasmid and viral DNA molecules. These processes are utilized in gene cloning.

The Diversity of Cloning Vectors Although gene cloning is relatively new, it has nevertheless developed into a very sophisticated technology. Today a wide variety of different cloning vectors are available. Almost all of these are derived from naturally occurring plasmids or viruses, but most have been modified in various·ways so that each one is suited for a particular type of cloning experiment. SCOPE AND IMPORTANCE OF BIOTECHNOLOGY

Biotechnology as explained above has its newest roots in the science of molecular biology and microbiology. Advances in these two areas have been exploited in a variety of ways-both for production of industrially important biochemicals (including enzymes) and for basic studies in molecular biology. Therefore, a new commerical environment has been created, in which many famous scientists including some Nobel laureates (like Walter Gilbert) opted to work for bio!echnology companies. Scores of these companies are created and closed down every year. Tn USA. alone there are more than 200 such companies including Genen tech , Cetus, Hybritech, Biogen, etc. USA., Japan and Europe are leaders in biotechnology research and development. Varied commerical projects are being undertaken by these companies and a range of these programmes is evident from a list of programme listed in table 1.3.

Table 1.3. Some ofthe biotechnological programmes being undertaken by several biotechnology companies. 1. 2. 3. 4. 5. 6.

Automated bioscreening. Genetical improvement of pharmaceutical micro-organisms. Engineering of a series of organisms for specific industrial uses. Developing immobilized cell and enzyme systems for chef!1ical process industries. Improved production of vitamin B 12 . ManufactUring fructose from inexpensive forms of glucose.

6

Biotechnology

7. Bioprocessing alkenes to valuable oxides and glycols. 8. Production of ethanol by continuous fermentation. 9. Upgrading hydrocarbons microbiologically. 10, Production of xanthan gum in oil fields for enhanced crude oil recovery. 11. Production of human insulin microbiologically. 12. Production of human interferons microbiologically. 13. Developing a vaccine to prevent colibacillosis, a widespread disease of newborn calves and piglets. 14. Production of monoclonal antibodies for organ transplant tissue typing. 15. Production of diagnositic kits for toxoplasmosis identification. 16. Production of plants resistant to herbicides, viruses, insects and other pests. 17. Production of photosynthetically efficient plants. 18. Production of transgenic animals as biocreactors for producing valuable drugs. 19. Production of biopesticides and biofertilizers and 20. Human gene therapy.

Tissue Culture Techniques in Biotechnology An important aspect of all biotechnology processes is the culture of either the microorganisms or plant and animal cells (or protoplasts in case of plants) or tissues and organs in artificial media. These possibilities led to Significant advances and novel possibilities. While microbes in culture are used in recombinant DNA technology and in a variety of industrial processes, plant cells and tissues are used for a variety of genetic manipulations. For example, anther culture is used for haploid breeding; gametic and somatic cell/tissue cultures are used for tapping gametoclonal and somaclonal variation or for production of artificial seeds. Transformation of protoplasts in culture leads to the production of useful transgenic plants. Embryo culture technique has also helped in extending the range of distant hybridization for plant breeding purposes. Similarly animal cells (e.g. egg cells) are used for multiplication of superior livestock using a variety of techniques like cloning of superior embryonic cells, transformation of cultured cells leading to the production of transgenic animals and in vitro fertilization and transfer of embryos to surrogate mothers. Gene Technology as a Tool for Biotechnology Most biotechnology companies make use of gene technology or genetic engineering, which involves recombinant DNA and gene cloning. More recently, extensive use of newly discovered polymerase chain reaction (peR) has also been made for gene technology. This gene technology has become a major thrust area of present day researches and some of the developed countries are encouraging researches in this field as a matter of national priority. Hybridoma and Monoclonal Antibodies in Biotechnology Hybridoma technique and monoclonal antibodies is another very important area in which rapid progress has been made, so that it is being extensively utilized for human health care, and a variety of other purposes. Enzyme conjugated antibodies are being used for detection of viruses both in plants and animals (including humans) using ELISA (enzyme linked immunosorbent assay) tests. Immunotoxins are being produced from gene fusions so that the toxic drugs, meant for killing tumor cells may be carried to the target sites with the help of specific antibodies. Biotechnology in Medicine In the field of medicine, insulin and interferon synthesized by bacteria have already been released for sale. A large number of vaccines for immunization against dreadly diseases, DNA probes and monoclonal antibodies (including ELISA tests) for diagnosis of various diseases human growth hormones and other pharmaceutical drugs for treatment of diseases are being released or are in the process of being released. In 1988, in an experiment to introduce in the human body, the lymphocytes containing a bacterial gene

Introduction

7

has been approved for patients who were in the terminal stages of cancer and had no chance to survive. During 1990-92, patients suffering with some lethal diseases were subjected to gene therapy. These patients are doing well. DNA fingerprinting and auto-antibody fingerprinting techniques are also proving a great boon in forensic medicine for identification of criminals like murderers and rapists through the study of DNA or antibodies from blood and semen stains, urine, tears, saliva, perspiration or hair roots etc.

Biotechnology and Protein (or Enzyme) Engineering Another very important area of biotechnology is protein engineering, that will lead to the production of superior enzymes and storage proteins. In this area, a protein engineer first prepares a computer aided protein model for a specific function and then prepares a synthetic gene that will produce this desired protein in a predictable manner. Thus, in future, proteins will be engineered in the desired manner. Biotechnology has also provided us with a remarkable technique in the form of immobilized enzyme systems, which allowed the production of a variety of substances e.g. production of high-fructose com syrup (as a sweetening agent for the soft drink industry) using an immobilized enzyme, glucose isomerase. The market for these immobilized enzymes now is of the order of billions of dollars per year and supports multibillion dollar industries, because the cost involved in the production of these enzyme systems is only a fraction of the value of the products manufactured.

Biotechnology and Metabolic Engineering One of the major objectives of biotechnology research is the use of living systems for the production of metabolites at the industrial scale. However, cell's metabolic networks that evolved in nature, are not optimized for industrial production of these metabolites. In such cases, performance of metabolic pathways are being manipulated, so that the metabolites are over-produced. The opportunity to introduce heterologous genes and regulatory elements made 'metabolic engineering', a very fascinating area of research. However, there is a variety of limitations in metabolic engineering, that need to be overcome. For instance, when alterations are made (by genetic manipulations) for flux distributions at key branchpoints (called nodes) of a metabolic pathway, this may be opposed by mechanisms evolved in the cell for its optimal growth. This is described as 'network rigidity' and efforts are being made to overcome problems like this.

Biotechnology in Agriculture Biotechnology has also revolutionized research activities in the area of agriculture which includes the following: (i) plant cell, tissue and organ culture; (ii) genetic engineering leading to transformation followed by regeneration of plants to give transgenic plants carrying desirable traits like disease resistance, insect resistance and herbicide resistance; eventually this may also be used for increasing photosynthetic efficiency, nitrogen fixing ability, improved storage proteins, hybrid crops, crops for food processing etc; (iii) somatic hybrids between sexually incompatible species, permitting transfer of desirable traits from wild or .unrelated crop species to our crop plants; (iv) transgenic animals produced in mice, pigs, goats, chicken, cows, etc; it is suggested that some of these will eventually be used as bioreactors to produce drugs through their milk, blood or urine-this area has sometimes been described as molecular farming.

Biotechnology and Industrial Microbiology Industrial microbiology is yet another area, receiving major attention of biotechnologists. A number of pharmaceutical drugs and chemicals are being produced, or will be produced in future utilizing techniques of biotechnology (including genetic engineering) to increase substantially both the quality and the quantity of these drugs and chemicals.

Biotechnology and Environment Biotechnology is also being used for dealing with environmental problems. Fears are also being expressed about the implication of advances in biotechnology in terms of release of harmful organisms developed through recombinant DNA technology. In view of this, rules and laws have been framed from time to time to safeguard against the risks, which the recombinant DNA technology poses to the clean and friendly environment.

Biotechnology

8

Biotechnological methods have been devised for some environmental problems like the following: (i) pollution control, (ii) depletion of natural resources for non-renewable energy, (iii) restoration of degraded lands and (iv) biodiversity conservation. For instance, microbes are being developed to be used as biopesticides, biofertilizers, biosensors etc., and for recovery of metals, cleaning of spilled oils and for a variety of other purposes. They are also used for biomonitoring in industries, where employees are exposed to a variety of risks. Biomass is being produced and used as a renewable source of energy, by capturing solar energy. Tissue culture and genetic engineering mycorrhizae (VAM fungi), root nodulation (both in legumes and non-legumes, using bacteria like rhizobia and actinomycetes fungus like Frankia) are also being used for reclamation of degraded lands.

Biotechnology and Intellectual Property Rights In recent ~/ears, there have also been discussions on the protection of intellectual property rights (IPR) emanating from the use of biotechnology. It is also emphasized that protection of these rights may also in its turn affect the development of biotechnology. The intellectual property rights include patents, trade secrets, trademarks and copyrights, which can be protected through a variety of laws in different countries. However, not all developments in biotechnology can be protected as intellectual property rights. For instance, techniques used in medical science (bypass heart surgery, organ transplant, artificial limbs, use of drugs, antibiotics and vaccines etc.) are not patentable. For plant biotechnology also, a variety of culture methods, biological control of pests and weeds are not patented. There are other examples of the products of biotechnology, which can be patented. These include products like modified antibiotics, hormones and enzymes, synthetic steroids, immobilized enzymes, organ specific drug delivery, heart valves, artificial teeth, plastic bags for blood storage etc. A variety of products in the field of agriculture and animal husbandary are also patentable. There are also international agreements made to enforce protection of intellectual property rights generated in one country and needing protection in other countries. COMMERCIAUZATION OF MOLECUlAR BIOTECHNOLOGY

The ultimate objective of all biotechnology research is the development of Gommerical products. Consequently, molecular biotechnology is driven, to a great extent, by economics. Not only does financial investment currently sustain molecualr biotechnology, but clearly the expectation of financial gain was responsible for the considerable interest and excitement during the initial stages of its development. By nightfall on October 15, 1980, the principal shareholders of Genentech stock were worth millions of dollars. The unprecedented enthusiastic public response to Genentech encouraged others to follow. Between 1980 and 1983, about 200 small biotechnology companies were founded in the United States with the help of tax incentives and funding from both stock market speculation and private investment. Like Herbert Boyer, who was first a research scientist at the University of California at San Francisco and then a vice president of Genentech, university professors started many of the early companies. By 1985, there were over 400 biotechnology companies in the United States, including many with names that contained variants of the word "gene" to emphasize their expertise in gene cloning: Biogen, Amgen, Calgene, Engenics, Genex, and Cangene. Today, there are over 1,500 biotechnology companies in the United States and more than 3,000 worldwide. In addition, all large multinational chemical and pharmaceutical companies including Monsanto, Du Pont, Upjohn, American Cyanamid, Eli Lilly, SmithKline Beecham, Merck, Novartis, and Hoffmann-LaRoche, to name but a few, have made significant research commitments to molecular biotechnology. During the rapid proliferation of the biotechnology business in the 1980s, small companies were absorbed by larger ones, strategic mergers took place and joint ventures were undertaken. For example, in 1991, 60% of Genentech was sold to Hoffmann-LaRoche for $2.1 billion. And, inevitably for various reasons, there were a number of bankruptcies. This state of flux is a characteristic feature of the biotechnology industry. By the mid-1990s, more than a dozen new drugs that were produced by recombinant DNA technology had been certified for use with humans; over 100 are in the process of being tested in human trials and more than 500 other recombinant pharmaceutical products are being developed. Similarly, many new

9

Introduction

molecular biotechnology products for enhancing crop and livestock yields have been created and are being marketed. The annual earnings of the molecular biotechnology industry have increased from about $60 billion per year. However, generally speaking, profits for biotechnology companies have been elusive. Notwithstanding these losses, the continuing enthusiasm of investors for biotechnology indicates, at least in their estimation, that molecular biotechnology will have a pervasive impact on society. New, independent molecular biotechnology companies have alwqys been specialized and tend to stress the use of a particular aspect of recombinant DNA technology which is often reflected in their names. For example, after the formation of companies dedicated to the cloning of commercially important genes, severa~ U.S. molecular biotechnology companies, including Immunex, ImmuLogic, ImmunoGen, Immunomedics, medImmune and Immune Response were formed to produce genetically engineered antibodies for treating infectious diseases, cancer and other disorders in humans. Much of the commercial development of molecular biotechnology has been centred in the United States. In other countries where investment capital is not readily available and where there is less emphasis on entrepreneurial activity, large corporations and governments have taken an active role in creating indigenous molecular biotechnology initiatives. In Japan, biotechnology was declared a "strategic industry" and made a national priority by the government. Instead of a plethora of small independent companies formed with venture capital, large Japanese corporations, which lacked basic researchers with training in recombinant DNA technology, initially formed collaborations with U.S. universities and companies. Now that they have acquired the necessary expertise, major Japanese corporations are more directly involved in molecular biotechnology research and product development. The European biotechnology industry has been developing steadily. In 1995, there were over 600 European biotechnology companies. In economically less developed countries, governments have played a significant role as the initiators of molecular biotechnology. The motivation for this activity lies in the strong belief that molecular biotechnology is the "last great technological revolution of the twentieth century." No country wants to be left out of the gains expected from this technology. Now that molecular biotechnology is nearing the end of its second decade, it has become the engine powering the development of many novel products. Although at first some scientists believed that molecular biotechnology was an esoteric technical development with marginal practical utility, now it is mainstream. Undoubtedly, the next 10 years of commerical molecular biotechnology will be exciting, although specific predictions are risky because new technical innovations rapidly replace existing technologies. CONCERNS AND CONSEQUENCES

Molecular biotechnology ought to contribute unprecedental benefits to humanity. It should• provide opportunities to accurately diagnose and prevent or cure a wide range of infectious and genetic diseases, • significantly increase crop yields by creating plants that are resistant to insect predation, fungal and viral diseases, and environmental stresses such as short-term drought and excessive heat, • develop microorganisms that will produce chemicals, antibiotics polymers, amino acids, enzymes, and various food additives, • develop livestock and other animals that have enhanced genetically determined attri~utes and • facilitate the removal of pollutants and waste materials from the environment. Although it is exciting and important to emphasize the positive aspects of new advances, there are also social concerns and consequences that must be addressed. Because molecular biotechnology is so broadly based, its potential impact on society must be considered. For example, • Will some genetically engineered organisms be harmful either to other organisms or to environment? • Will the development and use of genetically engineered organisms reduce natural genetic diversity? • Should humans be genetically engineered? • Will diagnostic procedures undermine individual privacy?

10

Biotechnology

• Should genetically engineered animals be patentable? • Will financial support for molecular biotechnology constrain the development of other important technologies? • Will the emphasis on commercial success mean that the benefits of molecular biotechnology will be available only to the wealthy? • Will agricultural molecular biotechnology undermine traditional farming practices? • Will medical therapies based on molecular biotechnology supersede equally effective traditional treatments? and • Will the quest for patents inhibit the free exchange of ideas among research scientists? These and many other issues have been considered by government commissions, discussed extensively at conferences, and thoughtfully debated and analyzed by individuals in popular and academic publications. On this baSis, rules and regulations have been formulated, guidelines established and policies created. There has been an active and extensive participation by both scientists and the general public in deciding how molecular biotechnology should proceed, although some controversies still remain. Molecular biotechnology, with much fuss and fanfare, has become a comprehensive scientific venture, both commerically and academically, in a remarkably short time. A number of new scientific and business publications are devoted to molecular biotechnology. Both graduate and undergraduate programs and courses have been created at many universities throughout the world to teach molecular biotechnology. The enthusiasm expressed in a 1987 U.S. Office to Technology Assessment document may not have been exaggerated in the statement that molecular biotechnology is "a new scientific revolution that could change the lives and futures of .... citizens as dramatically as did the Industrial Revolution two centuries ago and the computer revolution today. The ability to manipulate genetic material to achieve specified outcomes in living organisms .... promises major changes in many aspects of moderm life. "

2 CELL ORGANISATION

..

The cell is the smallest but complete expression of the fundamental structure and function of all organisms and that is why it is also called a unit of biological activity. It is delimited by a semi-permeable membrane and capable of self reproduction in a suitable non-living medium. The body of all living organisms (bacteria, blue green algae, plants and animals) except viruses has cellular organization and many contain one or many cells. The organisms with only one cell in their body are called unicellular organisms. (e.g. bacteria, blue green algae, some algae, protozoans etc). The organisms having many cells in their body are called multicellular organisms (e.g. most plants and animals). Every multicellular organism contains only one type of cells, either prokaryotic cells or eukaryotic cells. It was Robert Hooke who first of all in 1665 observed under a microscope honeycomb like structures in cork and he applied the term 'cell' (L.cella- small room) for the same. Previously it was believed that this is only one component of the cell which is cell wall. But in 1831, one more peculiar structure was observed by Robert Brown. He gave the name 'nucleus'. Later in 1835, the word 'sarcode'wasproposed for the jelly like material present inside the cell by Dujardin. In 1840, Purkinje replaced 'sarcode' by protoplasm which is now used universally. VIRUSES

Virus particles outside their host cells consist of a core of nucleic acid, either DNA or RNA, surrounded by a coat of protein. All degrees of complexity exist in viruses, from simple particles consisting of core with a single nucleic acid molecule and a coat of protein molecules of a single type to more complex particles with coats made up of more than 50 different kinds of proteins. Few viruses infecting animal cells such as the influenza and herpes viruses, are surrounded by an outer membrane derived from the plasma membrane of their hosts. The nucleic acid molecules of viral particles may be either linear or circular. Some of the linear molecules consist of a single nucleotide chain, while others are in double helical form. The viruses infecting plant cells usually contain linear RNA molecules, viruses infecting animal or bacterial cells may contain either RNA or DNA molecules, in either linear or circular form. The protein coat enclosing the nucleic acid core, depending on virus is either rod, spherical or lollipop shaped. The coat of the much studied tobacco mosaic virus, for example is a rod shaped structure about 15 by 300 nm long, built up from more than 2000 identical protein units. The RNA molecule of this virus winds into a helix extending through the axis of the rod. The viruses infecting bacteria, called bacteriophages, are among the most complex viral particles known. Best studied of these are the bacteriophages attacking the bacterium Escherichia coli. The "T-even" E.coli bacteriophages (T 2 , T4 and T6) have a polyhedral head enclosing a DNA core and a tai I containing several different proteins. The tail is complex in structure and consists of a collar at the point of attachment of the head, a cylindrical sheath and a base plate. The base plate carries six long hair like extensions, the tail fibers. 11

12

Biotechnology

The life cycle of the T-even bacteriophages illustrates the general pattern by which virus particles infect their host cells. Free bacteriophage particles randomly colloide with bacterial cells. If T-even cell virus particle colloides with Ecoli cell, the tail fibers 'recognize' and bind specific sites on the bacterial cell wall. The head and tail sheath then contract and inject the DNA core into the bacterial cell, while the proteins of virus remain outside. Viruses are best classified as non-living matter when they are outside their host cells. In this form. they carry out none of the activities of the life and are inert except for the capacity to attach specifically DNA core to their host cells. They can be crystallized in thIs non-living form and stored indefinitely without change or damage. The viral nucleic acid molecule head protien subunits carries only the information required to direct the host cell machinery to make more viral particles and is active in this function only when inside the host cell. Thus, a virus particle probably represents gollar - - - - - - " ~!"".(-- sheath nothing more or less than a fragment of a nucleoid or chromosome derived from a once living cell that is reduced to a set of coded directions for making additional particles of the same kind. PROKARYOTIC CELLS

Prokaryotes are small, single cell organisms. usually less than a micrometer and generally not larger than 311m. Their structural organization is simpler than that of the eukaryotes. They do not have a nucleus, and they are lacking in many membranous structures found in more complex cell types.

Prokaryotes are divided into two major groups, the eubacteria and archebacteria. Both groups share many properties that clearly separate them from eukaryotes. Most species of bacteria are Fig. 2.1. Virus. eubacteria. Eubacteria can be divided into two major groups: The non photosynthetic and photosynthetic bacteria. The bacterium Ecoli is used as an example of non-photosynthetic eubacteria.

Non-photosynthetic Eubacteria Ecoli is a bacterium that commonly inhabits the intestinal tract of humans and other animals. It is a cylindrical cell about 2 11m long and 1 11m in diameter with a volume of about 1.6 11m3. On its surface are a number of filamentous appendages called flagella, usually six, by which it rapidly propels itself. One cm3 (about one gm) of packed Ecoli contains 50 x 109 cells. An individual cell grows by increasing its length while maintaining a constant diameter. The cell divides into two daughter cells by forming a partition through the middle of the cylinder. The genetic constitution of E-coli allows the organisms to grow and divide in a medium containing only a few kinds of inorganic ions and a source of organic carbon, for example, the sugar glucose. Thus DNA of this bacterial cell contains genes for all of the enzymes needed for the synthesis of all the amino acids, nucleosides, fatty acids and other components needed to make macro-molecules, using only a simple organic molecule such as glucose and inorganic salts as a starting material.

13

Cell Organisation

In glucose containing medium, Ecoli doubles in size and divides every 40 minutes at the optimum temperature of 37°C.

#. /;;

.1-) .

~t~~:n~~~~~~~~f~e~:i;f~:~

~~:/:r;'\

7

~---plasma

EA£gf~§~ ~-------1I+-tt-~~~~;~';~~lt~ e(i

membrane

cell wall

occurs because provision of useful nutrient receives the cell of photosynthetic------jt\-\ the need to synthesize these membranes .•,~;...:,.~::,::."!.,,:, ~ ~~i..;J">;'" • '---l+-- ribosomes components. By addition of a rich variety of nutrients to the medium, Ecoli cell approaches the upper limit in its reproductive rate, doubling all of its contents and dividing every 20 minutes. The upper limit of growth Fig. 2.2. Diagram of a blue·green alga. rate is probably set by the maximum rate at which Ecoli can synthesize macro-molecules with a generation time of only 20 minutes, .and a single cell could give rise to more than 3 x 1011 cells in just 12 hours. Prokaryotic cells usually grow and reproduce much more rapidly than eukaryotes. For instance, for a bleb mammalian cell, seven hours is about the shortest generation time or cell cycle time-the time a cell needs to go from one cell division to the next. Some protozoa which are unicellular eukaryotes, have generation times as short as two hours in nutritionally rich medium, and one DNA kind of yeast cell can divide every 75 minutes; but none of the eukaryotes approach the. rapid proliferation rates common among prokaryotes. Prokaryotes are structurally simple cells. Most types have a rigid cell wall made of polysaccharides, peptides and lipids laid down outside the cell. A few types for e.g., a small bacteria known as mycoplasma lack an extracellular wall. The rigid wall of rod shaped bacteria maintains the cylindrical shape of the • •••• • • •• • cell. Other species of bacteria have walls that produce a • • • :., ./"')!..~ac~ole spherical or a spiral form. The cell wall provides mechanical V~. protection, particularly against o~otic pressure. •• • •••• • •• ... •• • • .04 Immediately inside the cell wall of E.coli is the plasma • •••~ •••• • ••• • • llThe plasma :• ... • •• membrane whih c completeIy encloses the ce. • •• e • • •• . . . . . : . . . • • membrane consists of a double layer of lipid molecules . • • • ••• • . •• ...'. with many associated protein molecules. In contrast to the • • • • • • •• • • cell wall, which is porous and therefore penetrable bymolecules and ions, the plasma membrane severely restricts Fig. 2.3. A schematic diagram of typical PPLO cell. the diffusion of molecules and ions in and out of the cell. Thus the membrane serves the critical role of retaining desired substances inside the cell, although it also limits diffusion into the cell of environmental substances necessary to sustain cell metabolism. Certain

· . \ .:

•• • •

..

• ••

••

14

Biotechnology

specialized proteins bound to the lipid bilayer of plasma membrane greatly enhance the inward passage of inorganic ions, sugars, amino acids, nucleosides and other dissolved materials which are useful to the cell. Other proteins bound in the plasma membrane of a bacterial cell catalyze the process by which the energy inorganic molecules are converted into a chemically unstable form. The intracellular contents of prokaryotes such as E-coli are present in two major structural parts-the nucleotide and the cytoplasm. The nucleotide consists of a single DNA molecule (the chromosome) condensed into a irregularly shaped fibrous network, which occupies a few percent of the total cell volume. It is thought that the nucleotide is attached at one point to the plasma membrane. This attachment of the chromosome to the membrane may help both in the control of chromosome replication and in the separation of daughter chromosomes during the cell division. The cytoplasm of Ecoli contains approximately 25,000 tiny particles called ribosomes, floating in a solution called the cytosol. Each ribosome is a machine for synthesizing proteins. The cytosol, which contains a large variety of ions, small organic molecules and enzymes, is the site where the cell carries out most of its metabolic activities. Much has been learned about the molecular biology of the cell from the study of prokaryotes, in particular from the study of Ecoli. In part, bacteria were chosen as research materials because they are functionally and structurally far less complex than any of the eukaryotic cells. In addition, the fast growth rate and low number of nutritional requirements of bacteria such as Ecoli constitute a great practical advantage for research because large number of cells can be obtained in a few hours with a simple, inexpensive culture medium. metabolites

cytoplasm

ribosomes

plasma membrane

cell wall plasma membrane

cell wall matrix

RNA .,..".~...

orotein body

ribosomes protein flagella

SOlubl~e-~¥ilDltffiBsmmm~~· RNA

A

B

granules reserve food

structural granule

C Fig. 2.4. Prokaryotic cells. A-PPLO (pleuropneumonia-like organism), B-Bacterium, C-Blue-green alga.

Ecoli is not the smallest type of cell known. Some bacteria, the mycoplasmas have volume as small of a.2/lm2, compared ta.il. minimum volume for Ecoli of 1.6/lm3 . Mycoplasmas lack cell walls and their chromosomes can be as small as one fifth of the chromosome in Ecoli. These are the smallest chromosomes known among bacteria. The mycoplasmas were identified about 1900 as the cause of respiratory diseases in animals and gained attention during World War II as the causative agent of pneumonia among U.S. Army recruits. Mycoplasmas are sometimes referred to as PPLO, which stands to pleuropneumonia like organism. Due to their small size and small amount of DNA, the mycoplasmas are no doubt genetically and functionally less complex than Ecoli. However, they require a nutritionally complicated medium for growth and grow slowly and hence are less convenient to use in research. Nevertheless, the study of mycoplasmas has intensified during recent years, and these simplest of known cells may well provide unique insight into principles of cell organization and operation. Mycoplasmas are sometimes referred to as the minimum cell because they approach the minimum genetic and molecular complexity necessary to sustain the life and reproduction of a cell.

Cell Organisation

15

Photosynthetic Bacteria The photosynthetic bacteria probably arose from non-photosynthetic bacteria very early in the course of evolution, perhaps as early as 3.1 billion years ago. Most photosynthetic bacteria are obligate photoautotrophs. Photoautotroph means requiring only light, water, inorganic ions and CO 2 ; obligate means that for growth, light is necessary because these bacteria cannot use organic compounds like sugars as an alternative source of energy. Photosynthetic bacteria are widely distributed in fresh and salt water and in soil. The enormous mass of photosynthetic bacteria growing in the oceans generates much of the oxygen in the Earth's atmosphere. Whenever seen under an electron microscope, a photosynthetic bacterium is enclosed by a rigid wall and, immediately inside the wall by aplasma membrane. As in other bacteria, the cytoplasm is rich in ribosomes and a nucleoid is present. In contrast to other kinds of bacteria, however, photosynthetic bactelia often have extensive internal membranes that contain light-absorbing pigments and the machinery for photosynthesis. Photosynthesis is the process by which the energy of light is captured and used to synthesize sugar, starting with carbon dioxide and water. EUKARYOTIC CEllS

Eukaryotic cells may be unicellular organisms such as protozoans and unicellular algae, or they may be cells that make up the tissues and organs of multicellular organisms. Though eukaryotic cells have different shape, size and physiology but all the cells are typically composed of plasma membrane, cytoplasm and membranous organelles, viz. mitochondria, ribosomes, endoplasmic reticulum, Golgi complex etc. and a true nucleus.

chromosome

mitochondrion

nudeus rough ER smooth ER Golgi complex

Go/gi complex vacuole

golgi-derived vesides (lysosomes)

microfilaments rough ER ribosomes

--'~~~.L---Iysosome

Fig. 2.5. A generalized eukaryotic cell.

Shape The shape of the cells varies from one type of cells to the other. Some cells, such as amoebae and leucocytes, change shape. Some other cells possess a typical shape, which is more or less specific and fixed

Biotechnology

16

viz. sperrnatozooids, erythrocytes, epithelial cells, nerve cells and muscle cells. The shape of the cells varies mainly due to the functional adaptation, surface tension, viscosity of the protoplasm, mechanical action exerted by the adjoining cells and rigidity of the cell membrane. Thus, the cells become cuboidal, polygonal, columnar, flat or plate like structures. Nerve cells, which serve for transmitting electrical impulses over long distances, possess long extensions. These extensions may, sometimes, be several centimeters in length. Muscle cells are also elongated so that force of action may be exerted properly in one direction. Epithelial cells are flattened. The supporting cells of the plant possess thick walls. When masses of cells are packed together, they take the shape of polyhedral solids, having many faces. Thus shape allows close pack of the cells, although regular polyhedral of four, six and twelve sides can be packed even without interstics. 14sided polyhedron satisfies the condition of minimal surface. After observing the cells taking into consideration three dimensions and reconstruction, it is revealed that many cells of animals and plants are on an average very close to fourteen faces.

Numbers Protozoa are single celled while other animals are multicellular. Rotifiers consist of a few hundred cells. Hydra, too, is made up of a few thousand of cells. In higher animals, generally, there are millions and trillions of cells. For example, man has about 26 trillions of cells in the body. In the human brain, the grey matter alone is made up of 92 billion cells. The size of the animal depends on the number of cells and not on the size of cells. Swift moving and active animals, like insects and birds, possess fewer cells per unit volume in comparision to the sluggish and lethargic animals. Size The size of the different cells ranges within broad limits. Some plant and animal cells are visible to the naked eyes, such as eggs of certain birds have a diameter of several centimetres. But great majority of cells are visible under the microscope. The smallest living cells are found among bacteria and viruses where the size ranges from .1 to .11l (Ill = O.OOlmm). Organisms like pleuropneumonia, so called "elementary bodies" have been observed with a diameter of 100 Ilm. These appear to be a resting form of the bacteria which may grow into bodies of 250 Ilm in diameter during the active metabolism of the organism. it would seem that 200-250 Ilm in diameter is the lower limit for the size of inactive, living cells. This lower limit may be set by minimum number and size of the component necessary for independent cellular existence. Usually the vast majority of cells lie in the range of 5 to 20 Il in diameter. The length of unicellular diatoms is about 100 Il. And the size of Amoeba proteus is 1 mm (1000 Il) in length. The size of human RBC is 7-81lm in diameter. In human beings, the volume of nerve cells varies from 200 113 and 15000 1l3 . The long nerve is also an example of the extremes in cell sizes, the range of which is quite remarkable in the living world.

A

TYPICAL CELL

Under the ordinary microscope, only few cell organelles like mitochondria, Golgi bodies, chlorc>plasts and nucleus are visible. However, under the electron microscope, several other cytoplasmic orga11elles such as ribosomes, lysosomes, endoplasmic reticulum, nuclear membrane, plasma membrane etc. are seen. Some important cellular components are being described here under the following heads:

Plasma Membrane The outer most boundary of the animal cell is called "plasma membrane". It is invisible under a light microscope but under the electron microscope, it appears to be composed of two dense layers which are called the outer dense layer and inner dense layer. Both these layers are about 20A in thickness, and are separated from each other by a less dense area of about 35A in thickness. By this way, the total thickness of plasma membrane is about 75A. Before the advance of the electron microscope, people used to think of plasma membrane as been stretched tightly over the cell. But now we know that single plasma membrane can have as many as 3000 microvilli. Another peculiarity of the structure of the membrane is that, it remains connected with the endoplasmic reticulum.

17

Cell Organisation chloroplasts

I

mitochondria

plasma m~mbrance

Goigi complex

Fig. 2.6. Structures typically seen in thin-sectioned plant cells.

The membrane itself may be perforated with tiny holes through which certain materials may cross. Also, as we will see shortly, materials actually move across the membranous material itself. There may also be other kinds of molecules associated with the membrane. For example, various sorts .of carbohydrates may be found attached to the outer side of the membrane, specific carbohydrates that determine the cell type. The underside of the membrane may be attached to a sort of internal support (skeleton) for the cell called the microtrabecular lattice. In a word, plasma membranes are more than fluid filled sacs .. They are living responding structures. Thus, the more membrane area an organism possesses. the greater its control over its internal environment. The microtrabecular lattice It has been recently discovered that the cell organelles may not float freely in an amorphous cytoplasm. Instead, they are held in place by a complex bridge work called the microtrabecular lattice, a web like system of microtubules and microfilaments that forms an internal cellular framework upon which many organelles are suspended. It appears as a maze like network of hollow fibres, extending throughout the cell, . connecting and suspending the organelles in a kind of three-dimensional web. Researches are already hard at work unlocking the secretes of this gland network.

Biotechnology

18

There is also important evidence that the lattice holds even enzymes in place. It has been suggested that precise spatial arrangement of enzymes would increase their efficiency by encouraging a specific sequence of interactions. For example, enzyme B might be near enzyme A, so that it might more easily interact with the product of A, enzyme C would be near B and so forth. Such a structural organization presumably would be important over random enzyme movement through the cell.

Cell Walls Plant cells have cell walls; animal cells do not. Cell walls are non-living, rather inflexible, highly permeable and strengthened by mats composed of cellulose fibres and other compounds in a tough and complex matrix. This is why trees are not limp.

nucleus

nuclear pore ~':-""-~~---l

\~_--:--:~~_r--;nuclear

envelope

~~~~~~~-7--nucledus i..-._ _,,",--mito-

chondnon

.I';---'-~~--Lperoxi­ some

o -" I

microtubule

.:.""-- ' - - -

rough endoplasmic reticulum (REA)

Fig. 2.7. Ultrastructure of a typical animal ceIl as seen in the electron microscope.

The cell walls of plants are commerically important in a number of ways. For example, we count cell walls to hold up our own walls as we frame our houses with wood. Also, it is for the cellulose in cell walls that we have revealed. Vast areas of our forests in response to wheedling commercials are designed to increase our demand for "disposable" paper commodities. The cellulose of plant cell walls is also valuable as a major component of cellulOid, rayon, cotton and hemp (Hemp was once provided by a legally cultivating plant called Cannabis Sativa, later known as "killer weed" or reefs). Another component of cell walls, lignin, was long considered a totally usel~ss by-product of paper manufacturing. However,

Cell Organisation

19

researchers worked hard to find ways to alter so that it could be sold. Furthermore, there is evidence that it is confluent with the plasma membrane. The hypothesis is partly based on notions of how the nucleus may have arisen. This arrangement would result in an open channel from the nucleus to the outside of the cell. The cell might thus be able to easily transport products manufactured in the nucleus directly to the outside. But more important, with such a connection, the nucleus might be able to react quickly to changes in the cell environment.

Golgi Bodies In 1898, Camillo Golgi, an Italian cytologist, was experimenting with some cell-staining procedures and discovered that when he used certain stains such as sHiver nitrate, "peculiar bodies" appeared in the cells. These structures had never been noticed before, but when other workers looked for them using the same stains, they turned up in a variety of cells. However, because these could not be seen in living cells, there was a great argument over whether they really were cell structures, or were just artificial or debris produced by the staining process itself. The electron microscope resolved the debate. Indeed, these strange bodies did exist, and appropriately enough they were named Golgi bodies. It was found that they had a characteristic and identifiable structure no matter what kind of cell they found in. In every case, they appeared as a group of tiny flattened vesicles, lying roughly parallel to each other, somewhat like pancakes. Even after their existence was confirmed, an argument continued over their function. What did they do for a living? We now know that they serve as a sort of packaging centre of the cell. They have been linked to manufacturing, warehousing and shipping centres (as well as Swiss finishing schools). Their role is indeed complex. Products formed by the endoplasmic reticulum are stored (and in some cases modified) in the Golgi complex. The complex also manufactures many polysaccharides including ones that will be secreted by the cell. Enzymes and other proteins as well as certain carbohydrates, are collected in these bodies and packaged into sacs or vesicles. In this way, they are kept apart from the rest of the cell. In some cases, the packages break away from the Golgi complex and move to the plasma membrane where the enclosed molecules are excreted from the cell. Lysosomes Lysosomes are somewhat spherical cytoplasmic organelles and are in general, distinctly unimpressive bodies. It is believed that lysosomes are packets of digestive enzymes that are synthesized by the cell and packaged by the Golgi bodies. The packaging is important because if these enzymes were floating free in the cells cytoplasm, the cell itself would be digested. Christian de Duve, who discovered the lysosomes, called them "suicide bags", and the drammatic description is not entirely univarrented, since they can actually destroy the cell that bears them. So why would cells have ever developed such a risk to themselves? In some cases, the destruction of cells is beneficial to the organism. For example, the cells could be old and not functioning well, or they might be in a part of the body that was undergOing reduction as a part of a normal developmental process, such as in the webbing between the fingers of a developing embryo. Lysosomes'may also help dispose off unwanted mitochondria, red blood cells or bacteria (fragments of all these have been found within the organelles). Interestingly, malfunctioning lysosomes have been associated with a number of human diseases, including cancer. Rupturing lysosomes have also been accused of contributing to the ageing process. Mitochondria These may be filamentous or granular structures. The size changes, depending on the physiological conditions of the cells. Mitochondria are usually .5)1 to 1 )1 in diameter and range in length from 2 )1 to 7 )1. The number in cells, such as in liver cells, mitochondria may be over a thousand. Electron microscope has revealed the basic structure of the mitochondria. Each mitochondrion is enclosed in double membrane. The outer membrane forms an uninterrupted boundary but the inner membrane is continously extended into folds, which project into the inner space of the mitochondrion. The inner folds are known as cristae. The inner space of the mitochondrion is filled with a flUid, which is rich in enzymes. Other enzymes are found in the membranes.

Biotechnology

20-

The mitochondrial membranes are similar to those of the cell membranes. They are also made up of double layers of phospholipid molecules sandwiched between layers of protein molecules. Similar to cell membranes, mitochondrial membranes can also expand and contract. outer membrane

peri mitochondrial space matrix

inner membrane

cristae

head piece

cristae

A

B

Fig 2.8. A Mitochondrion. A-Sectional View, 8-A part of crista magnified.

Mitochondria render oxidation of food substances. They change the potential energy of different food materials into a form of energy, which can be used by the cell for its various activities. It is, therefore, regarded that mitochondria may gather in most regions of the cells. Due to their functions, mitochondria are also called power house of the cell.

Endoplasmic Reticulum The term 'microsome' was introduced to the modem cytology by Claude (1943) to represent one of the sub-microscopic cellular components isolated by centrifugation. These are now known to be broken parts of the endoplasMic reticulum after the studies of Kollman (1953). Porter (1945) was the first man to describe their electron microscopic structure in cultured cells. He described them as lace like reticulum. There are membrane bounded sacs in the form of double membrane or cisternae. The term cisternae was introduced by Sjostrand (1953) to describe long and elongated rod like parts of the endoplasmic reticulum measuring SO-200A. A. Wiess (1953), described the vesicles of the endoplasmic reticulum which usually has a diameter of 2s-sooA while Bradfield (1913) came across another type of endoplasmic reticulum and termed them as tubules, measuring 50 to 100A in diameter. On the basis of the presence or absence of ribosomes or RNA particles, the endoplasmic reticulum (E.R.) is distinguished in two varieties. The rough walled endoplasmic reticulum, and smooth walled endoplasmic reticulum are also known as granular or a-~ type of endoplasmic reticulum. Rough Walled Endoplasmic Reticulum Rough walled endoplasmic reticulum (R.E.R.) is that variety which bears the ribosomes at the external surface of the cisternae. The distribution of the ribosomes can be circular, spircil, or rosette type. The particles may be induced to leave the surface of the cisternae or the capacity to associate again with the particles. Usually the rough walled endoplasmic reticulum is richly distributed in those cells which are engaged in the synthesis of the proteins. Smooth Walled Endoplasmic Reticulum The other large division of the system owes its identity in parts to the absence of the particles and therefore commonly referred to as the smooth surfaced agranular form. like the RER, the Smooth walled

Cell Organisation

21

endoplasmic reticulum (SER) shows a characteristic morphology which is tubular rather than cisternae. The tubules have the diameter nearly of 15-100 m~. As studied by Fawcett (1960), SER is richly distributed in those cells which are engaged in the synthesis of steroids. Continuity between the SER of a particular cell type possess a characteristic patterns of structure. Thus it is typical for rat liver cells to show groups of eight or ten slender profiles in paralleled array.

Ribosomes These are tiny, darkly staining particles present either attached to ER or freely distributed in cytoplasm. Ribosomes were first discovered in 1941 by Claude, who called them microsomes, Palade (1956) named them as ribosomes. Usually the membrane bound ribosomes (membrane piece + ribosomes) are called the microsomes. Ribosomes are very significant at the sites of protein synthesis. They are called engines used by cell in protein synthesis. In cells which are highly active in protein synthesis, simple ribosomes get transformed into clusters called polyribosomes. The number and concentration of ribosomes are related to the metabolic state of the cell. Measurement of the sedimentation co-efficient by ultracentrifugation has revealed the presence of two sizes in ribosomes. In bacteria they have a coefficient of 70S whereas in eukaryotic cells, they have about 80S. Ribosomes are generally spheroidal and have a size of 250A x 150A. Each spheroid has two subunits, a larger (140-160A) and a smaller (50-60A) one. The smaller one is attached in the form of a cap to the larger sub-unit. Chemical analysis of ribosomes reveals the presence of RNA and proteins.

Peroxisomes These are also bounded by a single membrane. These contain enzymes for the break-down of hydrogen peroxide to form water and oxygen. These are protective organelles of the cell.

Crystals and Oil Droplets In certain plant cells, the food or waste material is found deposited in the form of crystals. The oil droplets are round in both plant and anifnal cells, which appear as tiny glistening white spheres. These serve as a reserve supply of energy rich fuel.

Cytosol, Hyaloplasm or ground substance The material that remains suspended under conditions sufficient to sediment even the ribosome is referred to by biochemists as the soluble fraction or cytosol. This fraction usually contains some material lost from the organelles in the course of homogenization and centrifugation. However, it also contains other molecules, including some enzymes, that are not known to be part of the intracellular structures identifiable in the microscope. Such molecules are usually thought to derive from the hyaloplasm (also called ground substance, cell matrix, or cell sap), which was originally defined by microscopists as the apparently structureless medium that occupies the space between the visible oIJganelles. With the improved techniques, less and less of the cell appears structureless. Most investigators cbntinue to believe that there dbes exist in the cytoplasm a "soluble phase"-a solution containing inorganic ions and small molecules, materials in transit from one cell region to another and dissolved or suspended enzymes that function as individual molecules or perhaps as parts of small groups of molecules rather than as components of large, microscopically recognizable organelles. But there is a lively debate going on over proposals that many components once thought to be suspended in this solution are actually bound, perhaps loosely, in structured arrangements that do not survive the disruption involved in cell fractionation.

Plastids The plastids are differentiated bits of protoplasm- "organs" of areas of metabolic activity associated with particular functions. They have a limiting, apparently semi-permeable membrane and complex internal structure. They are often coloured and usually conspfcuous. In size they are small and generally may occur in cells. They are variable in shape, but rounded types are most common. Spherical, ovoid, discoid,

22

Biotechnology

granular, rod like plastids all occur frequently. Large plastids of peculiar shape are present in many algae and rarely elsewhere. They may be found in all living cells of plants and probably are present in every cell in its early stages of development. Later they become restricted to certain cells and are abundant only in those which have specialized functions, such as photosynthesis, storage and colour manifestation.

Origin Plastids are present in large numbers in young meristematic cells where they are minute, the smallest being at the limit of microscopic visibility. At this stage, when they are known as proplastids, they are rounded bodies that do not resemble plastids. As the cell grows, proplastids multiply freely and mature plastids gradually develop. Increase in number by division continues at all stages but less frequently in mature plastids. Probably plastids arise only from pre-existing plastids.

ria

,plasmodes, mata

Types of Plastids Several fairly distinct types of plastids "1ar fundamentaI plasma membrane occur, but aII are 0 f sImI nature. They fail into two chief classes: coloured plastids, chromoplasts and colourless plastids, leucoplasts. Green chlorophyll bearing chromoplasts are known Fig. 2.9. A typical plant cell. as chloroplasts and are commonly set apart as a third major type because of their important function in food manufacturing. This leaves the term chromoplast to cover all coloured plastids except chloroplasts, and this is its general use.

Chloroplasts

.

The chloroplasts of the higher giants are mostly uniform in size and chiefly flattened, ellipsoid or disc like in shape. They are numerous, from a few to many in a cell-and small, averaging 5 micra in diameter. They multiply by constriction, and may at other times change shape, appearing as though semi-liquid. The chlorophyll is wholly or largely restricted to numerous small granules known as grana. Apparently these deeply coloured bodies are commonly so arranged and so closely packed that the plastid appears structurally homogenous. Sometimes an obscure layering can be seen. Chloroplasts of shaded leaves are somewhat larger than those of leaves in full light on the same plant and their chlorophyll content is possibly greater per unit volume.

Chromoplasts Non-green chromoplasts range in colour through yellow, orange and yellow red. The colour is gi~n chiefly by xanthophyll, carotin and carofinoids. Chromoplasts show great variety in shape but are chiefly irregular, granular, angular, acicular and forked types occur. The irregular and pointed shapes are believed to be caused in part by the presence of coloured substancp.s, especially carotene and carotenoids in crystalline form, as in the root of Daucus. The functions of these plastids are obscure. They are associated with colours of flowers and fruits but occur also in other regions such as roots commonly represent transformed chloroplasts, but may form directly from smallieucoplasts.

Cell Organisation

23

l.eucoplasts The term' leucoplasts' covers various types of colourless plastids. The early stages of all plastids are called leucoplasts but such young plastids are best called proplastids, and only mature colourless plastids should be called leucoplasts. Like chromplasts, leucoplasts vary in shape; extreme forms are rod like. They change shape readily and are probably always highly plastic. They are concerned with food storage and doubtless have other unknown functions. This type of leucoplasts associated with starch grains formation in storage regions is known as amyloplasts. Leucoplasts related to the formation and storage of oils and fatty substances are known as elaioplasts. These are at least in part amyloplast that vary at functions at certain times. The leucoplasts of hair and other epidermal cells are probably degenerate or dormant plastids of other types. That all plastids are alike in nature is clear from the readiness with INhich one type is transformed into another. For example, the chloroplasts of young fruits and of developing petals may become the chromoplasts of a ripe fruit and of the mature flower respectively, the leucoplasts of the potato tuber become chloroplasts on exposure to light. Vacuoles Vacuoles are fluid filled sacs found in cells of both plants and animals as well as in microscopic organisms called protists. It is in plant cells, however, where they reach their greatest development; infact, they may be the most conspicous bodies in the plant cell. There are a number of types of vacuoles, each with a different function. Some, for example, are highly active in the cell's metabolism, while others are simple storage vessels. In plants, the vacuoles are filled with a "cell sap" that can change volume through osmosis. Infact, it is the pressure of the swelling vacuole that forces the plasma membrane against the cell wall and makes the plant tissue firm. A plant wilts when there is not enough fluid to keep its huge vacuoles filled. Besides water, the vacuole sap may contain sugars, proteins, pigments and organiC acids. It is these acids that give oranges and lemons their sour taste. Some single celled organisms (such as the Paramecium) contain contractile vacuoles that enable them to SQueeze waste material or excess water but through the plasma membrane. Such vacuoles may appear and disappear in response to the organism's needs, or they may be relatively permanent structures. The latter help us distinguish one species of these tiny organisms from another. Microtubules . Microtubules, as you might expect, look like tiny tubes. Their constituent protein called tubulin, occurs in doublet spheres whose arrangement forms the tubes or cylinders. Microtubules form a core, not only of flagella, cillia and sperm tails, but also the star-shaped 'asters' and the spindle fibres that appear at mitosis. Centrioles Centrioles are small cylindrical bodies, barely visible under a light microscope, that lie just outside the nucleus in an area of specialized cytoplasm. They are normally found in cells of animals, algae and some fungi; they are absent in the cells of flowering plants. They are associated complex ways with cell division and cell movement. Centriole is made up of a rounded cylinder of about 3000-soooA in length and lS00-1800A in diameter. Each centriole consists of a stack of nine fibres arranged around a central axis. Each fibre is comprised of three secondary fibres. All these fibres are interconnected and are enc10sed in a thick membrane. Cilia and Flagella Cilia and flagella are hair like extensions that project from the surface of certain kinds of cells. They differ only in length; cilia are about 10 to 30 micrometers while flagella may extend to thousands of micrometers. Both make 'beating' movements, and both may function in moving the cell along through the fluid cilia, in addition, some substances across the surface of a stationary cell. As examples, a sperm cell

24

Biotechnology

swims by beating its whip like flagellum, and the beating cilia that line your breathing passages help sweep away airborne debris (unless you have killed them by smoking). Both cilia and flagella contain microtubules that form a 9+2 arrangement. Basically, the structure involves a circle of nine parts of microtubules surounding two Jingle microtubules. (The arrangement on centrioles is similar, but they lack the central pair). . The 9+2 arrangement of cilia and flagella has generated much speculation about their origin. For example, some researchers believe that each cilium develops as the extension of one of a pair of centrioles.

The Nucleus Perhaps the most prominent structure in most cells is the nucleus. It also plays a central role in the life of the cell in that it is involved in a number of critical processes, such as regulation and reproduction. The nucleus is surrounded by a double membranous covering, a fact that suggests two hypotheses regarding the origin of the nucleus, as you may recall. The nuclear membrane or the nuclear envelope is perforated by nuclear pores. Such pores facilitate the movement of molecules between the nucleus and cytoplasm. The nucleus of the meristematic cell appears to show two types of resulting nuclei in different plants. One type is known as the 'solid nucleus' and the other as the "pro-chromosomal" or over circular type. The prochromosomal type of nucleus consists of a vesicle of sap around the periphery of which is the whole of the stainable material of the nucleus, the chromatin, included in relatively small bodies called prochromosomes. One or more nucleoli are also present. The "solid" type of resting nucleus is devoid of free sap and after fixation, has the appearance of a uniform meshwork of chromatin filling the whole nucleus except for the space occupied by one or more nucleoi. The latter, at least in the early stages, are fluid. This type of nucleus is found in members of the Liliaceae, in Vicia and Osmunda (the royal fern). The chromatin meshwork is frequently referred to as a 'reticulum' composed of 'karyotin'. The nucleus plays an important role in governing the metabolic processes which go on within the cell, resulting in cell division, growth and differentiation. The nucleus also contains the hereditary characters which are passed on from parent to the offspring. Inside the nucleus is a fluid matric in which a number of different types of bodies are suspended. When cells are appropriately stained, a net like structure becomes visible within the nucleus. This material is called chromatin (from Greek: chromo-colour) because of its affinity for the stains. At a certain period in the cell's cycle, the chromatin shortens and thickens and forms chromosomes, the structures that include sequences of genes. Undoubtedly the meristematic cell is bounded by a thin cell wall and contains the protoplast. The latter consists of a relatively large central nucleus and the surrounding cytoplasm. Within the cytoplasm are suspended specialized protoplasmic bodies called plastids or chromatophores, and non-living inclusions in the form of minute granules and droplets.

The protoplasmic substance Observation of living cell indicates clearly that the protoplasm is not homogeneous, but its physical nature is still rather a matter for conjecture. There is a little doubt, however, that is a colloidal system, usually with many of the properties of a liquid, being infact a liquid (son in which are dispersed either liqUid or solid particles which are either aggregates of small molecules or are very large molecules. The particles dispersed in the continuous liquid phase of the protoplasm are extremely minute, and often only just visible with the most powerful microscope. Many of the properties of the protoplasm are the outcome of its colloidal nature. Chemically, the dry substance of the protoplasm appears to consist mainly of proteins with appreciable amounts of fats and fatty substances, and of carbohydrates, and smaller amounts of other organic compounds and of mineral matter. Analysis of the protoplasm always, of course, includes any substances present in it at the time, that is both living and non-living protoplasmic inclusions. But it must be remembered that

Cell Organisation

methods of chemical analysis bring about the death of the protoplasm, and so the analysis is really of dead and not living protoplasm. The analysis does, however, show the presence of substances (fat like compounds and proteins) which are capable of forming the disperse phase in a colloidal system. The evidence of chemical analysis and of observation on the physical properties of the protoplasm suggest that in the protoplasm, we have a continuous phase of water containing proteins and fatty substances, and dispersed in this are granules (solid or liquid) of protein, either alone or united with other substances, the whole being of the consistancy of a slightly "viscous liquid. The surface layer of protoplasm differs from the inner part, and in a system such as that outlined above, we might expect to find the outer most layers with concentration of fatty substance. Any such surface layer is certainly of a complex nature and of vital importance, as its properties will govern the entry of substances into, and their exit, from the protoplasm. Cytoplasm In mature living cells, the cytoplasm (that part of the protoplasm distinct from the nucleus) forms a lining to a cell wall. Internally the cytoplasm is in contact with the content of the vacuole, and externally with the cell wall and the liquid with which it is saturated. The cytoplasm is not homogeneous. On physical ground, we should expect the surface layers of the cytoplasm to differ in composition and structure from the inner most parts. A knowledge of the laws of surface tension leads us to expect an accumulation of the fat like component of the cytoplasm at the surface. The differentiated layers frequently visible in animal cells and called the ectoplasm, but rarely observed in plant cells from the plasma membranes. Although the especially differentiated plasma membranes are but rarely visible in plant cells, there is experimental evidence of their widespread existence. The inner most part of the cytoplasm (endoplasm) contains specialized protoplasmic bodies and non-living inclusions. Table 2.1. Comparison between Plant and Animal Cells Plant Cell

Animal Cell

1. Generally larger than the animal cell. 2. Plant cells are surrounded by a rigid cell wall of cellulose. 3. Majority of the mature plant cells have a large central sap vacuole. 4. Plastids, especially chloroplasts are found usually in cells of green plants. 5. In the cell division, the division of cytoplasm of plant cells takes place by formation of a partition called cell plate.

1.· Usually smaller than the plant cell. 2. Cellulose wall is not found in animal cell. 3. Vacuoles in animal cells are small. 4. Chloroplast is not found in animal cell except in some protozoans. 5. Animal cells divide by a constriction during cell division.

Table 2.2. Function of Cellular Organelles Cell Organelles

Functions

Cell or Plasma membrane Serves as a differentially permeable membrane through which extra-cellular substances may be selectively sampled and cell products may be liberated. Gives external form, provides mechanical support and thus protects the cell. Cell Wall (in plants) Thick cellulose wall around the cell membrane gives strength and rigidity to the cell. Cytoplasm Provides greatly expanded surface. EndoplasmiC retieulum area for various biochemical reactions which normally takes place at or across membrane surfaces.

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Biotechnology

Ribosomes Mitochondria

Centrioles (usually not seen in plant cells) Golgi body or apparatus (known as dictyosomes in plants) Lysosomes (in animals) Plastids (in plants) Vacuoles Hyaloplasm Cilia and flagella Nucleus Nucleoplasm Nucleolus Chromosomes

Sites of protein synthesis and known as "Protein factories of the cell". Oxidation of food substances, changes potential energy into a form of energy to be used by the cell. Energy production (Krebs' cycle, electron transport chain, beta oxidation of fatty acids etc.) Power house of cell. Form poles for the cell division. Capable of replication usually. Produce cellular secretions

Produce intracellular digestive enzymes which help in disposal of bacteria and other foreign bodies. If ruptured, may cause cell destruction. Serve for storage of starch, pigments and other cellular products. Photosynthesis takes place in chloroplasts. Store excess water, waste products, soluble pigments etc. Contains enzymes for glycolysis and structural materials, viz, sugars, amino acids, water, vitamins, nuc!eotides etc. Serve to move the animals or to move the material. Provides selective continuity. Contain materials, for building DNA and messenger molecules which serve as intermediates between nucleus and cytoplasm. May synthesize ribosomes. Disappears during cellular replication. Function uncertain. Bearers of hereditary instructions. Regulation of cellular processes (evident only during nuclear division). Control the nucleus and metabolism.

Table 2.3. Comparison between Prokaryotic and Eukaryotic Cells Prokaryotic

Eukaryotic

1. Generally smaller than the eukaryotic (mostly

1. Usually larger than the prokaryotic cells (mostly

1 to 10 ~m) 2. They are never assoCiated to form group

10 to 100 ~m) 2. They may be associated in chains, filaments or groupings or multicellular. 3. They have plasma membrane which is devoid of cellulose deposition. 4. Structure like septum, mesosomes etc. are absent.

3. They have cell wall like plant cells. 4. Occasionally outer wall projects into an invagination of the plasma membrane, forming a septum that may further develop mesosomes. 5. Protoplasm is differentiated into cytoplasm and nucleoid. 6. Nuclear material is not bounded by nuclear membrane. 7. DNA not associated with histone proteins in chromosomes. 8. DNA is circular. 9. One linkage group.

5. Protoplasm is differentiated into cytoplasm and nucleoplasm. 6. Definite nuclear membrane encloses nuclear material. 7. DNA complexed with histone proteins chromosomes. .,.8. DNA is filamentous. 9. Two or more linkage groups.

27

Cell Organisation

10. It lacks well defined cytoplasmic organelles such as E.R., Golgi body, Mitochondria etc. except for photosynthetic membranes in some bacteria 11. Ribosomes are of 70S type. 12. They reproduce in amitotic way, no mitosis takes place. 13. Respiratory and photosynthetic enzymes are located in the plasma membrane. 14. 15. 16. 17. 18. 19.

10. Well defined cytoplasmic organelles such as Mitochondria, Golgi body, E.R. etc. are present.

11. Animal and plant cells have 80S ribosomes. 12. Mitotic division is of common occurrence.

13. Respiratory and photosynthetic enzymes are located in the mitochondria and chloroplast respectively . No nucleolus. 14. One or more nucleoli. Bacteria and cyanobacteria belong to this 15. This category includes protists, fungi, plants, and category. animals. 8+ DNA content ranges from 750,000 base 16. 8+ DNA content ranges from about 1.5 x 107 pairs to 5 x 106 base pairs to 1.5 x 1011 base pairs. Genes generally lack introns. 17. Most genes contain introns. lack microtubules, micro filaments and 18. Contain microtubules, microfilaments and interintermediate filaments. mediate filaments. Sterols usually not present in the plasma 19. Sterols present in the plasma membrane. membrane.

3 CHROMOSOMES There are certain thread like filamentous bodies in karyolymph or nucleoplasm which appear as thread like coloured structures. These were first termed as chromosomes by W Waldeyer in 1988. Chromosomes are not visible in the interphase nucleus or metabolically active nucleus due to their high water content. DUring cell division, the chromosomes undergo condensation, and in this, they become thick and short. Permanently condensed chromosomes are known to occur in various protists, fungi and other simple organisms. Chromosomes become all the more important because of their role in variation, mutation, heredity and evolution. They are usually considered as a nuclear component with a special organization, individuality and function. They are capable of duplication and maintaining their morphologic and physiologic properties through successive cell division. The morphological study of chromosomes has been done by Hertz (1935), Kuwanda (1939), Geitter (1940), and Kaufmann (1948). The detailed morphology of the chromosomes varies from cell to cell and major changes are associated with the cell division process. Regardless of differences in their detailed morphology, both direct and indirect evidences make it clear that each chromosome is an individual entity which, barring accidents, is retained throughout the life of the cell. The stainable materials of chromosomes are called chromatin or chromatin fibres. These fibres are complexes of DNA and proteins for the most part, although some minor amount of RNA may also be present. The chromatin fibre may be considered the basic structural unit of the eukaryotic chromosome. VIRAL CHROMOSOMES

Viruses are acellula~ organisms and are said to be the infection particles present outside the living cells. Each virus is composed of a central core of nucleic acid surrounded by a protein coat called capsid. Only one type of nucleic acid is present in a virus. The viruses contain single or double stranded DNA or RNA. The chromosome of bacteriophage lamda consists of linear molecule of double stranded DNA of 17.5 11m length, having about 48,000 nucleotide pairs. In the case ofT2 and T4 phages, the chromosomes are composed of linear double stranded DNA molecules. There are about 6,000 pairs of nucleotides in one chromosome. A single stranded DNA of 0x 174 bacteriophage contains about 5375 nucleotides. It has cohesive ends. Likewise, the chromosome of MS2 phage is made up of a single stranded RNA having 3569 nucleotides. Inside the cell of the host, the viral chromosome is either integrated into the host chromosome or it exists free in cytoplasm. The linear molecule of viral DNA shows the follOwing characteristic features proving their transformation from the original circular one.

(a) Cohesive Ends The linear molecule of DNA is composed of two anti-parallel strands. The 5' ends of these duplex DNAs project as a single strand beyond 3' ends of anti-parallel strands. These single strands, formed of about 12 nucleotides are complementary with the 3' ends forming circles.

28

\

29

Chromosomes

(b) Terminal Repitition The terminal ends of the linear molecule of DNA have repititive bases and the 3' end of DNA bears a base sequence that is repeated at the 5' end of the strand.

(c) Modified Bases In the linear molecule of DNA, some of the nitrogenous bases are modified in such a way they project viral DNA from degeneration by host endonuclease. For example, in T-even phages, hydroxymethylcytosine which is often glycosylated, occurs in place of cytosine. (d) Circular Permutation The circular permutated linear DNAs have also been recognised forming the identical circular molecules. Such viral DNAs contain a population of molecules whose sequences are circular permutations of each other.

1 nick

1 additional nick

Fig. 3.1. A schematic drawing of the folded supercoiled E.co/i chromosome, showing only 15 of the 40 to 50 loops attached to a putative protein core (shaded area and the opening of loops by nicks). PROKARYOTIC CHROMOSOMES

The prokaryotes usually have only one chromosome, and it bears little morphological resemblance to eukaryotic chromosomes. Among prokaryotes, there is considerable variation in genome length bearing genes. The genome length is the . " smallest in RNA viruses. In this case, the organism is provided with only a few genes in its chromosome. The number of genes may be as high as 150 in sqme larger bacteriophage g#nomes. In Escherichia coli , about3000 and 4000 genes are organized into its one circular chromosome. The chromosome exists as a highly folded and coiled structure dispersed throughout the cell. The folded nature of the chromosome is due to the incorporation of RNA with DNA. The interaction of RNA with a single molecule of DNA results in the looping of DNA molecule. causing the reduction of th e structure . There are about 50 loops in the chromosome of E coli. These loops are highly Fig. :3 ..!. A" electron micrograph of an E. coli chromosome showing the multiple loop s emerging from a central region. twisted or supercoiled structures

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with about four million nucleotide pairs. Its molecular weight is about 2.8 x 109 . During replication of DNA, the coiling must be relaxed. DNA gyrose is necessary for the unwinding of the coils. It is apparent from these studies that the functional prokaryotic c:1romosome is not simply a naked strand of DNA, it is the supercoiled structure that is a functional one.

I--

(a)unfolded double helix of DNA

(b) looping caused by RNA "connectors" (in red)

(c) supercoiled folded chormosome

Fig. 3.3. Folded prokaryotic chromosomes. EUKARYOTIC CHROMOSOMES

A eukaryotic chromosome contains a single DNA molecule of enormous length. For example, the largest chromosome in the Drosophila meianogaster genome has a DNA content of about 65,000, kb (6.5 x 107 nucleotide pairs), which is equivalent to a continuous linear duplex about 22 mm long. The DNA of all eukaryotic chromosomes is associated with numerous protein molecules in a stable ordered aggregate called chromatin. Some of the proteins present in chromatin determine the chromosome structure and the changes in structure that occur in the division cycle of the cell. Other chromatin proteins appear to have important roles in regulating chromosome function. In eukaryotes, the chromosomes are well organized and even more complex in structure than that of prokaryotic chromosomes. Eukaryotic chromosomes belong both to animals and plants. The chromosomes are well distinct and clearly visible in the tissue of any eukaryotic organism at an actively dividing state. In plants, the root tips, the shoot tips, the leaf, young vegetative filament and flowers are the main sources of material for chromosome study. In animals, the very young larvae, larval tails, salivary glands, bone marrow, stratum germinativum of epidermis and goands are the main sources for the routine work. MORPHOLOGY

It is not possible to give a generalized structure of chromosomes in lower organisms like l;>acteria, viruses etc. but in higher organisms (animals and plants), the morphology of chromosomes is essentially similar except some variations in shape and size depending upon the divisional stage it is in. In many types of resting nuclei, the chromosomes are unfixable. Therefore, these have been' studied best during mitosis. It is usual to study the morphology of chromosomes during the metaphase or anaphase of mitotic division because in these two stages, the chromosomes have reached their maximum contraction attaihing a length, that under ordinary environmental conditions, remains remarkably constant from cell to cell. Shape

As stated above, the chromosomes come in picture during the cell division so their definite shape is usually observed either during the metaphase or anaphase. The shape of the chromosomes is variable from phase to phase in the continuous process of cell division. During interphase, the chromosomes are too thin to be seen under light power microscope. During prophase, they become short and thick with a distinct and clear zone-the centromere. The centromere creates a constriction-the primary constriction in the chromosome. It divides the chromosome into two parts, each part is called chromosome arm. On the basis of the respective position of centromere, four types of chromosomes are distinguished as follows:

31

Chromosomes

Table 3.1. Difference between Prokaryotic and Eukaryotic Chromosomes. Prokaryotic Chromosome . (Prochromosome)

Eukaryotic Chromosome (Chromosome)

1. It is a primitive structure, also called prochromosome. 2. A prokaryotic cell has a single chromosome equivalent. 3 . The chromosome is attached to a replicating structure like mesosome . 4 . DNA is circular. 5 . DNA is not associated with histone proteins. It is therefore called naked. 6. Coiling is caused by bands of RNA or polyamines. 7. It is in contact with plasma membrane through mesosome. 8 . Prokaryotic chromosome is embedded directly in the cytoplasm. 9. A nuclear envelope is absent around the thromosome. 10. Chromosomes are compact in the metabolic cell. 11 . There is a single replicon.

1 . Eukaryotic chromosome is the typical chromosome. 2. The eukaryotic cell possesses two to several chromosomes. 3 . A mesosome like structure is absent 4 . DNA is linear. 5 . DNA is associated with histones. 6. Coiling is due to scaffolding and histone proteins. 7 . The chromosomes are seldom in contact with plasmalemma. 8 . Eukaryotic chromosomes do not lie in direct contact with cytoplasm. 9 . A nuclear envelope surrounds the chromosomes complex. 10. Chromosomes occur in the form of chromatin fibres in the metabolic cell. 12. The eukaryotic cell has several replicons.

(a) Acrocentric

Here the centromere is located near the extremity of one end of the chromosome, giving rod shape to the chromosome. One arm of the chromosome is very long and other is very short. Stich chromosomes are found in locust (all), IVth chromosomes of Drosophila and human beings.

(b) Sub-metacentric When the centromere is located .in between the end and middle part of the chromosome, it is considered as sub-· metacen tric . Such chromosomes contain unequal arms and assume L or J shape during anaphasic movement. Some chromosomes in human beings belong to this category. (c) Metacentric Here, the centromere is located in the middle region of chromosomes giving the V-shape and two isochromatic arms of equal length to the chromosome. Such a chromosome is referred to as metacentric chromosome. Amphibians, Tri[/ium and Tradescantia have metacentric·chromosomes.

secondary constriction

chromatids

centromere FIg.

3.4. (a) External view of a metacentric chromosome. (b) Internal . structure of a metacentric chromosome.

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(d) Telocentric When the centromere is located at the end of the chromosome, it is said to be the telocentric chromosome. Its both free ends develop centromeres, the stage is called the bi or dicentric. Telocentric chromosome,> are very rare and most unstable. Marks in 1957 has reported such chromosomes in some .species of telocentric protozoans, mammals and plants.

'\

~o\.

sub-metacentric

metacentnc

acrocentric

Size

The size of chromosomes is a relatively constant feature in a particular species; although some narration of size may be detected in the chromosome of different tissues or zones within Fig. 3.5. Morphological variations. single organism. In plant Mediola, the root tip chromosomes are 50% bigger than the shoot tip chromosomes. The chromosomes may vary greatly in closely related genera. Generally, it is seen that the organism with less number of chromosomes contains large sized chromosomes. The length of a chromosome may vary form 0 .21l to 50ll and the diameter varying between 0.21l to 21l. In some animals,there Is symmetrical karyotypes i.e. have all chromosomes of the same size. Asymmetrical karyotype refers to the chromosomes of differ~nt sizes. This difference may be gradual as in human beings or there may be two or three different size groups as in Vacca arkansana. Generally, plant chromosomes are bigger than the animal ones and chromosomes of monocots are bigger than those of dicots and other plants. In human beings, the approximate length of the chromosome is 41l to 61l. Trillium (plant) contains largest chromosomes that may attain 30-32 microns length. Among anirnals, grasshoppers, Crickets, mantids, newts and salamanders have large chromosomes. Variation in size of the chromosomes can be induced by a number of environmental agents: (i) at low temperature, dividing cells have shorter and more compact chromosomes than those dividing at high temperature; (ii) on treating cells wjth colchicine, the chromosomes become short and (iii) rapid and repeated divisions tend to result in smaller chromosomes.

Number The number of chromosomes varies from species to species but it remains constant for a particular species. The basic number of chromosomes in the somatic cells of an individual is referred to as the sqmatic or zygotic or diploid number. Certain cells and tissues may contain multiples of this number and anheferred to as polyploids. The somatic number of chromosomes becomes half during gametogenesis. The gametes with half number of chromosomes are said to possess one genome that can be expressed as the genetic or haploid or In' number. The number of haploid chromosomes in most animals and plants lies between 6 and 25. The smallest number of chromosomes recorded so far, is found in Ascaris megalocephala, here In' is one only. There are four chromosomes in 'Mesotoma' (flatworm) and Ophryotrocha (polychaete). In human beings there are 46 chromosomes. The greatest number of chromosomes in animals is 254 in the hermit crab with the exception of Au locantha (radiolarian) where the number is as high as 1600 (Belar, 1928).

Diploid number of chromosomes in cells of some organisms (A) Animal cells

Ascaris Mosquito Drosophila

2

-

6 8

Paramecium Hydra vulgaris Earthworm

30-40 32 32

O:m

Goat Guin~pig

60 60 64

33

Chromosomes

Musca Locust Planaria Bu/o Opossum Cockroach Triturus Grasshopper Hyla Frog (Indian) Frog (British) Axolotl

120"

13S? 14 = 16 = 22 22 230"

24S? 24 24 24 24 26 28

Alligator Honeybee Cat Hog Pig Mouse Rattus Rhesus monkey Rabbit Homo (Man) Chimpanzee Sheep Helix Bombyx

= = = =

32 32, 16 38 40 40 40 42 42 44 46 48 54 54 56

Horse Donkey Dog Gallus Duck Columba Turkey Gold fish Carp

.

Crow Crayfish Hermit crab Amoeba Aulocantha (Radiolarian)

= = = = =

66 66 78 78 78· 80 81,82 94 104 200 208 254 500 1600

(B) Plant cells

Broad bean Evening primrose Garden pea Sweet pea Pink bread mold Slime mold Barley Rye Snapdrogon Aspergillus mold Garden onion Yeast Cabbage Raddish Orange Com Banana

Bean Rice Tomato

12 14 14 14 14 14 14 14 16 16 16 18 = 18 18 18 20 22 22 22 24

Yellow pine Scarlet Wheat (summer) Cherry Green algae (Chalamydomanas) Sunflower Apple Pear Wheat bread Coffee Potato Tobacco Plum Cotton Sugarcane

24 24 28 32 32 34 34 34 42 44 48 48 48 52 80

KINDS OF CHROMOSOMES

The eukaryotic chromosomes have been placed under two categories: autosomes and sex chromosomes. Autosomes are the chromosomes bearing geQes for the somatic characters. They have nothing to do with the sex determination. In human beings, there are twenty pairs of autosomes. Sex chromosomes are concerned with the sex-that is why they take part in the determination and even formation of sex. The sex chromosomes are variously called allosomes or hetero-chromosomes. They are usually two in number. The number of sex-chromosomes is not affected by the number of autosomes. Sex chromosomes are of the two types: X chromosomes and Y chromosomes. X chromosome is usually a longer and functional

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chromosome whereas Y chromosome is usually quite short and genetically inert chromosome. In most of the cases, females have XX chromosomes and males have XY chromosomes. The characters by which a set of the chromosomes of a species is identified, are called their karyotypes. When the karyovjpes of a species are represented by a diagram, then such a diagram is called idiogram. STRUCTURE

Eukaryotic chromosomes are well distinct at various stages of division due to having many more DNA than prokaryotes. Metaphasic as well as anaphasic chromosomes are well defined elongated bodies having the following parts:

Pellicle Each chromosome apparently consists of an outer membranous pellicle which encloses a homogeneous matrix. Presumably, the matrix and pellicle are composed of an achromatic or non-genetic material. These are not stainable with dyes, that is why their true structure and function are not known. But recent researches have shown clearly the absence of any definite pellicle around the chromosomes.

Chromatids and Chromonemata Each chromosome is made up of two chromatids or genonema, and each chromatid is further composed of two fibrils called chromonemata or chromonematal fibrils. These fibrils remain coiled with each other. The coils may of the following two types: (i) Paranemic coils In this, both the coiled chromonemata are not relationally coiled around each other and the sub-units can easily be separated. This type of coiling is more common in mitotic chromosomes.

(ii) Plectonemic coils In this, the chromonemal sub-units coil around each other and remain interwined. Their separation is not easy. If the subunits are drawn out, they tum into relational coils. This type of coiling is more common in meiotic chromosomes. Each chromonema is a continuous nucleoprotein fibrea bundle of fibres upon which the genes are assumed to be arranged

paranemic coils

chromonemal threads

FIg. 3.6. Diagramatlc representatIon of paranemlc alld plectummllc cuiiillg.

in a linear fashion. If splitting has not yet occurred at this stage, there will be a single chromonema in each chromatid, and if it has already taken place, there would be two or more. At metaphase, the chromonemata are spirally coiled. This was first noted by Baranetsky in 1880, but it was not realized until much later that spiralization was a universal feature of chromosome structure. At least in certain chromosomes, these appear to have a coiled structure. The thread which forms the major spiral itself is a minor spiral. But it is uncertain that how far this coiled structure is present in the somatic mitosis. In meiotic chromosome the major coil contains about 10 to 30 gyers and a minor coil which is perpendicular to the major coil contains many more gyres. At first, it was uncertain whether the direction of coiling was constant for a particular chromosome or chromosomal region. The number and pitch of major and minor coils may be modified, subjecting the living cell to heat or chemical starvation or by any other physical or chemical factors. In mitotic chromosomes, a helical structure similar to the major coil of meiotic chromosomes has been described. This is the somatic or standard coils.

Chromosomes

35

Some parts of chromonemal fibres or chromatids or chromosomes are thickly coiled, whereas others are less coiled and some parts are not coiled at all. Degree of staining of chromosome parts perhaps depends upon the extent of coiling. There is lack of coiling at the centromE.re region.

(iii) Chromomeres

=:

Sometimes dark staining granular bodies are seen in the nucleoplasm of even resting cells. But, generally such bodies are distributed over chromosomes at intervals along its length. These are the chromomeres and the thin regions between the bead like chromomeres ~ are the inter-chromomer~s. The ~ chromomeres were descnbed by Balbiani (1876) and Pfitzner (1881). A Their dark nature indicates that chromomeres a'e pe,haps those pam ~ of chromosomes which are not totally ~ uncoiled during the interphase. -=--Chromomeres are seen very clearly in C o some special chromosomes like Fig. 3.7. Kinetochore. salivary gland chromosomes of diptera or lamp-brush chromosomes of amphibian oocytes. The chromomeres found on these giant chromosomes are bigger and are known as chromeoles. Recently, it has been postulated that the chromomeres are the region of the super imposed coils.

~

(iv) Centromere or kinetochore or primary constriction Schrader described the centromere as a regionally modified and thickened part of the chromosome where the chromonemal fibres of a chromosome are joined together. The chromosomes are able to orient themselves properly only due to their centromeres. Actually, it is the site at which the spindle fibres shorten, causing the chromosomes to move towarqs the poles. The position of the centromere is constant for a particular chromosome_ Centromere is also " ,i responsible for the shape of the chromosome. On the basis of the location of centro meres on chromosomes, four categories of chromosomes are

~~

~~

recognized-acrocentric, subm'etacentric, metacentric and telocentric (already explained). C The centromere, which is a granule in the clear B A zone in primary constriction, is ovoid, non-stainable Fig. 3.B. Kinetochore structure. structure with a large diameter, as in Maize or may be like tiny stainable granule as in Tradescantia. Centromere has a clear zone in which the fibrils remain uncoiled or less coiled than those in the rest of the chromosome. Centromere is made up of three zones or layers present in duplicate: outer, middle proximal arm region with ~ and inner. The outer, moderately dense special \diVision cycle ~_______ layer is called the centromere plate; the inner dense layer is formed from compact chromatin fibre and the middle one is the most transparent of the three. In flowering sister chromatids outer mid inner centric plants, there seems to be no layered -----.,,,..-----J' chromosome centromere at all. The middle zone zones maintains the relation of the chromosome Fig. 3.9. Diagram showing the organization of the centromere C. to the spindle. During cell division,

V

~

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particularly at metaphase, spindle fibres can be seen inserted into the centromere. Thus, we see that the centromeres are merely the hold-fasts for spindle fibres. However, in a few plants and Chromatid animals, the centromeres are non-localized and the spindle fibres at the time of cell division may attach at any piace. The centromeres thus serve both as the sites of association of sister chromatids and as the attachment sites for microtubules of the mitotic spindle. They consist of specific DNA sequences to which a number of centromere-associated proteins bind, forming Spindle a specialized structure called the kinetochore. The binding of fibers microtubules to kinetochore proteins mediates the attachment of chromosomes to the mitotic spindle. Proteins associated with the kinetochore then act as "molecular motors" that drive the movement of chromosomes along the spindle fibres, segregating the chromosomes to daughter nuclei. Centromeric DNA sequences have been defined best in yeasts, where their function can be assayed by following the segregation of plasmids at mitosis. Plasmids that contain functional centromeres segregate like chromosomes and are equally distributed to daughter cells following mitosis. In the absence of a functional centromere, however, the plasmid does not segregate Fig. 3.10. The centromere of a metaphase chromosome. properly, and many daugi)ter cells fail to inherit plasmid DNA.

Transform yeast

o Mitosis missegregate

Some daughter cells lose plasmid and require leucine for growth Fig. 3.11. Assay

Plasmids

rID

1:Q)';:'~~~~~

AU daughter cells inherit plasmid and can grow on medium lacking leucine 01 a centromere in yeast.

37

Chromosomes

Assays of this type have enabled determination of the sequences required for centromere function. Such experiments first showed that the centromere sequences of the well-studied yeast Saccharomyces cerevisiae are contained in approximately 125 base pairs consisting of three sequence elements: two short sequences of 8 and 25 base pairs separated by 78 to 86 base pairs of very AT-rich DNA. The short centromere sequences defined in S. cerevisiae, however, do not appear to reflect the situation in other eukaryotes. More recent studies have defined the centromeres of the fission yeast Schizosaccharomyces pombe by a similar functional approach. Although S. cereuisiae and S. pombe are both yeasts, they appear to be as divergent from each other as either is from humans and are quite different in many aspects of their cell biology. These two yeast species thus provide complementary models for simple and easily studied eukaryotic cells. The centromeres of S. pombe span 50 to 100 kb of DNA; they are approximately a thousand times larger than those of S. cerevisiae. They consist of a centrai core of 7000 base pairs of single-copy DNA flanked by tandem repeats of three sets of repetitive sequences. Not only the central core but also the flanking repeated sequences are required for centromere function, so the centromeres of S.pombe appear to be considerably more complex than those of S. cerevisiae. The centromeres of higher eukaryotes have not yet been defined by functional studies but mammalian centromeres are characterized by extensive regions of heterochromatin consisting of highly repetitive satellite DNA sequences. In humans and other primates, the primary centromeric sequence is a satellite DNA, as well as the potential activities of other repetitive sequences in mammalian centromeres remains to be established. Consistent with their large size, mammalian centromeres form large kinetochores that bond 30 to 40 microtubules, whereas only single microtubules bind to the centromeres of S.cerevisiae. Depending upon the presence or absence and number of centromeres, the chromosomes are classified as follows:

(a) Acrocentric chromosomes Acrocentric chromosomes are those lacking centromere. Such chromosomes are of rare occurrence. These may be developed due to chromosomal aberration specially in deletion or deficiency. Chromosomes without centromere do not attach with the spindle fibres and are lost before the division is completed. (b) Centric chromosomes A B In this, the chromosomes are provided with one or more centromeres. The centromere may occupy any position along the chromosome. On the basis of the number of centromeres, 10 A centric chromosomes are further categorized into: Monocentric: It is most common type of centric B ----~--~~~~~chromosomes. In this, each chromosome has only one A I A t B centromere. The shape of a chromosome is best studied in --~~~OGOI====== monocentric condition. A I 'I A A • Bi or dicentric: In bi or dicentric chromosome, two centromeres are present per chromosome, whatever their position ~ may be along the chromosomes, but when both the centromeres Fig. 3.12. Formation of iso and te/o-centric are located on either ends of the chromosomes, they are said to chromosome. be the telocentric chromosomes. Polycentric: When more than two centromeres are present on the same chromosome, they are said to be polycentriC chromosomes. In such chromosomes, all the centromeres are not located in one position but lie along the length of chromosomes. This is the diffuse nature of the cel)i:r9mere. Polycentric chromosomes are observed in,hemipterans, homopterans, AscariS and many plants. '

____A ____~:!:>-~B~-6

(v) Secondary constriction Besides primary constriction, the chromosome may develop one or more secondary constrictions which differ from the former by the absence of marked angular deviation of the chromosomal segments. The secondary constrictions have been termed as olisthero zones by Ressende in 1945. These constrictions

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are constant in their position, so that these can be used as useful markers. There are two types of secondary constrictions-secondary constriction I and secondary constriction II. Secondary constriction I is associated with the formation of nucleolus and this specialized region is referred to as nucleolar organizer. The chromatin of this region is uncoiled and penetrates the nucleolus and that is why this part is stained lightly. The nucleolus is developed in the post-mitotic reconstriction phase. The nucleolar organizer represents about 0.3% of the total amount of nuclear DNA. It contains repeated sequences of DNA bases and codes for RNA. The nucleolus diminishes in volume during prophase and disappears in the cytoplasm. Spirogyra is the exception, and in this the nucleolus maintains its identity and stainability even during cell division. Secondary constriction II is the satellite or trabant. It remains attached to the rest of the body by a thread of chromatin. In human beings, this constriction is found on the long arm of chromosomes numbering 1, 10, 13, 16 and Y. Chromosomes having satellites are also called SAT chromosomes. The shape and size of the satellite remain constant. Steward and Bomford (1949) reported that the secondary constriction has same diameter as the remainder chromosome. Resinde (1940) assumed that the constriction possesses a spiral structure similar to the rest of the chromosome, but the chromatin contained therein exhibits negative heteropycnosis.

(vi) Telomeres When the chromosome breaks down into pieces, they reunite in such a way that the Original terminal ends are left behind as they do not fuse. The chromosomal ends or extremites are known as the telomeres. This term was contained by Muller in 1938. The telomere has a polarity that prevents other chromosomal segments to be fused with it. If the chromosomal end is broken off either spontaneously or by induction, the broken telomere is usually lost from the nucleus in subsequent cell division because it lacks centromere. The broken end of the remaining free chromosome is unstable and may unite with another broken end of the chromosome in the vicinity. However, the broken end or telomere will not unite with the normal end of the chromosome. The sequences at the ends of eukaryotic chromosomes, called telomeres, play critical roles in chromosome replication and maintenance. Telomeres were initially recognized as distinct structures because broken chromosomes were highly unstable in eukaryotic cells, implying that specific sequences are required at normal chromosomal termini. This was subsequently demonstrated by experiments in which telomeres from the protozoan Tetrahymena were added to the ends of linear molecules of yeast plasmid DNA. The addition of these telomeric DNA sequences allowed these plasmids to replicate as linear chromosome-like molecules in yeasts, demonstrating directly that telomeres are required for the replication of linear DNA molecules. The telomere DNA sequences of a variety of eukaryotes are similar, consisting of repeats of a simplesequence DNA containing clusters of G residues on one strand. For example, the sequence of telomere repeats in humans and other mammals is AGGGTT, and the telomere repeat in Tetrahymena is GGGGTT. These sequences are repeated hundreds or thousands of times, thus spanning up to several kilobases. Telomeres playa critical role in replication of the ends of linear DNA molecules. DNA polymerase is able to extend a growing DNA chai~ but cannot initiate synthesis of a new chain at the terminus of a linear DNA molecule. Consequently, the ends of linear chromosomes cannot be replicated by the normal action of DNA polymerase. This problem has been solved by the evolution of a special mechanism, involving reverse transcriptase activity, to replicate telomeric DNA sequences. In meiotic prohase, the telomeres are often attracted to the centriole and seen to migrate to the nuclear membrane near the centrosome or centriole. This behaviour results in what is often described as a bouquet stage. HETEROCHROMATIN AND EUCHROMATIN

In most species of animals and plants, it is seen that certain segments of chromosomes, at various stages of mitosis and meiosis are more or less condensed than the rest of the karyotype. This refers to the heteropycnosis. The interphase chromosomes cannot be distinguished individually, Heteropycnosis may

Chromosomes

39

be positive (over condensation) or negative (under condensation). The chromatin material which remains in a highly condensed stage throughout the divisional cycle is called heterochromatin and that the remains less condensed and becomes extended or less coiled, is termed euchromatin. The term heterochromatin was originally introduced by Heitz in 1928. Heterochromatin is associated with tight folding and coiling of the chromosome fibre and that is why it stains deeply. The heterochromatin is considered as genetically c: inert substance but it controls the metabolism of 0 .0, c: the chromosome, biosynthesis of the nucleic acic !!! 0 chromomere .0, .2 and the energy metabolism. The DNA oj !!! iii (.) heterochromatin is genetically inert and does not chromonema E e ~ .s::: transcribe in RNA for protein synthesis. Y E (.) :::l e Q) chromosome of Drosophila melanogaster is quite .s::: (.) :::l chromatids a long heterochromatic element and is relatively Q) inert genetically. Euchromatin stains less deeply Euchromatin forms the major portion of the chromosomes. It is rich in DNA and is considered as genetically active substance. Its DNA synthesizes mRNA during interphase. That is, much euchromatin and a large nucleolus are observed in synthetically active cells. Chromatin can be transformed from centromere heterochromatin to euchromatin and vice versa. Fig. 3.13. Structure of a typical chromosome. A striking example of such a change is seen when human lymphocytes are activated in vitro. Normally, the nucleus of a peripheral blood lymphocyte contains a high proportion of heterochromatin. When stimulated with phytohaemagglutinin, however, the nucleus enlarges, most of the heterochromatin is converted to euchromatin and the nucleolus increases in size. Concomitantly, the nucleus begins to synthesize much RNA. Two main types of heterochromatin are sometimes distinguished, constitutive heterochromatin and facultative heterochromatin.

Constitutive heterochromatin It is a permanantly condensed, genetically conservative late replicating material found in all cell types all the time. It tends to have a constant position on the homologous chromosomes. Constitutive heterochromatin is highly repetitive and becomes inactive during protein synthesis. This heterochromatin is found in specific regions of the chromosomes such as: (iJ Centromere The most common site for constitutive heterochromatin is the kinetochore found on all the chromosomes in most species studied so far. Centromeric heterochromatin remains condensed in interphase as well in mitosis, replicates late in the S phase and contains highly repetitive sequences of DNA. This DNA is also known as satellite DNA. The centrometric heterochromatin apparently plays a role in the movement of the chromosome along with spindle. It provides points of attachment with the nuclear membrane, and also gives strength to centromere. Y chromosome is entirely heterochromatic but in the autosome taken from the salivary gland of Drosophila, heterochromatin occurs in the form of large bands at the centromere. The chromatin

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40 around the centromere is compact and resistant to destaining. This is also known as alpha constitutive heterochromatin. Beta constitutive heterochromatin is less resistant to destaining and is relatively diffused forming the rest of the centromeric heterochromatin. Beta chromatin has a capacity to impart the variability of phenotypic expression to the euchromatic genes (Hannah 1951).

intercalary -~~~~ heterochromatin

(ii) Telomeres

Telomeres are the terminal ends of a chromosome. It has been reported that the telomeres contain constitutive heterochromatin that helps in protecting the inner active genes. The chromatin also maintains the individual integrity of each chromosome. (iii) Nucleolar organizer Nucleolar organizer is the secondary constriction found on the specific chromosomes. It consists of euchromatin and appears as destained gaps which are seen associated with the nucleolus at telophase. It contains the genes for the synthesis of RNA.

(iv) Intercalary heterochromatin

secondary constriction I I

echromatin

primary constriction

It is found in the form of bands between the euchromatic segments. The bands are clearly visible in the polytene chromosomes of Drosophila melanogaster. The intercalary heterochromatin bands in identifying the mammalian chromosomes. primary

Facultative Heterochromatin constriction Facultative heterochromatin contains active genes, but may become condensed and genetically inactive in response to physiological and constitutive developmental conditions, and it may revert to a euchromatic state at heterochromatin certain times. Facultative heterochromatin becomes the sex chromatin body early in embryogenesis. A well known instance of this heterochromatin FIg. 3.14. Metacentrlc chromosome involves the mammalian X-chromosome. There is only one Xwith constitutIVe hetero· chromosome in males and this chromosome is euchromatic along most chromatin of its length. Females have two X-chromosomes, but only one of these remains euchromatic during the life of each cell, while the second X-chromosome becomes condensed heterochromatic very early during embryonic development. The heterochromatic X-chromosome is seen as a dense blob in the nucleus of non-dividing cells or interphase nucleus. This blob is called a Barr body. Females have one Barr body nucleolus per nucleus, this being the second X-chromosome. Males have no organizer Barr body, since they have only X chromosome, which remains euchromatic. Facultative heterochromatin comprises about 2.5% of the genome. It also appears in the neutrophils in the form of Drumstick , body attached to one of the lobes of the nucleus. !'-- centromere CHROMOSOME BANDING

The chromosomes have a unique staining pattern allowing their specific identification. It is based on the differential staining techniques of the chromosomes, resulting in stained and unstained areas in the form of bands. These methods are now called chromosome banding techniques. Banding patterns can also be obtained by a diversity of treatments, including heating, digestion with acid, alkali or salt and .. treatment with a variety of compounds. FIg, 3.15, Nuclear orgamzer

Chromosomes

41

3

2

6

5

4

p.

•,

•,

2

7

8

10

9

II

I;

pi

q

m

.? 13

19

14

20

§i

. :

15

21

Iv

12

16

22

17

y

16

x

Fig. 3.16. Diagram showing the banding pattern of human chromosomes.

Methods for chromosomal banding have been given under the following heads: (i) C-Banding One of the first chromosome banding techniques was discovered by Pardue and Gall during the development of the in situ hybridization procedure. They observed the unique staining pattern after the

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42

heat denaturation of chromosomes followed by their staining with the Giemsa staining method. In this, the centromeric regions of mitotic chromosomes takes up stain. These regions are composed of highly repetitive constitutive heterochromatin (the basis for naming it C-banding). This staining pattern is referred to as Cbanding. The C bands apparently develop because much more stainable chromatin remain in this region after preliminary treatment of extract DNA and protein. Since there is no little DNA and protein remaining after extraction elsewhere along the chromosome, very little stain becomes absorbed ~md most of the chromosome looks pale. (ii) Q-Banding A group of Swedish researchers, led by Tobjorn Caspersson, developed another chromosome banding technique which provided even greater staining differentiation of metaphase chromosomes. When the chromosomes are treated with the flurochrome quinacrine mustared, bands with differential brightness are produced. These bands are rich in adenine and thymine bases. The guanine-cytosine regions remain unstained. (iii) G-Banding Another banding technique has been developed by Tau-chiuh Hsue and Frances E. Arrighi. The bands produced by this method are called G-bands. These are stained with Giemsa and other non-fluorescent stains. Another technique results in the reverse G-band staining pattern, called R-band pattern. These are reciprocal of Q bands and G-bands. G-bands appear in the areas which are S-rich proteins. G-banding is the most useful chromosome.

(iv) R-Banding These bands appear when chromosomes are incubated in a buffer at high temperature and stained with Giemsa stain. R-bands correspond to the region on chromosomes having sulphur free proteins. These are reciprocal of G-bands. The variety of staining reactions under different conditions reflects the heterogeneity and complexity of chromosome composition. The variations in nucleic acid composition that exit along the longitudinal axis of any given chromosome, with its altemating section of heterochromatin and euchromatin, suggest functional divesity. Heterochromatin is considerably more folded and collapsed, one fold against another, resulting in a condensed appearance and deep stainability because there is so much more material per unit area to absorb stains. The mechanism that leads to different degrees of folding under intensive study is present. ULTRA STRUCTURE OF CHROMOSOME

As already stated, chromosomes are visible only during cell division. The chromosomes at metaphase and anaphase do not show any interval structure under the microscope beyond the centromere, constrictions, satellite etc. But by special treatment, the chromosome was observed to comprise a coiled filament chromosome 1x4000 AO chromatid

2x2000 AO

1/2chromatid 4x4000 AO

114 chromatid

8x500Ao

1/8 chromatid

16x200 -250Ao

111{> microfibril chromatid subfibral DNA double helfix

32x100 -125 AO 64x20 -40 AO

Fig. 3.17. Scheme of chromosome according to the multiple strand hypotheSIS.

43

Chromosomes

lengthwise. There exists a lot of corlfusion regarding the number and exact nature of these fibrils. Various models, have been proposed to explain the ultra structure of chromosomes.

40A

1. Multi-stranded Model According to this hypothesis, each chromosome is composed of several fibrils which are made up of DNA and DNA protein molecules. There are two chromatids forming a chromosome. Each chromatid is further composed of two elementary chromosome chromonemal fibres. In all, there are four chromonemal fibres protein fibril in a chromosome. In mosquito, prophase c~romosomes seem to contain at least 16 chromonemata. In leptotene chromosome of Tradescantia are found 8 fibrils in each chromatid. The salivary gland chromosomes of dipteran larva contain 1000 to nucleoprotein 200A fibril 100A 1600 chromonemal fibrils. Each fibril is supposed to contain Fig. 3.18. Multistranded model. double helix of DNA molecule to which protein is found associated. The number of DNA helices in each fibril varies from species to species. Steffensen (1952) stated that there may be as many as 64 double helices of DNA arranged in a parallel way. These DNA helices are twisted like the strands of a rope. In the year 1961, Ris proposed a model, the DNA molecule which is about 20A thick, becomes associated with histone protein, forming a fibril of nucleoprotein of about 40A thickness. The further association of every two such nucleoprotein fibril results in the formation of elementary chromosome fibril of about 100A thickness. There are about 16-32 elementary chromosome fibrils which are arranged in pairs and form one chromatid. Several studies failed to prove the multistranded view, but presented significant evidence in favour of unineme model or single stranded model.

2. Unineme Model According to the Unineme model, the chromosome is one long coiled or folded DNA molecule which is associated with histone protein or nucleoprotein. In 1957, Taylor proposed a centipede model to explain the unineme nature of the chromosome. According to his model, there is a long protein backbone from which DNA coilings branch off just like the legs of a centipede. But in 1965, Taylor modified his mcxIei based on the Freese' madel. According to this model, there are two proteinous backbones, instead of one. The DNA chains stretch between the back bones like the steps of a ladder. In 1965, Dl!lpraw proposed a folded fibre model in favour of the Unineme model. According to this model, a chromosome

protein

Fig. 3.19. Centipede model 0/ Taylor.

Fig. 3.20. Freese-Taylor model

44

•

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consists of a single long chain of DNA which is first wrapped in the nucleoprotein and then it is coiled to form / .,.,\ proteins 250-300A thick fibre . The fibre then (:t('r'~;" ~/ DNA becomes folded back longitudinally and transversely and thus interwined and forms the body of chromatid. This has been confirmed by Mc Devmott (1968), Lampert (1969), and Fedric (1969). The DNA protein molecule of both the chromatids of the chromosome remain held at the centromere. The folded fibre model applies to both the interphase and the metaphase chromosomes. The folded ~ : . .. .. . fibre model has been proved by various .•.•..:.:. .. ... . cytochemical analysis and electron :' ..•'...... • microscopic observation. '. 3. Nucleosome Model It is well known that the eukaryotic Fig. 3.21. Folded Model of Dupraw. chromosomes are composed of DNA and protein. Approximately 13-20 percent of mammalian chromosome is DNA, and the remainder consists of proteins and a varying but small amount of RNA. Histones ate the common proteins associated with DNA. 'In 1973, Woodcock observed chromatin fibre under the electron microscope. In 1974, Roger Korenberg proposed a model of the chromatin fibre as a fle.xibly jointed chain, resembling beads on a string forming a nuinber of repeating units. In 1975, Oudest and his colleagues suggested the name nuc1eosomes or v-lxx1ies for the repeating unit. The nucleosome appears to be the basic structure of chromatin. a.nucleosome unit

DNA

-200AO-200 basp

pairs of DNA

Fig. 3.22. A mOdel of a chromatin fibre with histone HI removed. The nucleosomes are constituted of two molecules each of histones H2o, H2b, H3 and H4 plus the DNA.

Each nucleosome consists of a spiral of DNA wrapped around an oetomer of histone molecules forming the core particle which is called the platysome (J. T. Finch, 1977). DNA makes about 13/. turns around an octomer (core DNA) yielding a flattened wedge shape of the dimensions 57A x 110A. Each turn of DNA is 80-100 nucleotide base pairs long, making the total nucleosome approximately 140-200 base pair long. The number of nucleotide pairs in the nucleosomes may vary from 154 (Aspergillus) to 241 (sperm of sea urchin). Each nucleosome is hooked to the next with 15 to 100 nucleotide pair of linker DNA or internuc1eosomal DNA that can be folded and packed into a tiny volume, very much like a flexibly jointed chain. A nucleosome has a diameter of almost 200A. One of the DNA enzyme makes single-strand nicks about every tenth base pair in the DNA associated with the nucleosomes.

45

Chromosomes

Histones are small proteins that contain between 100 and 200 amino acids and differ from most other proteins and in that form, 20 to 30 percent of the amino acids are lysine and arginine, both of which have a positive charge. The positive charges enable histone molecules to bind to DNA primariy by electrostatic attraction to the negatively charged phosphate

Linker DNA

Histones H2A, H2B. H3, H4

T

55

A

1 Core DNA

110 A

groups in the sugar phosphate Fig. 3.23. Diagram of a nucleosome core particle. The DNA molecule is wound one backbone of DNA Placing and three·fourths turns around a histone octomer. If Hl were present, it chromatin in a solution with would bind to the octomer surface and to the linkers, causing the linkers to a high salt concentration (2 cross. molar NaCl) to eliminate the electrostatic attraction causes the histones to dissociate from the DNA Histones also bind tightly to each other, both DNA-histone and histone-histone binding are important for chromatin structure. The octomer of proteins consists of two molecules each of four different histone (tetramers). These histones are H 2A, H 2B, H3 and H 4. The flat tetramers of histone are stacked one on top of the other. These histones have finger like projections around DNA These projections contain NH terminal regions. Fifth class of histones is termed as HI which seems to playa different role in finishing the packaging of the nucl~wsQme . This histone protein holds the two ends of DNA in a nucleosome, but it does not form the integral part of a nucleosome.

Table 3.2. Categories of the properties of histone protein Histone type

Lysine arginine content

HI HzA H2B H3 H4

lysine rich Slightly lysine-rich Slightly lysine-rich Arginine-rich Arginine-rich

Molecular weight (daltons)

21,000 14,500 13,700 15,300 11,300

ISOCHROMOSOMES

A

A

B C

B C

A

B C

F G

D

E

F : G

G

F E D D

Another meiotic error that leads to unbalanced genetic material is the formation of an isochromosome, or a chromosome with identical arms. This occurs when, during division, the centromeres part in the wrong plane, and in this, the depiction is horizontal rather than vertical. RING 'CHROMOSOMES

D E

E F G

Fig. 3.24. Isochromosorrles

'lOve

Chromosomes shaped like rings form in lout of 25,000 identical arms. Th~y form when chromatids divide conceptions. They can involve any chromosome. Ring chromosomes along the wrong plane {in may arise when telomeres are lost, leaving sticky ends that tend to close this depiction, horizontally up. Exposure to radiation can form rings. rather than vertically. Ring chromosomes can produce symptoms when they add genetic material. For example, a small ring chromosome of DNA from chromosome 22 causes cat eye syndrome. Affected children have vertical

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pupils, are mentally retarded, have heart and urinary tract anomalies and have skin growing over the anus. They have 47 chromosomes-the normal two chromosome 22 and a ring.

GIANT CHROMOSOMES The giant chromosomes are strictly confined to certain type of cells of the organism. These chromosomes attain their largest size in the spherical nuclei of their respective czlls. There are two types of giant chromosomes: 1. Polytene Chromosomes left arm of Balbiani in 1881 was the first to observe salivary gland chromosomes in the salivary glands of chironomous larva and that is why these are known as . salivary gland chromosomes. The term polytene chromosomes is more preferable than the general salivary gland chromosomes and was given by Koller due to the presence of many chrornonemal fibrils in them. Such chromoSomes also exist frequently in the other tissues, such as the living cells of the gut, Malpighian tubules, muscle cells, fat cells and in several Fig. 3.25. Polytene chromosome. species of protozoans and plants. The plant species with large polytene chromosomes are Phaseolus, Rhinanthus and Tropaeolum. In plants they occur in endosperm cells, antipodial cells of embryo sac and suspensor cells of the embryo. Size The salivary gland chromosomes are the largest chromosomes known that are readily available for cytogenetical study. In Drosophila melanogaster, these chromosomes, observed in the late larval (3rd instar) stages are over 100 times the length of the somatic metaphase chromosomes, which measure only about 7.5 ~. According to Bridges (1938), these chromosomes.measure upto 1,180~ or even upto 2000~ in Drosophila melanogaster. In 1922, Beeman reported a pair of synapsed chromosomes attaining the size of 270~ in length and about 20~-in diameter. Those of a related genus, Rhyncosciara, are even larger and may reach even greater dimensions as a result of pathological disturbances. Structure The structure of the salivary gland chromosome is of great cytogenetic interest. The salivary gland chromosomes show somatic pairing at interphase because of their ml,1lti-stranded giant nature. Another characteristic feature of the Polytene chromosomes lies in their number. The number of these chromosomes in the salivary gland cells always appear to be half of the normal somatic cells. Along the ltmgth of the chromosome, there is a series of dark bands alternating with other clear zones called interbands. The dark bands are heterochromatic in nature and stain intensely and are Feulgen positive. Further more, they absorb ultraviolet light at 600A. In Drosophila, about 5,000 bands have been observed on the four chromosomes of this species. Burke Judd and colleagues have reported about 1000 bands only on the 'X' chromosome of Drosophila. These bands may be considered as discs, since they occupy the whole diameter of the chromosome. The banding pattern is distinctive for each homologous pair of chromosomes in any given species. Individual bands are sometimes called chromomeres, a generalized term which describes

Chromosomes

.

47

lateral condensatiorls of material along the axis of the chromosome: They ~re of varying size. The longer bands have more complicated structure. They qften form 9uplets, two bands located next to each other and of identical thickness and shape. The interbands are of fibrillar aspect, do not stain with basic dyes, are Feulgen negative and absorb very little ultra violet light. Furthermore, they present greater elasticity than'the region of the bands. The .' constancy in situation and dist6bution of the A discs or bands in two homologous (paired) B chromosomes is notable. In the case of Drosophila melanogaster, the chromosomes 0f each polytene nucleus, when flattened, appear as five long strands and one quite short, attached to a central mass known as the ehromocenter; to which the single large nucleolus is attached. Of the six • strands, the short one represents the IV ' chromosome, and the larger one represents . the X chromosome, while the remaining four are the limbs of V shaped II and III c chromo~omes,the Vth being quite small and 0 almost completely included in chromocenter., Fig, 3.26, Puffing in polytene chr~'!I~some, In salivary gland nuclei from female larVae, the strand representing the 'X' is double like the others, while in nuclei from, male indiVidual, it is single. Y chromosome is 'also found within the chromocenter and is therefore seen as a separate strand. • . ' Chromocenter occurs in all species of Drosophila and its size depend upon whether the prOximal heterochromatic segments are extensive or not. In some groups of Diptera, of the families 'Sciaridae' and 'Chromonidal' , chromocenter is absent. "

Puffs, Balbianic rings and Gene activity The m~st important morphological peculiarity of salivary gland chromosome is the presence of bands and interbands. For many years, it was postulated that each band on the Drosophila chromosomes represented one gene. Geneticsts counted approximately'SOOO bands on the four chromosomes of this species, and this number seemed to agree quite 'well with the estimated number of genes in Drosophila. liowever, Brewer, Pauan, Beerman n, Me ehelke and others have found that at certain stages of larval development, some specific bands of the polytene chromosome show an enlargement. These enlarged bands are considered as ultimate Units of heredity-the genes at work. These active genes take the form of puffs scattered here and there along the salivary gland chromosomes: Actually"the puffs are the expanded expression of the compacte9 chromatin regions which expand and take part in transcription. It has been observed that in the interband regions of polytene chromosome in Drosophila melanogaster, Z-DNA which is a normal cOl'Tlponent of chtomos9mes is present. The restriction of Z-DNA to interband regions prompts several interesting seculations. The presence of Z-DNA in these regions, traditionally thought to be .devoid of genes, may be rel?ted to the control of chromosomal replication and/or gene activity. Beermann and U. Clever (1964) have found that the pUffs produce RNA. The RNA of different puffs differ from each other in chemical composition. The fine structure of the individual1:iand can differ with respect to puffs that are in one location on a chromosome. in one tissue and in another location on the same chromosome at another time or in another ,

~, l

.'

48

Biotechnology

tissue. The band regions are developed by a mechanism which interband prevents transcription of these regions at all time. Each band region is merely a compact area of DNA which loosen up to form a puff at the ti~e of high genetic activity so 'that transcription is possible. The localized modifications in chromosome structure of various dipterans had been noted many years earlier but their possible significance was overlooked. The coherence of chromosome filaments is loosened at the puffed regions. The Joose ring always starts at a .single band. In small puffs, a particular band simply loses its sharp contour and presents a djffusea, out of focus appearance in the microscope. At other loci or at other times, a band may look as though it qad "exposed" into a large ring of loops around the chromosomes. Such nut like structures are called Balbiani rings, after E.G. Balbiani, who first described them in 1881. Puffing is thought to be due to the unfolding or uncoiling of individual chromosomal fibres in a band. Beermann (1952) postulated a particular sequence of puffs representing a corresponding pattern of gene activity. Of differential gene activation which does, in fact occur, one could predict that gene in a specific type of cell will regularly puff whereas the same gene in other tissue will not puff. It has been reported that the two different species of Chironomous produce secretion of a different nature. A gene of Fig. 3.27. A highly schematic uiew of the the same nature has been described in a group of four cells of process of puff formation in a salivary glands of Chironomous. Chironomous pallidiuittatus polytene chromosome .. produces a granular secretion. The closely related species Chironomous tentatus gives off a clear, nongranular secretion from the same cells. In hybrids of these two species, this natUre follows simple mendelian laws of heredity. Beermann and Cleuer (1964) were able to localize the difference in a group of fewer than 10 bands in one of the chromosomes of Chironomous. This chromosome is designated as N chromosome. The granule producing cells of Chironomous pallidiuittatus have a puff associated with this group of bands, a puff that is entirely absent at the corresponding loci of chromosome N in Chironomous ten tatus. In hybrids, the puff appears only on the chromosome coming from Chironomous pallidiuittatus parent, the hybrid produces a far smaller number of granules than the parent. Moreover, the size of the puff is positively co-related with the number of granules. This reveals quite dearly the association between the puff and a cellular (specific) prOduct. This study demonstrates a sjJecific relationship between puff-gene and the specific function of a cell. Regulation of puffing Ulrich Cleuer in 1960 showed that the ecdysone (moulting hormone) was directly involved in puff formation in Chironomous. Toe usual puffing was absent when the ecdysone was withheld from developing cells and the affected cells did not differentiate. When 'ecdysone was injected into such cells, puffing took place in exactly the same way as during normal moulting in larvae about to pupate. . The mechanism of puffing has been explained by the hypotheses: (i) The primary event in~olves detachment of associated proteins from DNA in the chromatin fibre, making the exposed DNA accessible for transcription. Puffing is, therefore, the first event in gene , action leading to transcription and ' (ii) Puffing belt may be the result of transcriptional activity of chromosomal DNA at a particular band . site. .

Polytene theory '" To explain the enormous increase in diameter of the chromosome, Painter proposed the polytene

Chromosomes

49

theory. He believed that the increase in diameter is due to an enlargement probably by a continuing duplication of the individual chromomeres but without a variable separation of individual chromonemata. It makes the giant chromosome a bundle of fibrils. This process is called endoduplication, endopolyploidy or endomitosis. In this, the nuclear membrane does not rupture. Single ordinary somatic chromosome contains four chromonemal fibrils. According to Painter, the multi-stranded chromosome may contain as many as 1024 chromonemal fibrils, while Beermann (1952) estimated the degree of polytene to be as high as 16,000 times. 2. Lampbrush Chromosome

Germ line chromosome with laterally projecting loops is matrix (RNA and protem) termed as Lampbrush chromoB somes. It was given this name because it is similar in appearance to the brushes used to clean lamp chimneys in centuries past. It was first observed in 1882 by Flemming. The name 'lampbrush' was given by Ruckert in 1892. These are found in the oocytic nuclei of vertebrates (sharks, amphibians, reptiles and birds) as well as in invertebrates (Sagitta, Sepia, Ehinaster and c A several species of insects). Grun Fig. 3.28. Diagramatic representation of lampbrush chromosome. (1958) has observed the lampbrush chromosomes in plants. However, most experimental work has been done with material taken from amphibian oocytes. Lampbrush chromosomes, at maximum development, are even larger than the largest salivary gland chromosomes, but unlike the later. They are no greater in number of strands than more typical chromosomes. The largest lampbrush chromosomes so far known are present in Urodela (amphibian), the animals with large DNA values. Organization At the stage of female meiotic prophase, the homologues are seen as synapsed pairs held together by chiasmata. Lampbrush chromosomes are generally described as having a central chromosomal axis from which project a series of lateral loops. Chromosomal axis The chromosomal axis contains two bivalent chromosomes each with two chromatids, so four chromatids in all are the present, and these chromatids further give rise to lateral loops. They are extensible and elastic, and as a chromosome is stretched, pair of loops become widely spaced along the axis but the bases of the loops don't open out appreciably. Biochemically, the chromosomal axis is composed of DNA and protein. The axis varies from 30-50A in diameter and is highly flexible. Centromeres The centromeres of lampbrush chromosomes are smooth, round and Feulgen positive, bearing no loops. In several species of Urodela, centromeres are conspicuous chromosome 'landmarks' as they are flat;tked by 'axial bars' formed by amalgamation of neighbOUring chromomeres.

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Biotechnology

Table 3.3. Difference between Polyteny and Polyploidy Polyteny 1. Homologous chromosomes undergo somatic pairing. 2. The products of polyteny remain attached to one another. 3. Polyteny produces hundreds of copies of the same chromosome. 4. Polytene chromosomes are visible in the interphase nucleus. 5. Polytene cells cannot multiply. They are destined to die.

Polyploidy 1. Pairing of homologous chromosomes does not occur in somatic cells. 2. Similar chromosomes remain separate from one another. 3. Polyploidy does not increase the number of chromosomes beyond 6-10 times. 4. Chromosomes are not visible in the interphase nucleus. 5. Polyploid cells behave like normal cells.

Telomeres Telomeres, which occupy ends of the chromosomes, have dual structures, each consisting of a small Feulgen-positive part embedded as, or closely applied to the surface of a smooth, round and Feulgennegative granules.

Chromomeres The chromosomal axis of lampbrush chromosome has a row of granules or chromomeres. These chromomeres are found in pairs. The chromomeres are connected by inter-chromomere fibres. In a bivalent chromosome, about 150-200 paired granules are present, each granule or chromomere may support a pair of lateral loops, which give the chromosome its distinctive appearance.

Lateral Loops Lateral loops project from the overwhelming majority of chromomeres. All are Feulgen negative. These loops give the chromosome its distinctive appearance. One to nine loops may arise from a single chromomere, each with an average size of 9 .5f..l in frog to 200f..l in newt. The number of pairs of loops gradually increases in meiosis till it reaches maximum in diplotene. As meiosis proceeds further, number of loops gradually decreases and the loops ultimately disappear due to disintegration rather than reabsorption back into the chromomeres. Each loop is thought to be composed of a loop . \ .' homolog 1 - homolog 2 . axis which is covered with matrix. The loop axis which is RNA being main axis of uncoiled ~NA produced homo logs double helix about 30-50A in thickness, is composed of one double helix of DNA whereas the matrix has protein combined Fig. 3.29. Lateral loop. with RNA. For deSCriptive convenience the matrixaccumulating loops may be classified into granular loops, the fusing loops, the lumpy loops, the giant fusing loops etc. Most loops are elastic and conform to one pattern and are termed normal loops. Large normal loops show signs of possessing an axis with many fine fibres projecting radially from it. (Callan, 1955). Each normal loop is asymmetric in the sense that one of its insertion in the chromosome is the base of fibres: as one follows round the loop from its base end, the fibres become progressively longer. These fibres are the RNA fibrils and make the loop markedly thicker on one side. Callan and Lloyd in 1960, postulated the master and slave hypothesis. According to these, each loop consists not of one gene, but of a number of duplicate copies containing repeating DNA sequences. There is a master copy of a particular gene in a chromomere which resembles to its identical slave gene copy. Callan further suggested that only slave copies take part in RNA synthesis.

51

Chromosomes

Functional significance There is a general agreement that these chromosomes are universally connected with synthetic activity in growing oocyte. Their morphology, the presence of RNA in the loops, the continuous production of 'nucleoli', all suggest that they are the states of intensive nucleoprotein metabolism. The considerable development, just at the time when synthesis of yolk is at its peak, suggests that they might playa role in the formation of deutoplasmic reserves. Gall and Callan (1962) found that although the giant granular loops of T. cristatus, chromosome XII synthesize RNA only in a restricted region, they synthesize protein throughout their lengths. Human Karyotype

The human autosomes are numbered 1 to 22. The sex chromosomes XX and XY are not numbered. In Drosophila, the sex chromosomes are numbered 1. The human autosomes were numbered and distinguished morphologically into seven groups, A to G, on the basis of relative size and position of ceTltromere : 1. Group A. It includes the chromosomes 1-3. These are largest in size and have median centromere and equal arms. 2. Group B. It includes the chromosomes 4 and 5. These are next largest in size and have sub-median centromere and unequal arms. 3. Group C. It includes the chromosomes 6-12. These are medium-sized and have sub-median centromere and unequal arms. 4. Group D. It includes the chromosomes 13-15. These are shorter than the chromosomes of group C, have centromere near the end and very unequal arms. They are sat chromosomes as they bear satellites. 5. Group E. It includes the chromosomes 16-18. These are short-sized and have median or sub-median centromere and equal or unequal arms. 6. Group F. It includes the chromosomes 19 and 20. These are short-sized and have median centromere and equal arms. 7. Group G. It includes the chromosomes 21 and 22. These are smallest in size, and have median centromere and equal arms. They are also sat chromosomes as they carry satellites. The X chromosome is placed in group C in view of its large size and sub-median centromere. The Y chromosome is placed in group G as it is very short and has terminal centromere. Its length may vary. CHROMOSOMES IN FiSHES

Chromosome number, along with conventional morphological criteria data from paleontology, behavioural patterns, ecology and genetic experiments provides a further tool for deciphering phylogeny in fishes. The latest summaries show that the diploid number varies from 18 to 104, but workers commonly disagree on the precise number of a given species. Chromosome numbers determined from mitotic chromosomal number that show so well, during stages of the egg cleavage, may differ radically within a single species, from those determined by studying male germ cells during spermatogenesis. The la.tter are said to be more accurate. It is to be expected, that is a close correlation by studying male germ cells during spermatogenesis. The latter are said to be more accurate. It is to be expected that a close correlation exists between chromosome morphology and structural organization, since evolutionary changes are initiated by changes in chromosomes. Surveys of chromosome number are less complete for fishes than for other groups of animals. It must be remembered, however, that fishes constitute a greater number and diversity of species than all other vertebrate~ combined. Serious work on fish chromosomes dates from the 1930s and this field of enquiry has received much of its importance from papers having appeared in the past 15 years. Approximately a hundred and thirty of an estimated sixteen to eighteen thousand species of living fishes have been studied by chromosome Cytologists. Growing interest in chromosome number and morphology in these animals promises to yield important contributions to evolutionary theory as well as to fish phylogeny.

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52

There is a general tendency for the chromosomes of related species and genera to show similarity in number and shape, but our knowledge of the subject is so incomplete that definite conclusions linking chromosomes and classification are premature at this time.

Table 3.4. Difference between Prokaryotic and Eukaryotic Chromosomes Prokaryotic Chromosomes

Eukaryotic Chromosomes

1. There is a single chromosome in one cell. 2. Chromosome is shorter, simpler and codes for far fewer proteins. It is called prochromosome. 3. Chromosome is not enclosed by a nu~lear envelope. 4. It lies in direct contact with the cytoplasm.

5. Chromosome is joined to the mesosome of plasma membrane. 6. Chromosomal DNA is circular in form. 7. DNA is not associated with proteins. 8. DNA replication starts at one point (origin).

9. Replication of chromosome goes on almost continuously. 10. Certain genes are 'leaky', i.e. transcribe some RNA even when they are turned off.

1. There are 2 to many chromosomes in one cell. 2. Chromosomes are longer, complex and code for far more proteins.

3. Chromosomes are enclosed by a nuclear envelope.

4. Chromosomes are separated from the cytoplasm by the nuclear envelope. 5. Chromosomes are far away from the plasma membrane. 6. Nuclear DNA is linear in form. 7. DNA is associated with proteins. 8. DNA replication begins at many points (origins). 9. Replication of chromosomes occurs only in the S phase of the cell cycle. 10. A technique (methylation) checks leakiness of the genes.

ARTIFICIAL CHROMOSOME

The research on circular and linear plasmids in yeast has provided all of the basic components of an artificial chromosome. The telomere sequences from yeast cells or from the protozoan Tetrahymena can be combined with yeast centromere sequences; to these are added DNA with selectable yeast genes and enough DNA from any source to make a total of more than 50 kb. (Smaller DNA segments do ...JrCAiL TEL not work as well.) This artificial chromosome TEL -ARS-Ieu rfE!Jreplicates in yeast cells and segregates almost perfectly (apprbximately 1 daughter cell in 1000 to 10, 000 fails to receive an artificial chromosome). During meiosis, the two sister chromatids of the artificial chromosome appear to separate correctly to produce haploid spores. Such studies strongly support the conclusion that yeast Fig. 3.30. Functional chromosomal elements from yeast. ch;:Omosomes and probably all eukaryotic chromosomes are linear, double-stranded DNA molecules with special sequences-including centromere (CEN), telomere (TEL), and replication origins (ARS)-that ensure replication and proper segregation.

4 NUCLEIC ACID If was Friedrich Meischer (1844-1895) who anticipated an approach to the biology of cell that has come

into its own only last twenty years. He was able to isolate the nucleus from the discarded bandages of pus cells for the first time. Furthermore, he obtained a material that possessed much stronger properties than protein and also contained large amount of phosphorus. Meischer called the material as nuclein. Later on, he worked more as nuclear material was found to contain a salt of the acidic nuclein and basic substance protamine. Altmann obtained essentially protein free nuclein and introduced the term nucleic acid. Fischer identified purines and pyrimidine bases in 1880. Kossel worked on chemistry of the nucleic acid and recognized that histones and protamines are associated with nucleic acids. Kossel was awarded the Nobel Prize for demonstrating the presence of the two pyrimidines and two purines in nucleic acids. Franklin W Stahl presented first evidence that nucleic acid forms the genetic material. FA. Levine (1931) stressed that there are two types of nucleic acids, i.e. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are found in all living organisms, whether plants, animals or viruses. They are longer and heaviest macromolecules ranging in molecular weight from about 30, 000 to several millions. Their large size is due to fact that in them thousand and even million of monomeric units are joined together by polymerization to form a single giant sized macromolecule. They occur in all living cells as a major component of the nucleus and also as a component of cytoplasmic structures such as ribosomes. LOCATION OF NUCLEIC ACID

Generally the DNA is supposed to be present only in nucleus associated with the chromosomes and RNA in the cytoplasm of the cells of all the organisms. But the recent observations, however, have proved that the DNA is not only associated with the nuclear regions of the cell but it is also associated with mitochondria, chloroplast, centrioles etc. and similarly, RNA is also present in the nuclear region particularly in nucleolus. CHEMICAL BACKGROUND OF NUCLEIC ACIDS

,

Nucleotides are the basic chemical sub-units of the nucleic acids, the structural relationship of the two

being analogous to that of amino acids and protein. Each nucleotide consists of three elements: (i) a heterocyclic ring containing nitrogen and referred to as the base, (ii) a five carbon sugar or pentose and (iii) phosphoric acid. On the removal of phosphoric acid, the remainder compound of the sub-unit is called a nucleoside. Now, let us discuss every component in detail: (a)Bases The bases are of common occurrence in nucleic acids and are five in number. The bases are of the organic type and are relatively hydrophobic or water shunning. By hydrolyses and paper chromatography, two types (Purines & Pyrimidines) of bases have been recorded. 53

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The pyrimidines are 6OH OH membered rings while the purines HOCH 2 consist of fused 5 or 6 membered rings. It is customary to designate the ring positions by the number one 1 through 9 for the purines and 1 through 6 for the pyrimidines. The two principal purine bases OH OH OH H are Adenine (A) and Guanine (G). The ribose sugar deoxyribose sugar pyrimidines are Uracil (U), Cytosine Fig. 4.1. Chenllcal formula of pentose sugars. (C) and Thymine (T), which differs from uracil only in having a methyl substituent in the C s position.

(b) Pentose Sugar Levine (1909) identified pentose sugar as a main constituent of nucleic acid structure. It exists in two forms, (i) ribose and (ii) deoxyribose. In the ribonucleotide series, the sugar is ribose. In the deoxyribonucleotide, it is deoxyribose. (c) Phosphoric Acid It is trifunctional and can form upto three ester bonds. This triply esterified phosphate does not appear to occur in nucleic acids, doubly esterified phosphate does occur and in fact provides the mode of linkage of the nucleotides to form the polymeric nucleic acids. The bond formed by a doubly esterified phosphate between the sugars of the different nucleotides is called the phosphodiester bond.

Ic

I

c

c

I

I

Ic-~UI -,~ o

c

I

Fig. 4.2. The phosphodiester linkage.

DEOXYRIBONUCLEIC ACID (DNA)

It was Furberg (1952) who suggested in a speculative way that DNA molecule is formed by the coiling of a single nucleic acid chain. An equally speculative model was proposed by Pauling & Corey (1953) in which three chains twisted to form a lon~axis, with the bases projecting outwards from a central core formed by the backbone phosphate group. In 1951 Chargajf has pointed out that DNA contains equal proportion of purine and pyrimidine. Dotty (1961) has emphasized much on the physical properties of DNA. Watson & Crick first made a plausible structural model for a self-duplicating molecule which fitted the physical and chemical facts. In honour of this work, Watson, Crick and Wilkins were awarded Noble Prize in 1962, and as a result, one strand of double stranded DNA is sometimes called Watson (W) and its complement is called Crick (C). Khorana (1968), noble laureate, devised chemical method for the specific synthesis of inter-nucleotide linkage and has applied them to the synthesis of ribopoly-nucleotides. The name for DNA is derived from the type of sugar it contains, its abundance is nuclei and its hydrophilic or water loving phosphates, which make the macromolecule sour in taste or acidic. DNA is present in the cells of all plants, animals, prokaryotes and in a number of viruses. In eukaryotes, it is combined with proteins to form nuc/eoproteins. In prokaryotes, it consists of a single giant molecule of DNA about 1000 microns long, without any association proteins. DNA is also found in certain cell organelles like mit.ochondria, plastids, centrioles etc.

DNA Contents The amount of DNA has been found to be constant in a species though it varies from species to species. For example, the amount of DNA in the diploid cells of fowl has been measured as 2.5 picograms, where thersperm of the same species contains just half (1.25 pg) of the DNA of the diploid cell. (The amount of DNA is usually measured by the microunit of the weight known as the picogram. One picogram (pg) is equal to 10-12 grams.

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One picogram of DNA has the molecule of 31 cm. long, so by taking the weight of DNA, amount in the nucleus and the size of the DNA molecule can be calculated easily. By employing this method, Dupraw and Bahs (1968) have calculated thatthe human diploid cells with the 5.6 pg. DNA amount contains 174 cm. long DNA molecule. Likewise they measured that the Trillium cells with 120 pg DNA contains 37 meter long DNA molecule and the polytenic chromosomes of the Drosophila with 293 pg DNA contents having 97 meters long DNA molecule. In polyploid organism, the amount of DNA increases proportionately. In tetraploid cell, it is double the amount of DNA found in the diploid cell of the same species. Minimum fluctuations have been reported in mammalian species whereas remarkable narrations have been observed in fishes , amphibians and avian species. STRUCTURE OF

DNA

DNA is the key molecule in the living world . It is most complex and the heaviest molecule of the cell, havihg a molecular weight of 108 to 1011. With the exception of some viruses, it is the hereditary material of all organisms from bacteria and protozoa to the higher plants and animals. Stahl presented first evidence that nucleic acid forms genetic material. Griffith, Avery, Macleod and McCarty proved the DNA as a genetic material. In DNA, about 400,000 nucleotides are present in one molecule. The average molecular weight of a nucleotide is 300. DNA is composed of two backbones or chains running in the antiparallel fashion . The two chains are interwined in a clockwise direction. In a double helix normally found in organisms, each successive base pair in the stack turns 36 in a clockwise direction. The double helix therefore makes a complete tum of 360, every ten base pairs like circular minor groove staircase . The diameter of the DNA molecule is about 20A or 2 nanometer (1 nanometer = 1 billionth of a meter) ; the base pair is about 3.4A thick and the distance for a complete turn is 34A. The twisting of the strands results in the formation of deep and shallow spiral grooves . . All the space between the back bone of one chain and the back bone of the other is, however, not uniformly filled in by the base pairs. Less of the space around the top of a base pair is filled in them around in the bottom, which creates an empty major area and an empty minor T ' T-11111I1 1LI - "area . .When we look at th~ double helix , all the err.pty m~jor areas ~Ao '\;:'~ . IIii'll/II combme to make up a major groove and all the empty mmor areas ~~ . . , combine to make up a minor groove. ",,-.. !II ., ';..'

.

MOLECUlAR WEIGHT OF

DNA

-
drofolate deficiency, then disruption of DNA synthesis and ultimately cell death) Inhibitors of DHF and their interaction is thus important for chemotherapy (treatment of bacterial, protozoan infections and leukemias). (e) From studies on staphylococcal nuclease enzyme (polypeptide with 14 amino acids and without disulfides) in which mutants were obtained to change amino acids at different positions, it became clear that contribution by any particular amino acid may not be constant but can depend on the presence of other amino acid substitutions at sites remote from the original mutation.

Purification of Enzyme Made Easier by Protein Engineering The presence of many arginine residues in enzyme protein would raise its pi (isoelectric pOint). If pH for native enzyme is 7, modified enzyme with arginine residues introduced into it at C (or N) terminal will have pH closer to 10.5. Then below pH 10.5, modified enzyme will carry net positive charge and will be retained by cation exchange resins while above pH 10.5, it carries no charge and would be eluted from cation exchange resin. Thus cell extract can now be taken at higher pH and most of the proteins would pass through the ion exchange column whereas modified enzyme would be retained. Subsequently, modified enzyme can be eluted by raising pH above 10.5. Protein Engineering Applications to Hormones Scientists have prepared artificial versions of calcitonin hormone that differed 66% of its components, but still worked. Natural calcitonin can be attacked by the body's defences, specifically by the antibodies and is rendered useless. Natural calcitonin is also susceptible to attack by the stomach's protein cutting enzymes. Synthetic calcitonin is resistant to both stomach enzymes and antibodies. It is simple to prepare and less expensive. Kaiser and his colleagues at Rockfeller University have already transformed a brain hormone, Beta-endorphin, which is the body's natural opium. Protein was extensively modified but active site was left intact and effective. There is an example of production of hormone made easier by protein engineering. There are efforts on to produce insulin by recombinant DNA technology. In this approach, gene for A chain of insulin and a gene for B chain of insulin or gene for proinsulin are joined with another gene (say tryp E or ~-gal structural gene). Recombinant products are formed as follows: ~ gal A chain ~ gal B chain or tryp E A chain tryp E, B chain or

tryp E Proinsulin

Separation of A chain or B chain from recombinant product is critical. By genetic engineering, AUG codon is introduced between gene for B gal or tryp E and insulin gene portion. This produced methionine between two proteins, e.g. f3 gal Methionine A chain. Further, cyanogen bromide treatment can cause cleavage of protein at methionine site and A chain or B chain can be separated. Later, A chain and B chain can be linked. In this example, genetic modification helps for recombinant protein production.

Protein Engineering Applications for Biopharmaceuticals Much protein engineering is aimed at biopharmaceuticals to alter pharmacological action. Making proteinic drugs which are more specific, more potent and coupling them to targeting mechanism so that effect will be on few cells or cell types, improving their survival time in patients, redUcing the side effects are some of the objectives of protein engineering in this field. For example, in the natural solutions used for therapy, insulin is mostly assembled as zinc containing hexamers. This self association may limit absorption. By making single amino acid change, Brange et. al (1988) were able to generate insulins which are monomeric at pharmaceutical concentrations. This improved the absorption by two to three times and biological activity was preserved. Similarly hybrid interferons, combining the aminoterminal half of IFN-a2 with the carboxy-terminal half of IFN--al have been prepared which show distinct antiviral properties from the two parental interferons. The commerical success will depend on many factors.s

24 VACCINE BIOTECHNOLOOGY Vaccination protects a recipient from pathogenic agents by establishing an immunological resistance to infection. An injected or oral vaccine induces the host to generate antibodies against the disease-causing organism; therefore, during future exposures, the infectious agent is inactivated (neutralized or killed), its proliferaion is prevented, a'1d the disease state is not established. Just over 200 years ago, in 1796, Dr. Edward Jenner experimentally tested the folklore-based notion that human infection with a mild cattle disease called cowpox would protect infected individuals against the human disease smallpox. Smallpox is an extremely virulent disease with a high death rate; if one survives, permanent disfigurement, mental derangement and blindness often follow. Jenner inoculated James Phipps, an 8-year-old with exudate from a cowpox pustule. In two separate trials after the initial vaccination, the body was fully protected against human smallpox. This country doctor had discovered the principle of vaccination. Communicable diseases such as tuberculosis, smallpox, cholera, typhus, bubonic plague and poliomyelitis have in the past been a scourge for humankind. With the advent of vaccination, antibiotics, and effective public health measures, these epidemic diseases have, for the most part, been brought under control. Occasionally, however, protective measures become ineffective, and devastating new outbreaks occur. In 1991, a cholera epidemic struck Peru, producing, over the next 3 years, approximately 1 million infections and several thousand deaths. Also, for many current human and animal diseases, there are no vaccines. Today, over 2 billion humans suffer from diseases that theoretically could be curtailed by vaccination. In addition, new diseases such as acquired immune deficiency syndrome (AIDS), for which a vaccine might be useful, continue to emerge. Modem vaccines typically consist of either a killed (inactivated) or a live, nonvirulent (attenuated) form of an infectious agent. Traditionally, the infectious agent is grown in culture, purified and either inactivated or attenuated without, of course, losing the ability to evoke an immune response that is effective against the virulent form of the infectious organism. Notwithstanding the considerable success that has been achieved in creating effective vaccines against diseases such as German measles, diphtheria, whooping cough, tetanus, smallpox and poliomyelitis, there are a number of limitations to the current mode of vaccine production: • Not all infectious ag~nts can be grown in culture, and so no vaccines have been developed for a number of diseases. • Production of animal and human viruses requires animal cell culture, which is expensive. • Both the yield and rate of production of animal and human viruses in culture are often quite low, thereby making vaccine production costly. • Extensive safety precautions are necessary to ensure that laboratory and production personnel are not exposed to a pathogenic agent. 352

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• Batches of vaccine may not be killed or may be insufficiently attenuated during the production process, ther~by introducing virulent organisms into the vaccine and inadvertently spreading the disease. • Attenuated strains may revert a possibility that requires continual testing to ensure that the reacquisition of virulence has not occurred. • Not all diseases (e.g. AIDS) are preventable through the use of traditional vaccines. • Most current vaccines have a limited shelf life and often require refrigeration to maintain potency. This requirement creates storage problems in countries with large, unelectrified rural areas. Within the last decade, recombinant DNA technology has provided a means of creating a new generation of vaccines that overcome the drawbacks of traditional vaccines. The availability of gene cloning has enabled researchers to contemplate various novel strategies for vaccine development: .• Virulence genes could be deleted from an infectious agent that retains the ability to stimulate an immunological response. In this case, the genetically engineering agent coula be used as a live vaccine without concern about reversion to virulence, because it is impossible for a whole gene to be reacquired spontaneously dUring growth in, pure culture. • Uve non-pathogenic carrier systems that carry discrete antigenic determinants of an unrelated pathogenic agent can be created. In this form, the carrier system facilitates the induction of a strong immunological response directed against the pathogenic agent. • For infectious agents that cannot be maintained in culture, the genes for the proteins that have c.ritical antigenic determinants can be isolated, cloned, and expressed in an alternative host system such as . Escherichia coli or a mammalian cell line. These cloned gene proteins can be formulated into a "subunit" vaccine. • There are some infectious agents that do not damage host cells directly; instead, the disease condition results when the host immune system attacks its own (infected) cells. For these diseases, it may be possible to create a targeted cell-specific killing system. Although not a true vaccine, this type of system attacks only infected cells, thereby removing the source of the adverse immunological response. In these cases, the gene for a fusion protein is constructed. First, one part of this fusion protein binds to an infected cell. Then, the other part kills the infected cell. Table 24.1. Human disease agents for which recombinant vaccines are currently being developed. Pathogenic agent Disease Viruses Varicella-zoster virus Chickenpox Cytomegalovirus Infection in infant and immunocompromised patients Hemorrhagic fever Dengue virus Hepatitis A virus High fever, liver damage Hepatitis B virus Long-term liver damage Herpes simplex virus type 2 Genital ulcers Acute respiratory disease Influenza A and B viruses Encephalitis Japanese encephalitis virus Parainfluenza virus Inflammation on the upper respiratory tract Rabies virus Encephalitis Respiratory syncytial virus Upper and lower respiratory tract lesions Rotavirus Acute infantile gastroenteritis Lesions of heart, kidney, and liver Yellow fever virus Human immunodeficiency virus AIDS Bacteria Vibrio cholerae Cholera Diarrheal disease E.coli enterotoxin strains

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Neisseria gonorrhoeae Haemophilus influenzae Mycobacterium leprae Neisseria meningitidis Bordetella pertussis Shigella strains Streptococcus group A Streptococcus group B Streptococcus pneumoniae Clostridium tetani Mycobacterium tuberculosis Salmonella typhi Parasites Onchocerca volvulus Leishmania spp. Plasmodium spp. Schistosoma mansoni Trypanosoma spp. Wuchereria bancrofti

Gonorrhea Meningitis, septicemic conditions Leprosy Meningitis Whooping cough Dysentery Scarlet fever, rheumatic fever, throat infection Sepsis, urogenital tract infection Pneumonia, meningitis Tetanus Tuberculosis Typhoid fever River blindness Internal and external lesions Malaria Schistosomiasis Sleeping sickness Filariasis

Because of less stringent regulatory requirements, the first vaccines that were produced by recombinant DNA techniques were for animal diseases such as foot-and-mouth disease, rabies, scours, and a diarrheal disease of piglets. In addition, many more animal vaccines are currently being developed. For human diseases, a large number of recombinant vaccines were to be made available by the year 2000. NATURE OF VACCINES

In a broader sense, the term vaccine is used for any biological material injected in the body to stimulate active immunity. Immunization of the body through vaccination is generally done for the prevention of a disease. The vaccine is administered in advance so as to give the body time to set active immunity, before invasion by the pathogen occurs. The objective of vaccination is to stimulate the production of antibodies without the person actually having to develop the disease. The vaccines may be of the following types:

Uving Organisms (with attenuated virulence) as Vaccines Some vaccines involve use of organisms, whose virulence is greatly attenuated or diminished. These organisms are unable to produce disease but induce immunity when injected in the host. There are at least five different ways of producing attenuated strains of pathogens: (i) Many successive passages of pathogen in some animals other than the usual host of the pathogen or in cultured cells, (ii) Selection of mutants of low virulence, (iii) Treatment of pathogens with chemicals, (iv) Cultivation of pathogens under unfavourable conditions such as high temperature and (v) Deletion or insertion of genes leading to attenuation of pathogens as tried for vaccinia virus. Some examples of vaccines prepared by the attenuation of virulence are given in table 24.2.

Table 24.2. Vaccines prepared by attenuation of virulence 1. 2. 3. 4. 5. 6.

Diseaslipathogen

Method of attenuation

Rabies Yellow fever Smallpox Polio B.e.G. (Bacillus of Calmette Guerien) Bacillus anthracis (Anthrax)

Cultivation in chick or duck embryo Cultivation in chick embryo Animal passage (calf); genetic engineering Mutant strain (sabin oral polio vaccine) Cultivation of organism on media containing bile Cultivation at 40-50DC.

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The concept of recombinant 'vaccinia' virus as alive vaccine gained momentum in the 1990s due to its earlier effective use as a smallpox vaccine. Its host range makes it suitable for livestock also. Following attributes of Vaccinia (used as an expression vector) made it attractive for va~cine development: 0) stimulation of humoral and cell mediated immunity, (ii) low cost, (iii) heat stability, (iv) simple method of administration, (v) visible proof of successful vaccination and (vi) long duration of effectiveness after a single dose.

Genetically Engineered Viruses as Vaccines Although in the past, attenuated strains of vaccinia virus were obtained by serial virus passage, genetic engineering of vaccinia has been used for this purpose in the 1980s and 1990s. Deletion of several genes (thymidine kinase, a growth factor, hemaglutinin, 13.8 kD secreted protein, and ribonucleotide reductase) decreases its virulence. Similarly insertion of some lymphokine genes (e.g. interleukin-2 or IL-2) also leads to decreased virulence. While reducing virulence or pathogenicity, immune response of the recombinant protein (coded by gene inserted in the virus for developing specific vaccine) must be increased. , More than one gene can also be stably integrated into attenuated vaccinia virus for developing recombinant virus to be used as a vaccine against multiple pathogens. A glycoprotein gene or rabies virus has been integrated and the resulting rabies virus vaccine has been field tested in Europe and U.S.A. giving promising results. Other vaccines against HIV are being developed using the gene for HIV-l envelope glycoprotein. In future, the widespread use of Vaccinia as a live vaccine will depend on improving safety, while achieving higher responses to the recombinant protein. Dead Organisms as Vaccines In some cases, the dead bodies of the pathogen, when infected into a host, stimulate the production of antibodies without causing any infection. This method has been used in immunizing against typhoid fever, whooping cough, blackleg in cattle and a few other diseases. Bacterial Toxins and Toxoids as Vaccines Exotoxins of most pathogenic bacteria are highly antigenic; they stimulate the production of antibodies (antitoxins) in animal or human body. These exotoxins are highly unstable and they lose toxicity during storage and are converted gradually into toxoids. Toxoids retain the antigenic power but they are not toxic. Therefore, they can be used in producing immunity against corresponding toxins. The transformation of toxins into toxoids is accelerated by heat, formaldehyde and other chemicals. Alum precipitated toxoids are highly antigenic. Toxoids have been used to develop immunity against several diseases including diphtheria, tetanus, gingrene, etc. Immunizing Sera as Vaccines An immunizing serum (plural 'sera') is the actual blood serum of a person or animal and contains an immunizing material or antibody. It is generally obtained by injecting a toxoid into an animal (usually a horse), permitting establishment of active immunity in the animal, and then withdrawing some of its blood. The corpuscles are removed from serum, which is then standardized as to the strength of antibodies and stored in sterile tubes or bottles. Immunizing sera are used mostly for cure rather than prevention. They may be given for prevention after exposure has taken place, but their immunizing effects are too shortlived to give any benefit, if administered much in advance of the disease. As a curative agent, the action of immunising serum is very rapid. Immunizing serum has been used in the cure of diphtheria and tetanus. Vaccines of Defined Chemical Nature I Since the chemical nature of many antigens is now known, it has been p~ssible to produce these antigens either by biological or by chemical means. Perhaps the first defined mol~cular species to be used as a vaccine were the polysaccharide vaccines for Neisseria meningitidis (meningitis) and Streptococcus pneumoniae (pneumonia). The pure polysaccharide preparations from the bacterial capsule could be used as an antigen in adults or children of over two years of age. Addition of some other chemicals or biological principles often increases the potency of the polysaccharide antigen. For example, Haemophilus injluenzae

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(influenza) B polysaccharide is very poor in inducing antibodies in the susceptible host on its own. However, its antigenic property is greatly enhanced if it is mixed up with diphtheria toxoid. Proteins and nucleic acids have also been identified as immunogenic molecules. An outer membrane protein-polysaccharide complex from meningococci bacteria in association with aluminium phosphate or hydroxide elicits immunogenic responss and hence the mixture can be used as a vaccine. Antigens produced using cloned genes, synthetic peptides and proteins along with certains adjuvants are also often used as vaccines. It has also been proposed that the nucleoprotein of influenza virus can be used as a universal vaccine for influenza. The advent of recombinant DNA and monoclonal technologies coupled with the recent advances in immunology, has opened a new era into the production of vaccines. It seems that isolated immunogens can now be produced in unlimited quantities and vaccine development can proceed without hindrance. Several vaccines based on the new technologies have been licensed for commercial production. Some of these include the following: (i) Yeast cells could be used for the production of vaccine for hepatitis B virus based on cloning the DNA sequence for the antigen; (ii) Escherichia coli vaccines could be produced for pigs and carried extra plasmids encoding the antigen for K88 strain of E.coli; (iii) Monoclonal antibody against K99 strain of E.coli could be produced for protection of calves. PREPARATIONS OF ACTIVE IMMUMZATION PRODUCTS

Preparations containing antigens or antigenic products or mixtures of antigens that induce active immunity are called vaccines. Vaccines are of two types: toxoids and suspension of microorganisms. Many bacterial pathogens produce disease conditions because of the chemical substance they produce called toxin. The toxins synthesized outside the cell are called as exotoxin and those inside the body are called as endotoxin. The exotoxins are highly toxic and thus they are never administered as such but as toxoids. Toxoids are preparations in which toxicity of the toxin is destroyed or reduced to a safe-level without altering the antigenic property of toxin. When immunization against whole organismsjs required, the vaccines are adminstered as suspensions of microorganisms. In this type of vaccines, either live or killed suspensions of microorganisms are used. Immunizati6n with live antigens is generally more effective than dead. Many times it is possible to isolate a strain of a pathogens (mutants) that has lost its virulence but not antigenic property, such type is called attenuated strains. The various types of vaccines used for active immunization are as follows: 1. Bacterial vaccine containing suspension of living bacteria or killed bacteria 2. Toxoid or toxin preparations 3. Viral vaccines and 4. Rickettssial vaccines. BACTERIAL VACCINE

This type of preparation is a living or killed suspension of bacteria. Bacterial vaccines are simple or mixed and univalent or polyvalent. Simple vaccines are prepared from one species only. Mixed vaccines are mixtures of two or more species. Univalent vaccines are those that contain single strain of one species and polyvalent vaccines are those that contain more than one strain of one species.

Killed Bacterial Vaccine

Cholera vaccine Pure culture ofsuitable strains of Vcholera are grown on solid media at 37°C for some days. The strains used are carefully selected for retention of antigenic property. After the incubation period, the organisms are harvested in saline solution, the suspension is centrifuged, the supernatant fluid is discarded and cells are resuspended in isotonic saline. The cells are killed by minimal heat treatment i.e., 1 hr at 56°C or by treatment with chemical bactericide. The approximate number of dead organisms in the suspension is determined by a total count method and the suspension is then diluted with bacterio~tatic saline to contain standard number of organisms. The final product is a polyvalent suspension of different strains.

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Official books such as Indian Pharmacopoeia (I.P) or British Pharmacopoeia (B.P) sets the standards for toxicity, sterility and requirements for storage and labelling.

Pertusis vaccine Suitable strains of Haemophilus pertusis are grown on solid or liquid medium for 24 to 72 hours. The organisms are halVested in saline, the suspension is centrifuged, the supernatant fluid is discarded and the cells are resuspended in isotonic saline containing the bactericide thiomersalate. The suspension is stored in cold for several months to reduce its toxicity and then a final dilution is made with thiomersalate solution. The product can be univalent or polyvalent killed vaccine. The B.P. or I.P. sets the standards for toxicity, sterility and requirements for storage and labelling.

Plague vaccine The preparation is a simple, killed vaccine of Pasteurella pestis. The organisms are grown on suitable medium and harvested when capsule production appears to be at maximum. The capsulated organisms are killed by halVesting them in formaldehyde-saline and kept at room temperature for 24 hrs before being diluted with phenol-saline to contain the standard number of microorganisms. The B.P. or I.P. sets the standards for toxicity, sterility and requirements for storage and labelling.

Typhoid vaccine These are mixed, polyvalent, killed vaccines of Salmonella typhi and Salmonella paratyphi A and B. The halVested bacteria are killed either by heat treatment or by the action of a bactericide and the suspensions diluted to the standard and mixed.

Living Bacterial Vaccine

BeG A pure culture of an authentic strain of attenuated Mycobacterium tuberculosis of Calmette and Guerin is grown on the suitable medium for not more than 14 days. The growth is harvested and suspended, at a suitable concentration, in a liquid medium, designed to preselVe the antigenicity and viability of vaccine. It is issued in freeze-dried form and liquid form. B.P. or I.P. sets the standards for skin-sensitizing potency, toxicity, virulence and sterility and states requirements for storage and labelling. TOXOID OR TOXIN PREPARATION

Diphtheria Vaccine A pure culture of Cornybacterium diphthriae is grown in suitable fluid culture medium for several days and a filtrate is obtained from this, which contains the specific exotoxin. The toxin is converted to the non-toxic, yet antigenically active, toxoid form and the vaccine issued as following preparations.

Formal toxoid (F. T.) A quantity of filtrate containing bacterial exotoxin is rendered non-toxic by adding a suitable concentration of formaldehyde solution and incubating the mixture at 37°C for 4 weeks or until the toxicity is completely removed.

Alum precipitated toxoid (AP. T) The formal toxoid is precipitated with sufficient potash alum, the precipitate is collected, washed alternately with salin.e solution and phosphate buffer and resuspended in thiomersalate--saline.

Purified, toxoid, aluminium phosphate (P. T.AP) Purified formal toxoid is absorbed onto a suspension of hydrated aluminium phosphate in saline. Thiomersalate is added as a bacteriostatic.

Purified toxoid aluminium hydroxide (P. T.AH) Purified formal toxoid is adsorbed onto a suspension of aluminium hydroxide in saline and thiomersalate is added.

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Toxoid antitoxin floccules (T.A.F) The toxicity of the filtrate containing exotoxin is reduced to a low value or completely destroyed, by the addition of formalin solution. A quantity of diphtheria antitoxin, which is about 80% of the toxoid, is added to the inactivated toxin. The mixture flocculates, and the reaction is allowed to proceed for few weeks before the floccules are separated, washed with water and resuspended in bacteriostatic-saline.

Staphylococcus Toxoid The filtrate from a culture of suitable toxigenic strain of S. aureus is prepared and the (X-toxin is treated with formaldehyde solution and rest of the preparation method is same as diphtheria F.T. Tetanus Toxoid The filtrate from the culture of Clostridium titani is used for the preparation of F.T, AP.T, P.T.T.P and P.T.AH tetanus toxoid. The method of preparation is similar to diphtheria toxoid. For the diphtheria toxoid, staphylococcus toxoid and tetanus toxoid preparation I.P or B.P sets the standards for potency, sterility, toxicity and requirement for storage and labelling. VIRAL VACCINE

Two tyrms of viral vaccines are available: inactivated (killed) vaccines and attenuated Oiving) vaccines. Virus, being intracellular parasites, can grow only within other living cells. Thus, viruses are cultivated in free living animals, fertile eggs or tissue cultures.

Influenza Vaccine The strains of influenza virus used in the preparation of the vaccine are those recommended by World Health Organization (W.H.O). Each strain is grown separately in the cells lining the allantoic cavity of 1013 day old fertile chicken-embryo. After incubation at optimum temperature for 2-3 days, the allantoic fluids are collected, cooled and inactivated by the addition of suitable concentration of formaldehyde solution. Inactivation is allowed to proceed for 2-3 days at cold temperature. Virus particles are collected by centrifugation and suspended in saline solution containing bactericide. Polyvalent vaccines are prepared by mixing different strains of the virus. B.P gives test for identification, toxicity, viral inactivation, hemagglutinating activity, sterility and requirements for storage and labelling. Poliomyelitis Vaccine Strains of poliomyelitis virus types 1, 2 and 3 are grown separately in the cultures of healthy kidney tissue obtained from suitable species of monkey. After a suitable incubation period, the tissue cells lyse and liberate virus. The virus suspension is harvested and passed through bacteria proof filter. After harvesting, but before filtration, the virus suspension is tested for presence of M. tuberculosis, hepatitis B virus and L.C.M virus. The TCIDso of each suspension is determined and if satisfactory, suspension is inactivated by the addition of suitable concentration of formaldehyde solution. Inactivation is effected at 37°C for usually not less than 12 days. A second filtration is made on the 6th day of inactivation to remove aggregates, which may protect the virus. Inoculating them into tissue cultures performs safety test for inactivation. Free formaldehyde is removed from blended vaccine by the addition of sodium metabisulphite. Rabies Vaccine Suitable animals are injected intra-cerebrally with fixed rabies viruses. The subsequently paralyzed animals are killed if they show typical symptoms of rabies lasting for 24 hours. The animal brains are removed and are suspended in the saline solution. Viruses are killed or attenuated by the treatment with phenol or other chemical substances. The product is diluted to contain a specified amount of brain material. Smallpox Vaccine It is prepared by growing vaccine viruses on chorio-allantoic membrane of fertile chicken egg. After a suitable incubation time, the membranes are separated, chilled, grounded to uniform suspension with 50% glycerol and with or without addition of bactericide. Preparation is issued either as freeze-dried or liquid preparation.

Vaccine Biotechnology

359 RICKETTSSIAL VACCINE

Yellow Fever Vaccine Fertile chicken eggs are incubated for 7 to 8 days and the embryos are injected with a strain of yellow fever viruses that are virulent for mice, but not for a man. After incubation for 3-4 days, the embryos are grounded and extracted with purified water. The resultant suspension is centrifuged and supernatant fluid is freeze-dried. The containers are filled with nitrogen before sealing.

Typhus Vaccine Fertiie chicken eggs are incubated for 7 days and the yoll< sacs are injected with virulent Rickettssia prowazeki. Following .1-2 weeks of incubation, the yolk sacs of the dead embryos are subjected to treatment to release rickettsia. The Envelope diSintegrated material is suspended in saline solution and formaldehyde solution is added in a Capsid concentration suitable to inactivate the suspension. N uc Iei c ac id Fat is removed from the yolk sac suspension by Fig. 24.1. Schematic representation of an animal virus. purification with solvent ether or trichlorotrifluroethane. The aqueous middle layers of the resultant mixture constitute the vaccine . SUBUNIT VACCINES

NH2

A

HSV glycoprotein 0

Exterior Membrane Interior

COOH Transmembrane domain

B

I H2N

Modified HSV glycoprotein 0

-1L______---l~ COOH

Vaccines generally consist of either killed or attenuated forms of the whole pathogenic agent. The antibodies elicited by these vaccines initiate an immune response to inactivate (neutrailize) pathogenic organisms by binding to proteins on the outer surface of the agent. So do vaccines need to contain the whole organism, or will specific portions of pathogenic organisms suffice? For disease-causing viruses, it has been shown that purified outer surface viral proteins, either capsid or envelope proteins, are alone sufficient for eliciting neutralizing antibodies in the host organism. Vaccines that use components of a pathogenic organism rather than the whole organism are called "subunit" vaccines; recombinant DNA technology is very well suited for developing new subunit vaccines.

Herpes Simplex Virus

Herpes simplex virus (HSV) has been implicated as a cancer-causing (oncogenic) agent-in addition Exterior to its more common roles in 'c ausing sexually Membrane transmitted disease, severe eye infections and encephalitis-so that prevention of HSV infection Interior by vaccination with either be killed or attenuated virus Fig. 24.2. A-Location in the cytoplasmic membrane of may put the recipient at risk for cancer. Thus, HSV·l gD with the transmembrane domain . protection againSt HSV would be best achieved by a B-Extracellular location of a soluble gD without subunit vaccine, which would not be oncogenic. the transmembrane domain.

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The primary requirement for creating any subunit vaccine is identification of the component{s) of the infectious agent that elicits antibodies that react against the intact form of the infectious agent. The HSV type 1 (HSV-1) envelope glycoprotein 0 (gO) is such a component, because, after injection into mice, it elicits antibodies that neutralize intact HSV. The HSV-1 gO gene was isolated and then cloned into a mammalian expression vector and expressed in Chinese hamster ovary (CHO) cells, which, unlike the Escherichia coli system, allows foreign proteins to be properly glycosylated. The complete sequence of the gO gene encodes a protein that normally is bound to the mammalian host cell membrane. However, a membrane-bound protein is more difficult to purify than a soluble protein, and so the gO gene was modified by removing the neucleotides encoding the C-terminal transmembrane binding domain. The modified gene was then transformed into CHO cells, where the product was glycosylated and secreted into the external medium since it could not be incorported into the cell membrane. In laboratory trials testing its neutralizing ability, the modified form of gO was effective against both HSV-1 and HSV-2.

Foot-and-Mouth Disease Foot-and-mouth disease virus (FMOV) has a devastating impact on cattle and swine and is extremely virulent, but it has been possible to keep the negative effects of this virus to a minimum by using formalinkilled FMOV preparations as a vaccine. Approximately 1 billion doses of this killed-virus vaccine are used worldwide each year. Research on FMOV found that the major antigenic determinant inducing neutralizing antibody is the capsid viral protein 1 (VP1). Although purified VP1 is a much less potent antigen than intact viral particles, it 'can still induce neutralizing antibodies by itself and therefore can protect animals from infection by FMOV. Thus, the gene for VP1 became a target for cloning. Tuberculosis . Tuberculosis, one of the most important infectious diseases worldwide, is caused by the bacterium Mycobacterium tuberculosis . This bacterium can form lesions in any tissue or organ, which leads to cell death. The lungs are most commonly affected . In addition, patients suffer fever and loss of body weight, and without treatment tuberculosis is often fatal. It is estimated that approximately 2 billion people are currently infected with this organism and that nearly 3 million deaths a year result from these infections. In an initial attempt to determine whether a safer and more effective subunit vaccine against tuberculosis might be developed, one group of researchers decided to first examine the immunoprotection that was provided by purified M. tuberculosis extracellular proteins. Following growth of the bacterium in liquid culture, six of the most abundant of the approximately 100 secreted proteins were purified. Each of these proteins was used separately and then in combination with the others to immunize guinea pigs. The immunized animals were then challenged with an aerosol containing approximately 200 cells of live M. tuberculosis-a large dose for these animals. The animals were observed for 9 to 10 weeks before their lungs and spleen were examined for the presence of disease-causing organisms. In these experiments, some of the purified protein combinations provided approximately the 3 same level of protection against weight loss, death and infection 2 4 of lungs and spleen as did the live BCG vaccine. A safe and efficacious subunit vaccine for the prevention of tuberculosis in humans can be developed once it is determined whether the Exterior protective M. tuberculosis proteins produced by recombinant Membrane DNA technology are as effective as secreted proteins. Interior Peptide Vaccines The question arises whether a small discrete portion (domain) of a protein can act as an effective subunit vaccine and induce the production of neutralizing antibodies. Intuitively, one would Fig. 24.3. Generalized envelope-bound protein with external epitopes (1 expect that only the portions or domains of a protein that are to 5) that might elicit an immune accessible to antibody binding, that is, those on the exterior surface response.

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of the virus, would be immunologically essential and that the inaccessible regions inside the virus particle may be ignored if they do not contribute to the conformation of the immunogenic domain. If this argument has validity, it is possible that short peptides that mimic epitopes (antigenic deh~rminants) will be immunogenic and could be used as vaccines (peptide vaccines). Chemically synthesized domains of FMDV VP1 were tested as potential peptide vaccines. Peptides correspondin~ to amino acids 141 to 160, 151 to 160 and 200 to 213. which are located near the C-terrninal end of VP1, and amino acids 9 to 24, 17 to 32, and 25 to 41, which are located near the N-terminal end of VP1, were each bound to a separate inert carrier protein (key-hole limpet hemocyanin)very .small pep tides are usually rapidly degraded unless they are bound to the surface of a larger carrier molecule-and injected into guinea pigs. A single inoculation with peptide 141 to 160 elicited sufficient antibody to protect the Short peptides animal against subsequent challenges with FMDV. By contrast, inoculation with complete VP1 or peptides 9 to Fig. 24 .. 4 S tructurei 0 ad pepti e vaccine composed 24, 17 to 32 or 25 to 41 yielded a lower level of neutralizing 01 identical short peptides bound to a carrier protein. antibodies. In an additional experiment, a longer peptide consisting of amino acids 141 to 158 joined to amino acids 200 to 213 by two proline residues elicited high levels of neutralizing antibodies in guinea pigs, even when it was injected without any carrier protein. This "two-peptide" molecule was more effective than either of the single peptides alone and prevented FMDV proliferation in cattle as well as in guinea pigs. . Although these results were promising, the amount (dose) of peptide material that had to be used to elicit an immunological response was approximately 1,000 times the amount of inactivated FMDV. To overcome this problem, DNA encoding FMDV VP1 peptide 142 to 160 was linked to the gene encoding a highly immunogenic carrier molecule, hepatitis B core protein (HBcAg). When the gene for this fusion protein was expressed in either E.coli or animal cells in culture, the protein molecules self-assembled into stable" 27 -nm particles," with the FMDV VP1 peptide located on the outer surface of the particle. These particles are highly immunogenic in laboratory animals. Therefore, HBcAg may be an effective carrier molecule for such short synthetic peptides. A comparison of the immunogenicity, in guinea pigs, of a variety of FMDV peptide vaccines, all of which contained the VP1 peptide 142-to160 sequence revealed that a fusion protein containing HBcAg and FMDV VP1 amino acids 142 to 160 was approximately 1/10 as immunogenic as inactivated FMDV particles, 35 times more immunogenic than a fusion protein containing E.coli p-galactosidase and FMDV VP1 amino acids 137 to 162 and 500 times more immunogenic than the free synthetic peptide composed of amino acids 142 to 160. Because synthetic peptides fused to HBcAg do not seem to interfere with the assembly of the 27-nm hepatitis B virus-like particles and because these particles are nearly as immunogenic as the intact virus from which the synthetic peptide was derived, this approach may become a general method for the delivery of peptide vaccines. Nevertheless, there are certain limitations to using short peptides as vaccines: • To be effective, an epitope must cortsistof a short stretch of contagious amino acids, which does not always occur naturally. • The peptide must be able to assume the same conformation that the epitope has in the intact viral particle. • A single epitope may not be sufficiently immunogenic. Obviously, more research needs to be done, but in the future, synthetic peptide vaccines could become highly specific, relatively inexpensive, safe and effective alternatives to traditional vaccines.

~

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Biotechnology ATTENUATED VACCINES

In some instances, genetic manipulation may be used to construct modified organisms (bacteria or viruses) that are used as live recombinant vaccines. These vaccines are either non-pathogenic organisms that have been engineered to carry and express antigenic determinants from a target pathogenic agent or engineered strains of pathogenic organisms in which the virulent genes have been modified or deleted. In these instances, as a part of a bacterium or a virus, the important antigenic determinants are presented to the immune system with a conformation that is very similar to the form of the antigen in the diseasecausing organism. By contract, purified antigen alone often lacks the native conformation and elicits a weak immunological response. Cholera If it is possible to develop a live vaccine, it is usually advantageous to do so, because live vaccines are generally much more effective than killed or subunit vaccines. The major requirement for a live vaccine is that no virulent forms be present in the inoculation material. With this objective in mind, a live cholera vaccine has been developed. Cholera is a fast-acting intestinal disease characterized by fever, dehydration, abdominal pain and diarrhoea. It is transmitted by drinking water contaminated with faecal matter. In developing countries, the threat of cholera is a real and significant health concern whenever water purification and sewage disposal systems are inadequate. The bacterium Vibrio cholerae, the causative agent of cholera, colonizes the small intestine and secretes large amounts of a hexameric enterotoxin, which is the actual pathogenic agent. This protein consists of one subunit, the A subunit, that has ADP-ribosylation activity and stimulates adenylate cyclase, and five identical B subunits that bind specifically to an intestinal mucosal cell receptor. The A subunit has two functional domains: the Al peptide, which contains the toxic activity and the A2 peptide, which joins the A subunit to the B subunits. The cholera vaccine that is currently used consists of phenol-killed V. cholerae. This vaccine generates only moderate protection, which normally lasts from about 3 to 6 months. However, other possible types of cholera vaccines have been examined. Previous studies had indicated that a subunit vaccine consisting of inactivated hexameric cholera enterotoxin was not effective in providing immunity against V. cholerae. Since V. cholerae colonizes the surface of the intestinal mucosa, it is thought that an effective cholera vaccine should probably be directed to this structure and should therefore be administered orally. With this in mind, a strain of V. cholerae in which part of the coding sequence for the Al peptide was deleted was created. This strain cannot produce enterotoxin; therefore, it is non-pathogenic and is a good candidate for a live vaccine. Specifically, in this experiment, a tetracycline resistance gene was incorporated into the Al peptide DNA sequence on the V. cholerae chromosome. This insertion inactivated the Al peptide activity and also . made the strain resistant to tetracycli!1e. Although the Al peptide sequence was disrupted, this strain is not acceptable as a vaccine because the inserted tetracycline resistance gene can excise spontaneously, thereby restoring enterotoxin activity. Consequently, it was necessary to engineer a strain carrying a defective Al peptide sequence that could not revert. 1. A plasmid containing the cloned DNA segment for the Al peptide was digested with the restriction enzymes Clal and Xbal, each of which cut only within the Al peptide-coding sequence of the insert. 2. To recircularize the plasmid, an Xbal linker was added to the Clal site and then cut with Xbai. 3. T4 DNA ligase was used to join the plasmid at the Xbal sites, thereby deleting a 550-base-pair (bp) segment from the middle of the Al peptide-coding region. This deletion removed 183 of the 94 amino acids of the Al peptide. 4. Then, by conjugation, the plasmid containing the deleted Al peptide-coding sequence was transferred into the V. cholerae strain carrying the tetracycline resistance gene within its Al peptide DNA sequence. 5. Recombination can occur between the remaining Al coding sequence on the plasmid and the Tetr gene-disrupted Al peptide gene on the chromosome. Such a double-crossover event replaces the chromosomal Al peptide-coding sequence with the homologous segment on the plasmid carrying the deletion.

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Chromosomal DNA

j

~ /

""

Conjugation

V. cholerae

/

of V. cholerae

Xballinker T4 DNA ligase

~

resistance " Tetracycline gene insert

Deleted Al peptide DNA sequence

Recombination /Chromosomal DNA

id

j

Chromosomal /DNA

Deleted Al peptide DNA sequence Fig. 24.5. Strategy for deleting part of the cholera toxin A J peptide DNA sequence from a strain of V. cholerae.

6. After growth for a number of generations, the extrachromosomal plasmid, which is unstable in V cholerae, was spontaneously lost. 7. Cells with an integrated defective Al peptide were selected on the basis of their tetracycline sensitivity. The desired cells no longer had the tetracycline resistance gene but carried the deleted AI peptide sequence. A stable strain with a deleted Al peptide sequence was selected in this way. This strain did not produce active enterotoxin but nevertheless retained all the other biochemical features of the pathogenic form of V cholerae. This strain is currently being evaluated in clinical trials to test its effectiveness as a cholera

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vaccine. The results so far have been equivocal. While this vaccine conferred nearly 90% protection against diarrhoeal disease in volunteers, it induced side effects in some of those who were tested. It is possible that this strain will require modification at an another chromosomal locus before it can be used as a vaccine. Salmonella Species Other attempts to engineer nonpathogenic strains of pathogenic bacteria that could be used as live vaccines have involved deletions in chromosomal regions that code for independent and essential functions. At least wo deletions are preferred, because the probability that both sets of functions can be simultaneously reacquired is very small. It is assumed that a "doubly deleted" strain would have a limited ability to proliferate after its injection as a vaccine, thereby curtailing its pathogenicity while allowing it to stimulate an immunological response. Strains of the Salmonella genes cause enteric fever, infant death, typhoid fever and food poisoning. Therefore, an effective vaccine against these organisms is needed. Deletions in the aro genes, which encode enzymes involved in the biosynthesis of aromatic compounds, and in the pur genes, which encode enzymes involved in purine metabolism, have been used to attenuate various Salmonella strains. These doubly deleted strains, which can be grown on a complete and enriched medium, generally establish only low-level infections; overall, their virulence is reduced by 106-fold or more. These attenuated Salmonella strains have been shown to be effective oral vaccines in mice, sheep, cattle, chickens and more recently, in humans. Leishmania Species Although the human immune system can respond to infections by protozoan parasites of the genus Leishmania, it has been difficult to develop an effective vaccine against these organisms. Attenuated strains of Leishmania are sometimes effective as vaccines; however, they often revert to virulence. Also, the attenuated parasite can persist for long periods in an infected but apparently asymptomatic individual. Such individuals can act as reservoirs for the parasite, which can be transferred to other people by an intermediate host. To overcome these problems, an attenuated strain of Leishmania that is unable to revert to virulence can be created by targeted deletion of an essential metabolic gene such as the one encoding dihydr%late reductase-thymidylate synthase. In one of these attenuated strains, Leishmania major El 0-5A3, the two dihydrofolate reductase-thymidylate synthase genes that are present in wild-type strains are replaced with the genes encoding resistance to the antibiotics G-418 and hygromycin. For growth in culture, it is necessary to add thymidine to the medium used to propagate the attenuated, but not the wild-type strain. In addition, unlike the wild type, the attenuated strain is unable to replicate in rnacrophages in tissue culture unless thymidine is added to the growth medium. Importantly, the attenuated strain survives for only a few days when inoculated into mice; in that time, it does not cause any disease. This period is sufficient to induce substantial immunity against leishmania lesions in BALA/c mice after administration of the wild-type parasite. Since the attenuated parasite did not establish a persistent infection or cause disease, even in the most susceptible strains of mice tested,it is considered to be a strong candidate vaccine. FollOwing additional experiments with animals, it should be possible to test whether this attenuated parasite is effective as a vaccine in humans.

25 PLANT BIOTECHNOLOGY Plant Biotechnology is based on techniques for the delivery into plant cells of functionally defined natural or synthetic nucleic acid sequences. Somatic plant cells can be competent for functional and stable integration of foreign DNA into their own genome as well as for development into differentiated tissue or a complete organism, thus yielding the biological basis for the production of chimeric or fully transgenic plants. Transfer of nucleic acids to plant cells and production of transgenic plants are routine tools for basic research on plant biology, as well as for plant gene identificaion and isolation and the analysis of gene regulation and function. Transgenic crop plants with newly added traits play an important role in the development of concepts for product quality improvement, integrated pest management and sustainable farming. PRINCIPLES

Physiology of the Recipient Cells Depending on their purposes, experiments require different levels of competence of a cell that has received genetic information for the analysis of gene regulation and function, competence for uptake and transient expression of the transferred DNA or RNA may be sufficient. For the production of transgenic tissue, a transformed cell must allow the replication of the transferred nucleic acids, either as episomes or as genomic inserts (non-integrative and integrative transformation respectively). For the production of fully transgenic plants, transgenic cells must be totipotent, that is, competent for regeneration into a complete, differentiated organism. The competence of a cell for integrative transformation and regeneration depends on its genotype and on its development and physiological state. The physiological state may be influenced by plant culture techniques, and it is possible to shift cells from a potentially competent to a competent state by mechanical or enzymatic wounding or by treatment with phytohormones.

Characteristics of Transferable Nucleic Acids Before being introduced into plant cells, nucleic acids are amenable to rearrangement by in vitro recombination techniques. Strong, constitutive expression signals of plant, bacterial or viral origin are available. Other promoter sequences confer tissue- or development-specific expression of the introduced genes, or they are inducible by various environmental stimuli. Regulatory and protein-encoding sequences are combined in transcriptional or translational gene fusions. These sequences are delivered to plant cells as parts of plasmid or cosmid vectors, in bacteriophages, or on yeast artificial chromosomes. In self-replicating vectors, components of plant virus genomes provide signals for extrachromosomal replication of the introduced DNA or RNA. Direct gene transfer methods also allow the introduction of native, non-cloned nucleic acids.

Characteristics of the Delivery Systems Transformation techniques for plants provide the means to let nucleic acids pass the cell wall, plasma

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membrane and nuclear envelope without hampering the viability of the target cell. Biological gene transfer systems use the plant pathogens Agrobacterium tumefaciens or A. rhizogenes as natural vectors. Direct (i.e. vectorless) gene transfer systems include chemical methods, electroporation, microinjection and biolistics. These most widely ana successfully used gene transfer techniques are compared in table 25.1, showing their advantages and disadvantages with respect to the production of transgenic plants. Table 25.1. Comparison of the most widely used methods for the delivery of nucleic acids into plants. Method

Advantages

Disadvantage

Agrobacteriummediated gene transfer

High efficiency Insertion of long, non-rearranged DNA stretches Mostly single- or few-copy integration Does not require special equipment Simple manipulations High efficiency Allows treatment of many samples in one experiment Chemical methods do not require special equipment

Limited host range Genetic informafion can be delivered only as T-DNA insert

Chemical methods and elect. ..;poration

Microinjection

Ballistic methods

Genotype independent High flexibility to reach different target structures Allows visual control of manipulations Genotype independent High flexibility to reach different target structures High promise for routine production of transgenic cereals

High degree of donor DNA rearrangement and multicopy integration events Chemical methods require protoplast culture and regeneration systems for production of tansgenic plants Enhanced risk of induction of somaclonal variation Sophisticated and expensive equipment required Much expertise required Sophisticated equipment required High degree of donor DNA rearrangement and multicopy integration events

There is no universal gene transfer metnod suitable for any kind of transformation program. Other gene transfer techniques are still being developed, as well as systems that combine components of more than one of the described methods. DNA DEUVERY METHODS

Agrobacterium-mediated Gene Transfer A. tumefaciens and A. rhizogenes are capable of transferring a stretch of DNA, the T-DNA, from their large tumor (Ti)- or root (Ri)-inducing plasmids, respectively to the nuclear genome of host plant cells. Several gene products of the T-DNA interfere with the regulation of the host cell development, thus leading to tumor or enhanced root hair formation. Virulence and host range of a bacterial strain depend on constitutively expressed bacterial chromosomal genes and of vir genes located on the Ti or Ri plasmids. Upon induction by plant factors (e.g., acetosyringone), the vir system mediate T-DNA excision, transfer to the plant cell and possibly integration into the nuclear genome. T-DNA transfer occurs in protoplasts or wounded organized tissue. Short border repeats of 24 bp flank the T-DNA; they are the only T-DNA components that need to remain intact for its transfer. Disarmed vectors have been developed for use in A. tumefaciens which, upon release of the T-DNA into the plant cell, do not cause tumor formation, since the native T-DNA genes from A. tumefaciens

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involved in disease symptom development were removed and can be replaced by genes of interest. In binary vectors, a modified T-DNA is located on a separate, Escherichia coli compatible plasmid and the necessary vir functions are provided in trans from a Ti-plasmid devoid of T-DNA. Agroinfection is Agrobacterium-mediated transfer to plants of cloned, functional (Le. replicating) viral DNA.

Chemical Methods and Electroporation Protoplasts, Le. cells depleted of the cell wall by enzymatic digestion can be isolated from callus material, cell suspension cultures and a wide range of differentiated plant tissues. For genetic transformation, protoplasts can be coincubated with the nucleic acids and chemical substances (e.g. polyethylen glycol, polyvinyl alcohol, calcium phosphate) that lead to reversible permeabilization of the plasma membrane. Nucleic acids are also delivered to protoplasts after encapsidation in,liposome vesicles followed by fusion with the plasma membrane or endocytosis. Changes in membrane permeability for gene transfer can also be induced by high voltage electric fields (electroporation). Electroporation is applicable to protoplasts, isolated cells and organized tissue.

Microinjection Microinjection allows the introduction of nucleic acids under microscopic control into subcellular compartments of protoplasts, isolated cells and cells in multicellular structures such as calli, meristems and embryos. Recipient cells or tissues are immobilized in sodium alginate, agarose, or poly-L-lysine, or by a holding capillary system. Nucleic acids can be delivered directly to the nucleus with the help of a glass capillary connected to a micromanipulator system. Various culture systems for single cells or small multicellular structures (single cell culture, hanging droplet culture, nurse culture) are used in combination with the microinjection technique.

Ballistic Methods In bOllistic transformation methods, nucleic acids associated with gold or tungsten particles in the micrometer range (microcarriers) are transported through cell walls. The microcarriers are accelerated directly or by the help of projectile carriers (macrocarriers). Motive forces are provided by gun powder, by compressed inert gases or by electric discharge through a small water droplet. Single cells, tissues or whole organisms are targeted inside a partially evacuated chamber. Various modifications of these basic principles were employed to meet the requirements of many different transformation problems, allowing the finetuning of the motive forces and the controlled delivery of the microprojectiles to the target. ANALYSIS OF TRANSGENIC PLANT MATERIAL

Analysis of Foreign Gene Products A number of gene expression vectors have been developed which confer a distinctive phenotype to the recipient cells and therefore allow the positive selection or visual screening of transformed cells, cell lineages and transgenic organisms. Activity of the foreign gene products are determined either in situ in transformed tissue or with in vitro enzyme assays of plant extracts. The gene products can be detected directly by immunological methods in vitro (Western blot) or in situ.

Physical Analysis of Foreign DNA The presence and physical organization of foreign DNA in plant cells is monitored by Southern blot analyses of genomic plant DNA and polymerase chain reaction (PCR) techniques. Integration of foreign DNA into plant nuclear genome occurs predominantly at random sites via illegitimate recombintion events. Depending on the transformation system used, structural rearrangements of integrated DNA and integration of multiple copies at one or several genetic loci can be frequent. As a rule, foreign DNA remains stably integrated for many generations. However, intrachromosomal homologous recombination between copies of introduced DNA can be a source of physical instability of the locus. Transcripts of the foreign genes are detected by Northern blot, RNase protection, or SI protection analysis. Transcription can be affected by neighbouring DNA sequences, leading to different expression

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levels in individual transgenic plants. Initially transcribed foreign DNA can be silenced; that is the transcription can be inactivated. CLONAL PROPAGATION OR MICROPROPAGATION

A variety of plant species can be conveniently propagated through the technique of cell, tissue and organ culture. This is popularly known as micropropagation or clonal propagation. The major benefits of this method are: (i) rapid multiplication of superior clones and maintenance of uniformity; (ii) multiplication of disease free plants; and (iii) multiplication of sexually derived sterile hybrids. In most cases, clonal propagation is achieved by placing sterilised shoot tips or axillary buds into a culture medium that is sufficient to induce formation of multiple buds. Stages of micropropagation procedure are: 1. Establishment of tissue in vitro 2. Multiplication of shoots 3. Root formation and conditioning of propagules prior to transfer to green house and 4. Growth in pots followed by field trials. A wide range of plants have now been regenerated through tissue culture. Few examples can beAsparagus, Banana, Bamboo, Chrysanthenum, Cardamom, Coffee, Eucalyptus, Grape, Vine, Ully, Pineapple, Papaya, Potato, Rose, Tomato, Vanilla etc. The cost of plant tissue culture, either for micropropagation or for production of bioactive compounds can be reduced by use of bioreactors. The production rate can be increased tens of thousands times. This is particularly important in western countries where cost of labour is high. For micropropagation, the organogenic or embryogenic tissue is multiplied in bioreactors and transferred to bioprocessor where propagules of different sizes are separated from each other. These propagules then grow and develop into plantlets in separate culture vessels which can be transplanted, stored or shipped. Cotyledons-somatic embryoids-root and shoot can be simultaneously obtained. PLANT CELL CULTURE METHODS

Plant cells and tissues can be grown in culture, especially on nutrient media. Different types of cultures are: 1. Organ culture--embryos, anthers, ovaries, buds, roots, flowers. Other plant organs can be cultured to study the morphogenesis or to grow the entire plant. 2. Meristem or tissue culture-shoot meristem, leaf, stem or other explant tissue for regeneration and multiplication of complete plants. 3. Callus culture-the initiation and culture of undifferentiated cell mass (callus) on agar media. Such cultures are initiated from an explant of seedling or other plant tissue source. 4. Cell culture-the culture of cells in liquid medium in vessels and their maintenance as shake cultures or as mass culture in fermenters. 5. Protoplast culture-the culture of protoplasts isolated from plant tissue or cultured cells.

Callus Culture Callus culture is the initial stage in the development of plant cell culture independently of the parent plant. If sterilised excised pieces of plant material are incubated on solid media containing full range of nutrients and plant growth substances (hormones) in suitable combination, an irregular outgrowth of the cell grows normally in one week and this cell mass is called a callus [analogy with the growth (callusing) occurring in wound healing of the plants.) It is possible, over a number of subcultures, to establish callus tissue growing independently and stocks of cultured plant cells are frequently kept in this form. The character of callus can be manipulated by altering the balance of hormones in the medium so that cell to cell adhesion is minimised to produce the so-called friable callus. Friable callus when shaken in liquid medium will slough off cells which grow to produce cell suspension culture. The establishment of such cell culture (cell line) from starting callus requires 2-4 months depending on the species and cell line. Cell line

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is usually maintained as a stock that is subcultured monthly on nutrient agar. Growth rate is faster in the developed cell line than that of the initial one.

Cell Culture Plant cell cultures differ in many respects from the culture of microorganisms. Plant cells in culture are derived from the complex eukaryotic organism and such cells are capable of existing in many different development states. Individual cells are large (20-50011) up to 5 X 105 times the volume of a bacterial cell. Doubling time is between 40-100 hrs. normally while the shortest is 15 hrs. for the tobacco cell line. Plant cell in culture is totipotent because it can give rise to the entire plant. Therefore, any biochemical character of the plant should be expressable if proper environment is maintained. This is theoretically true but practically so far difficult. Efforts are on to raise mutants from the plant cell cultures in the same way as it is done for microorganisms by exposing plant cells in culture suspension to mutagenes.

Table 25.2. Commercial uses of Plant ceU(fissue culture. Industry

Application

Economic Benefits Improved productivity

3. Citrus

Rapid multiplication of entire plant Biosynthesis of Chemicals, Propagation of medicinal plants Virus elimination

4. Coffee 5. Land reclamation

Disease-resistance breeding Mass propagation

6. Ornamental Horticulture

Mass propagation

7. Pineapple

Mass propagation

8. Strawberry

Mass propagation

1. Asparagus 2. Chemical and Pharmaceutical

Reduced production costs. Large number of plants for planting Improved quality and high productivity Disease resistance Availability of select clones of wild species for revegetation Reduced cost of Sp. virus elimination. Introduction of new selectants. Improved quality in higher volume. Rapid introduction of new strain.

Tissue culture is aiso referred to as micropropagation. A majority of tissue culture done through shoot tips are costly being labour-intensive.

Table 25.3. Examples of Plants propagated through tissue culture. Horticulture Agricultural crops Food Crops Sylviculture

Orchid, rubber plant, rose, glacinia, Gerbera, Dracaena, Chrysanthemum, African violet. Oil palm, citrus, Date palm, Jojoba. Beet, Cauliflower, Lettuce, Spinach, Sweet Potato. Douglas fir, Loblolly pine, Redwood.

Table 25.4. Products being studied in plant cell culture. Alkaloids

Ajmalicine Vinblastine Serpentine Codeine

Quinine

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Biotechnology Nicotine Vincristine Shikonin Ubiquinone Anthroquinone Digoxin Digitoxin Ginseng

Quinones

Cardiac glycosides Saponins

Table 25.5. Cell suspension culture producing products greater than intact plant.

1. 2. 3. 4. 5. 6. 7.

Product

Plant

Anthraquinone Ajmalicine serpentine Diosgenin Rosmarinic acid Ubiquinone 10 Glutathione Nicotine

Morinda citr%lia Catharanthus roseus Disoscorea deltoids Coleus blumei Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum

Cell culture suspension % dry wt. 18

1.8 2

25 0.04

1 5

Whole plant % dry wt

Ratio cell culture Whole plant

2.2 0.8 2 3 0.003 0.1 2.1

8.1 2.2 1 8.3 13 10 2.3

Embryo Rescue The recovery of hybrid plants in culture, is usually from wide sexual crosses, when endosperm tissues are deficient. The term 'embryo rescue' is used for embryo culture, ovule culture and ovary culture since in each case the objective is to rescue the embryo. During distant hybridization, often the embryo aborts at an early stage of development, so that no mature seed can be obtained. This problem can be overcome, if young hybrid embryos are excised and cultured on a synthetic medium. These culture embryos can directly develop into young seedlings. When embryos cannot be excised easily, whole ovules can be cultured and when ovule is too small to be removed, whole ovaries may be cultured.

Embryo culture Incompatibility, failure of pollen to fertilize an ovule is the major obstacle in conventional breeding programmes. Incompatibility may be due to failure of pollen tube to develop the abortion of the young embryo. This problem can be overcome by fertilizing the ovule in vitro. Pistils are cultured on nutrient media and after removing the part of the ovary wall, fertilization is effected by placing the pollen directly on the exposed ovule. Sometimes incompatibility may be due to failure of embryo to develop because nutrient starvation prevents the endosperm from developing. In this instance, plants can be recovered for breeding purpose by culturing the embryo on nutrient media. Immature seeds are surface sterilized and soaked in water for few hours. Embryos are then excised from immature seeds under asceptic conditions. The excised embryos are directly transferred to plate or tube containing synthetic nutrient medium. It is then provided with proper temperature, humidity and photoperiod. The frequency of excised embryos that gives rise to seedling varies greatly. Embryo culture is used largely for making interspecific and intergeneric crosses within tribe Triticeae of grass family (Poaceae). The hybrids rabed from embryo culture are used for (i) phylogenetic studies and genome analysis, (ii) transfer of useful agronomic traits from wild genera and species to the cultivated crops and (iii) to raise synthetic crops like triticale. Embryo culture has also been used for haploid production through distant hybridization followed by elimination of the chromosomes of one parent in the hybrid embryos (cultured). Example of this can be

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hybridizaiton of wheat or barley with Hordeum bulbosum leading to production of haploid wheat or haploid barley. Also haploid wheat plants are obtained through culture of hybrid embryos from wheat x maize crosses.

Ovule culture Ovules from members of the families like Malvaceae, Fabaceae, Cruciferae, Solanaceae, etc. after fertilization are successfully cultured to obtain successful crosses and mature embryos/seeds. Ovule culture is mainly tried only in those cases, where embryo aborts very early and embryo culture is not possible due to difficulty of its excision at a very early stage.

Ovary culture Technique for growing excised ovaries on culture medium was developed in several plants like wheat, barley, tobacco, tomato, beans etc. Ovary culture is often used either for in vitro pollination and fertilization or for embryo rescue when embryo culture and ovule culture are not possible or fail. Interspecific hybrids, using ovary culture are obtained for several genera of Brassica. For interspecific or intergeneric crosses, ovaries are excised at the zygote stage or at the two-celled proembryo stage and normal development is completed in vitro. The development of fruit may be promoted by application of chemicals like IAA or by addition of coconut milk to the medium. Ovary culture may also lead to the development of parthenogenetic haploids in wheat, barley and other crops. Ovary culture also has been used for a study of the understanding of physiology of fruit development.

Anther and Pollen Culture An essential requirement in many plant breeding programmes is plant stocks homozygous for genes of interest. Such stocks breed true but are difficult to prepare and several times hack crossing "vill be required. A much simpler alternative is to use anther or pollen culture. Plant regenerated from anther or pollen culture are haploid but it is easier to double the chromosome by colchicine treatment. Colchicine interferes with mitosis and effects chromosome doubling by preventing sister chromatids from separating at anaphase. Many of the agronomically important traits such as yields, grain-moisture contents etc. have obvious phenotype and their selection and quantitation is difficult in breeding programmes. Restriction fragment length polymorphism (RELP) may be useful here. Pollen grains are determinate cells whose normal development involves the formation of a pollen tube and male gametes. When placed on suitable nutrient media like MS medium, most pollen grains follow this development pathway but few grains will form callus instead. Instead of culturing isolated pollen grains, it is possible to culture the intact anthers containing developing pollen and this results in the formation of embryoids directly from pollen grains. These embryoids can be induced to develop into whole plants which are true haploids. As long as optimal conditions for donor plants and explants are provided, it is possible to obtain several hundred haploid plants from single anther. For successful anther culture, it is necessary to excise the flower buds at the correct time and usually this is at the time of the first mitotic division of the uninucleate microspore tetrads. Storage of pollen grains in cold for several days to several weeks (cold stress) before putting pollen grains on nutrient medium improves the frequency at which pollen grains develop into plants. Anther culture or pollen culture is a very popular method for production of haploids. Haploids have been obtained in more than 150 species belonging to 23 families of angiosperms. Many of them are economically important. Successful anther culture depends on: (i) conditions of donor plant; (ii) genotype of donar plant; (iii) the pretreatment; (iv) the development stage of anther; and (v) the culture medium and conditions during culture growth.

Endosperm Culture Tissue culture may be used for culturing endosperm, which is unique in its function of supplying nutrition to the developing embryos and in being triploid in its chromosome constitution. Triploid plants

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are useful for production of seedless fruits (e.g. apple, banana, watermelon etc.) and for production of trisomics for cytogenetic studies. Generally these triploids are obtained by crossing colchicine induced tetraploids with diploids followed by rescuring the triploid embryos. Endosperm culture is an alternate method. Steps of endosperm culture are: (i) immature or mature seeds are dissected under asceptic conditions and endosperm along with embryo are excised. (ii) the excised endosperms are cultured on a suitable medium and embryos are removed after initial growth. (iii) the initial callus phase is followed by 'embryogenesis' or 'shoot bud differentiation' and (iv) the shoots and roots may subsequently develop and complete triploid plants can be established for further use. THE GENE ADDITION APPROACH TO PlANT GENETIC ENGINEERING

Gene addition simply involves the use of cloning techniques to introduce into a plant one or more new genes coding for a useful characteristic that the plant lacks. A good example of the technique is provided by the development of plants that resist insect attack by synthesizing insecticides coded by cloned genes.

Plants that Make their Own Insecticides Plants are subject to predation by virtually all other types of organism-viruses, bacteria, fungi and animals-but in agricultural settings, the greatest problems are caused by insects. To reduce losses, crops are regularly sprayed with insecticides. Most conventional insecticides (e.g. pyrethroids and organophosphates) are relatively non-specific poisons that kill a broad spectrum of insects, not just the ones eating the crop. Because of their high toxicity, several of these insecticides also have potentially harmful side effects for other members of the local biosphere, including in some cases humans. These problems are exacerbated by the need to apply conventional insecticides to the surfaces of plants by spraying, which means that subsequent movement of the chemicals in the ecosystem cannot be controlled. Furthermore, insects that live within the plant or on the undersurfaces of leaves, can sometimes avoid the toxic effects altogether. What features would be displayed by the ideal insecticide? Clearly it must be toxic to the insects against which it is targeted, but if possible, this toxicity should be highly selective, so that the insecticide is harmless to other insects and is not poisonous to animals and to humans. The insecticide should be biodegradable, so that any residues that remain after the crop is harvested, or which are carried out of the field by rainwater, do not persist long enough to damage the environment. And it should be possible to apply the insecticide in such a way that all parts of the crop, not just the upper surfaces of the plants, are protected against insect attack. The ideal insecticide has not yet been discovered. The closest we have are the d-endotoxins produced by the soil bacterium Bacillus thuringiensis.

(a) The i1-endotoxins of Bacillus thuringiensis Insects not only eat plants: bacteria also form an occasional part of their diet. In response, several types of bacteia have evolved defence mechanisms against insect predation, an example being Bacillus thuringiensis which, during sporulation, forms intracellular crystalline bodies that contain an insecticidal protein called the a-endotoxin. The activated protein is highly poisonous to insects, some 80,000 times more toxic than organophosphate insecticides and is relatively selective, different strains of the bacterium synthesizing proteins effective against the larvae of different groups of insects. Table 25.6. The range of insects poisoned by the various types of B. thuringiensis aendotoxins. a-Endotoxin type

Effective against

CryI Cryll CryIII

Lepidoptera (moth and butterfly) larvae Lepidoptera and Diptera (two-winged) larvae Lepidoptera larvae Diptera larvae Nematode worms Nematode worms

CryN CryV CryVI

373

Plant Biotechnology

The a-endotoxin protein that a-Endotoxin gene accumulates in the bacterium is in fact an inactive precursor. After ingestion by the insect, this protoxin is cleaved by proteinases, resulting in shorter versions of the protein that display the toxic activity, binding to the inside of the insect's gut and damaging the surface epithelium so that the insect is unable to feed and consequently starves to death. Variation in the structure of these binding sites in different ~ inr~:einase digestion groups of insects is probably the ~e insect gut underlying cause of the high specificities displayed by the different types of a-endotoxin. B. thuringiensis toxins are not ~ Active toxin recent discoveries, the first patent for their use in crop protection having been granted in 1904. Over the years, there have been several attempts to market them as environmentally friendly insecticides, but their biodegradability acts as a Damages the gut disadvantage because it means that epithelial cells they must be reapplied at regular Fig. 25.1. Mode of action of a if-endotoxin. intervals during the growing season, increasing the farmer's costs. Current research is therefore aimed at developing a-endotoxins that do not require regular application. One approach is via protein engineering, modifying the structure of the toxin so that it is more stable. A second approach is to engineer the crop to synthesize its own toxin.

~ In._ proIo,'"

\

(b) Cloning a d-endotoxin gene in maize Maize is an example of a crop plant that is not served well by conventional insecticides. A major pest is the European com borer (Ostrinia nubilialisl, which tunnels into the plant from eggs laid on the undersurfaces of leaves, thereby evading the effects of insecticides applied by spraying. The first attempt at countering this pest by engineering maize plants to synthesize a-endotoxin was made by plant biotechnologists at a Ciba-Geigy laboratory in North Carolina. They worked with the CryIA(b) version of the toxin, which had previously been shown to be a 1155-amino acid protein, with the toxic activity residing in the segment from amino acids 29 to 607. Rather than isolating the natural gene, the Ciba-Geigy group made a shortened version, containing the first 648 codons, by artificial gene synthesis. This strategy enabled them to introduce modifications into the gene to improve its expression in maize plants. For example, the codons that were used in the artificial gene were those known to be preferred by maize, and the overall GC content of the gene was set at 65%, compared with the 38% GC content of the native bacterial version of the gene. The artificial gene was ligated into a cassette vector between a promoter and polyadenylation signal from cauliflower mosaic virus, and introduced into maize embryos by bombardment with DNA-coated microprojectiles. The embryos were grown into mature plants, and transforrnants identified by PCR analysis of DNA extracts, using primers specific for a segment of the artificial gene. The next step Was to use an immunological test to determine if a-endotuxin was being synthesized by the transform".!d plants. The results showed that the artificial gene was indeed active, but that the amounts

Biotechnology

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of a-endotoxin being produced (a) Synthesis of an artificial a-endotoxin gene varied from plant to plant, from 1155 about 250 to 1750 ng of toxin per mg of total protein. These B. thuringiensis gene differences were probably due to Artificial gene positional effects, the level of 607 29 expression of a gene cloned in a plant (or animal) often being Preferred codons and influenced by the exact location of GC content for maize the gene in the host chromosomes. For reasons that are not fully understood, genes inserted at some (b) Attachment of a promoter and polyadenylation signal positions are less well expressed than genes located at other positions. Promoter sequence Were the transformed plants able to resist the attentions of the com borers? This was assessed by field trials in which transformed and Polyadenylation sequence normal maize plants were artificially infested with larvae and the effects of predation measured over a (c) peR analysis of mature plants period of 6 weeks. Two criteria were used: the amount of damage suffered by the foliage of the 1. DNA size markers infested plants, and the lengths of the tunnels produced by the larvae 2. Result of PCR With DNA from boring into the plants. In both a transformed plant respects, ,the transformed plants 3, Result of PCR With DNA from gave better results than the normal a non-transformed plant ones, In particular, the average lengths of the larval tunnels was reduced from 40.7 cm with the controls to just 6.3 cm for the engineered plants, In real terms, this is Fig. 25.2. Important steps in the procedure used to obtain genetically engineered maize plants expressing an artificial ikndotoxin gene. a very significant level of resistance,

~

•

e\

Other .Gene Addition Projects Maize is not the only plant that has '------Gene inserted at pOint A - been engineered to produce a-endotoxin, poorly expressed similar projects having been carried out with rice, cotton, potato, tomato and other crops. Neither is this the only approach to Gene inserted at point B insect resistance. Equally successful results highly expressed have been obtained with genes coding for proteinase inhibitors, small polypeptides that disrupt the activities of enzymes in the Fig. 25.3. Positional effects. insect gut, preventing or slowing growth. Proteinase inhibitors are produced naturally by several types of plant, notably legumes such as cowpeas and common beans, and their genes have been successfully transferred to other crops which do not normally make significant amounts of these proteins. The inhibitors are particularly effective against beetle larvae

~

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375

that feed on seeds, and so may be a better alternative than a-endotoxin for plants whose seeds are stored for long periods. In many cases, the objective is to improve the plant's ability to withstand pests such as insects, fungi, bacteria and viruses, or to be able to resist the toxic effects of herbicides used to control weeds. Other projects are starting to explore the use of genetic engineering to improve the nutritional quality of crop plants, for example by increasing the content of essential amino acids and by changing the plant biochemistry so that more of the available nutrients can be utilized during digestion by humans or animals.

Table 25.7. Examples of gene addition projects with plants. Gene for

Source organism

Characteristic conferred on transformed plants

a-Endotoxin Proteinase inhibitor Chitinase Glucanase Ribosome-inactivating protein Ornithine carbamoyl transferase Virus coat proteins Satellite RNAs 2'-5' oligoadenylate synthetase Mutant form of 5-enolpyruvylshikimic acid 3-phosphate synthase Phosphinothricin acetyl transferase Ribonuclease Methionine-rich protein Thaumatin Monellin Stearyl-CoA desaturase

Bacillus thuringiensis Various legumes Rice Alfalfa Barley Pseudomonas syringae Various viruses Various viruses

Insect resistance Insect resistance Fungal resistance Fungal resistance Fungal resistance Bacterial resistance Virus resistance Virus resistance Virus resistance Herbicide resistance

Rat Salmonella typhimurium Streptomyces hygroscopicus Bacillus amylo/iquefaciens Brazil nuts Thaumatococcus danielli Thaumatococcus danielli Rat

Herbicide- resistance Male sterility Improved S content Sweetness Sweetness Higher mono unsaturated fatty acid content

GENE SUBTRACTION

Gene subtraction is a misnomer as the modification does not involve the actual removal of a gene, merely its inactivation. There are several ways by which a single, chosen gene could be inactivated in a living plant, the most successful so far in practical terms being the use of antisense technology. The example we will focus on is an important one as it has resulted in one of the first genetically engineered foodstuffs to be approved for sale to the general public.

The Principle behind Antisense Technology In an antisense experiment, the gene to be cloned is ligated into the vector in reverse orientation. This means that when the cloned 'gene' is transcribed, the RNA that is synthesized is the reverse complement of the mRNA produced from the normal version of the gene. We refer to this reverse complement as an antisense RNA, sometimes abbreviated to asRNA. An antisense RNA is able to prevent synthesis of the product of the gene it is directed against. The underlying mechanism is not altogether clear, but it almost certainly involves hybridization between the antisense and sense copeis of the RNA. It is possible that the block to expression arises because the resulting double-stranded RNA molecule is rapidly degraded by cellular ribonucleases or the explanation might be that the antisense RNA simply prevents ribosomes from attaching to the sense strand. Whatever the mechanism, synthesis of antisense RNA in a transformed plant is an effective way of carrying out gene substration.

Biotechnology

376 Antisense RNA and the Engineering of Fruit Ripening in Tomato At present, commerciallygrown tomatoes and other soft fruits are usually picked before they are completely ripe, to allow time for the fruits to be transported to the marketplace before they begin to spoil. This is essential if the process is to be economically viable, but there is a problem in that most immature fruits do not develop their full flavour if they are removed from the plant before they are fully ripe. The result is that mass-produced tomatoes often have a bland taste which makes them less attractive to the consumer. Two biotechnology companies, Calgene in the USA and ICI Seeds in the UK, have used antisense technology as a means of genetically engineering tomato

--------

Gene in the correct orientation

•

!

Promoter

_ _- - - - - - - m R N A

Gene In the reverse Orientation

•

!

Promoter

~antlsense

RNA

(reverse complement althe mRNA) Fig 25,4 Antisense RNA.

antisense

plants so that the fruit ripening process is slowed down. This enables the grower to leave the fruits on the plant until they ripen to the stage where the flavour has fully developed, there still being time to transport and market the crop before spoilage sets in.

RNi\

--_.-/

(a) The role of the polygalacturonase gene in tomato fruit ripening

Ribosomes cannot attach?

Degraded by ribonucleases? Fig. 255. Possible mechanisms for thiS inhibition oj gene expressIOn by antisense RNA

The timescale for development of a fruit is measured as the number of days or weeks after flowering. In tomato, this process takes approximately 8 weeks from start to finish, with the colour and flavour changes associated with ripening beginning after about 6 weeks. At about this time, a number of genes involved in the later stages of ripening are switched on, including one coding for the polygalacturonase enzyme. This

377

Plant Biotechnology

enzyme slowly breaks down the polygalact100 uronic acid component of the cell walls in the fruit pericarp, resulting in a gradual softening. The softening makes the fruit palatable, but if taken too far results in a squashy, spoilt tomato "0_ c attractive only to students with limited financial c1$ iii0 _ _ Polygalacturonase E resources.

How could antisense technology be used to achieve this result? 8 (b) Cloning the antisense polygalact°6 7 uronase 'gene' Weeks after flowering The experiment that we follo..v was carried Fig. 25.6. The increase in polygalacturonase gene expression seen out by the Piant Biotechnology Section of leI during the later stages of tomato frUit ripening. Seeds, together with scientists at the University of Nottingham in the mid-1980s. A 730-bp restriction fragment was obtained from the S'region of the normal polygalacturonase gene, representing just under half of the coding sequence. A plant Polygalacturonase gene polyadenylation signal was attached to the beginning of this fragment, a cauliflower mosaic virus promoter R R was ligated to the end and the construction was inserted into the Restriction ligation to control sequences Ti plasmid vector pBIN19. Once in the reverse orientation inside the plant, transcription from the cauliflower mosaic virus promoter should result in synthesis Polyadenylation signal of an antisense RNA complePromoter mentary to the first half of the polygalacturonase mRNA. • • Antisense Previous experiments with antisense polygalacturonase RNA had suggested that this would 'gene' be sufficient to reduce or even Fig. 25.7. Construction of an antisense polygalacturonase 'gene'. R, restriction prevent translation of the target site. mRNA. Transformation was carried out by introducing the recombinant pBIN19 molecules into Agrobacterium tumefaciens bacteria and then allowing the bacteria to infect tomato stem segments. Small amounts of callus material collected from the surfaces of these segments were tested for their ability to grow on an agar medium containing kanamycin (remember that pBIN19 carries a gene for kanamycin resistance). Resistant transformants were identified and allowed to develop into mature plants. The results of the experiment were assessed in a number of ways: 1. The presence of the antisense 'gene' in the DNA of the transformed plants was checked by Southern hybridization. 2. Expression of ,the antisense 'gene' was measured by Northern hybridization with a single-stranded DNA probe that would hybridize only the antisense RNA. 3. The effect of antisense RNA synthesis on the amount of polygalacturonase mRNA in the cells of ripening fruit was determined by northern hybridization with a second single-stranded DNA probe,

'\

I

Biotechnology

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.,

A. tumefaciens containing this one specific for the sense recombinant pBIN19 mRNA. These experiments Tomato stem showed that ripening fruit from transformed plants segment contained less polygalact./ / /-;,----/'". uronase mRNA than the / / /, , - - -' V .,fruits from normal plants. / / / " / - / /'/ ~Incubatefor 4. The amounts of polygalact~/ / / / /' several days uronase enzyme produced in Agar medium ~ the ripening fruits of Test lor transformed plants were ~ - - - =:. kanamYCin ----/' - - ..' , resistance estimated from the intensities Callus growths , : /", ~ _- _ :-. / / , • of the relevant bands after //'/' /' separation of fruit proteins by ,,////;/;/ , / //' / polyacrylamide gel electro, " " phoresis and by directly measuring the enzyme Fig. 25.8. Obtaining transformed plants by infection of stem segments with activities in the fruits. The recombinant A. tumefaciens. results showed that less enzyme was synthesized in transformed fruits. Most importantly, the transformed fruits, 100 although undergoing a gradual softening, could be stored for a prolonged period before beginning to spoil. This indicated that the antisense RNA had not completely inactivated Normal plants the polygalacturonase gene, but had "0 _ Ql rn nonetheless produced a sufficient reduction C r100-kb) DNA inserts. Also called YAC. Yeast episomal vector. A cloning vector for the yeast Saccharom yces cerevisiae that uses the 211m plasmid origin of replication and is maintained as an extrachromosomal nuclear DNA molecule. Yeast integrative plasmid (YIp). A yeast vector that relies on integration into the host chromosome for replication. Yeast replicative plasmid (YRp). A yeast vector that carries a chromosomal origin of replication.

Z The maximum lod score for a range of recombination fractions from 0 to 0.5. Zinc finger. Sequence-specific DNA binding proteins that contain domains that bind Zn2+. Zoo blot. Hybridization of a cloned human DNA sequence to DNA from various vertebrate organisms to determine whether the cloned DNA is evolutionarily conserved. Zmax.

INDEX Symbols i)-endotoxin, 372 30S initiation complex, 132 5-enolpyruvylshikimate-3-phosphate synthase, 383 70S initiation complex, 132

Adenylic acid, 59 Affinity chromatography, 186 Agrobacterium, 205, 212, 367 Agrobacteriumtumefaciens, 205, 217, 219, 262, 263, 264,366,377,380 Agromyza lantannae, 395 AIDS, 330 Alcaligenes, 383 Alcohol oxidase, 342 Alexandra, 310, 311 Alfred Hershey, 154 Alkaptonuria, 195 Allele, 162, 198 Allergies, 331 Allfrey, 68 Allolactose, 174 Allosomes, 33 Alpha constitutive heterochromatin, 40 Alpha-I-antitrypsin, 306 Alpha-amylase, 224 Alpha-foetoprotein (AFP), 275 Altmann, 53 ALU,I71 Alzheimer's disease, 307 Amino acid binding site, 97 Amino acyl synthetase, 97 Aminoacids, 121 Aminoacyl-tRNA synthetase, 129 Aminopeptidase, 132 Aminoscopy, 271 Amniocentesis, 198, 272, 293 Amoeba proteus, 16 Amplified, 253 Amyloplasts, 23

A A chain, 351 a-chain, 192 A Korenberg, 77 A. nidulans, 342 A rhizogenes, 366 A tumefaciens 205,206,207,262,263,366,380, 381 A Wiess, 20 AD. Hershey, 154 acceptor 98 Aconitase, 126 Actinomycin, 193 Actinomycin D, 182 Activating enzymes, 129 Activator, 168, 184 Activator gene, 167 ADA cDNA, 303 Adaptive enzyme, 174 Adaptor RNA, 96 Adenine, 56, 164 Adeno-associated virus, 299 Adenosine, 59 Adenosine deaminase, 292 Adenosine deaminase deficiency (ADD), 328 Adenosine monophosphate, 74 Adenosine phosphate, 59 Adenyl cyclase, 185 Adenylate cyclase, 362

425

426" Anaemia, 192 Andrew Gobea, 293 Anencephaly, 274 Anna Johnson, 316 Anthonomus grandis grandis, 381 Antibiotic, 193 Antibiotic-carrying plasmid, 217 Antibodies, 320 Anticodon arm, 97 Anticodon stems, 98 Anticodon, 97, 131 Antigen presentation, 323 Antigen, 320 Antigenic determinant, 320 Antiparallel, 59 Antisence technology, 375 Antisense RNA, 375, 382 Antonarakis, 280 Apolar, 61 Ara, 186, 187, 188 Arabidopsis thaliana, 262 Arabinose, 186 Archaebacteria, 12, 92 Arg, 167 Arlette, 310 Arlette Schweitzer, 310, 316 Arms, 96 Aro,364 ARS, 52 Arthur Kelman, 218 Arthur Korenberg, 83 Artificial chromosome, 52 Artificial insemination, 207, 315 Asbury, 60 Ascaris, 37 Ascaris megalocephala, 32 Ashanti, 293, 299 Ashanti DaSilva, 293 Aspergillus, 44 Aspergillus nidulans, 162, 342 ATP, 78 Attenuated, 358 Attenuated strains, 356 Attenuation, 179 Attenuator, 180 AUG,132 Aulocantha. 32 Aulosira Anabeana Tolypothrix, 393 Autoantibody fingerprinting, 7

Biotechnology

Autocatalytic, 68 Autologous cells, 299 Automation, 269 Autosomes, 33 Avers, 77 Avery, 3, 55, 73, 164 Avery Mac Leod, 154 Axial bars, 49 Azolla, 392, 394 Azospirillum, 385 Azotobacter, 385, 392, 393 Azotobacter chroococum, 393 B B cells, 322 ~-chain 192 ~ gal, 351 ~ galactosidase, 173, 175 B Lymphocytes, 322, 325 B memory cells, 325 B. Albert, 76 B. circulans, 345 B. japonicum, 385, 390 B. japonicum hup, 390 B. singer, 159 B. stearothermophilus, 347 B. subtilis, 348 B. thuringiensis, 206, 373, 379 Baby M, 316 Bacillus amyloliquefaciens, 252, 348 Bacillus circulans, 345 Bacillus magaterium, 393 Bacillus papilliae, 394 Bacillus stearothermophilus,. 333 Bacillus subtilis, 394 Bacillus thuringiensis 206,212,217,219,372,379, 394 Bacillus thuringiensis inserelensis, 394 Back mutation, 148 Bacteriophage lamda, 28 Bacteriophages, 4, 11, 163, 254 Bacterization, 392, 393 Baculoviruses, 343 Bahs,55 Balbiani, 35, 46 Balbiani rings, 48 Ballistic transformation, 367 Bam. 260 Bar, 383 Baranetsky. 34

Index Barchydanio rerio, 237 Bamstiel Wallace, 68 Barr body, 40 Barrt Barell, 167 Baruent, 143 Barziali & Thomas, 69 Baseplate, 11 Beadle, 166, 167 Beadly, 195 Becker muscular dystrophy, 305 Becker's MD, 280 Beeman, 46 Beermann,47,48,49 Begin, 137 Belar,32 Benign tumors, 313 Benzene ring, 57 Benzer, 165, 169 Beta constitutive heterochromatin, 40 Beta-galactosidase, 175 Betaglobin gene, 168 Bidirectional, 76 Bidirectional replication, 70 Binary fission, 254 Bio-pesticides, 8 Bioengineering, 3 Biofertilizer, 8, 392 Biogen,5 Bioherbicides, 395 Biolistic, 263 Biomonitoring, 8 Biosensors, 8 Biotechnology, 1, 3 Blender experiment, 154 Blood factor VIII, 216 Blood islands, 305 Blood typing, 268 Blue sclerotics, 200 Bomford,38 Bonds, 61 Bone marrow, 304 Bouquet stage, 38 Boyer, 3 Brachet, 121 Bradfield, 20 Bradyrhizobium, 385 Bradyrhizobium japonicum, 385 Bram,61 Branchet, 94

427 Brange, 351 Brassica,371 Breathnack, 168 Brener, 143 Brewer, 47 Bridges, 46, 190 Brittle bones, 200 Broda, 69 Broken, 169 Brunori, 107 Bubbles, 76 Burke judd, 46 Burnham, 162 C C-banding, 42 Cairn, 69, 72, 75, 76 Calcitonin, 351 Callan, 50, 51 Callus, 205, 225,368, 371 Calvert, 316 Camillo Golgi, 19 Cannabis sativa, 18 Capping, 100 Capsid,28 Carl Goran Heden, 1 Carol Greider, 81 Carotin, 22 Carotinoids, 22 Carrier, 286 Casperson, 94, 121 Cat eye syndrome, 45 Catabolite gene activator, 185 Catabolite repression, 185 Cell,ll Cell matrix, 21 Cell sap, 21 Cell wall, 11 Cellulolytic enzymes, 342 CEN,52 Cenalakis, 99 Central dogma, 122 Central dogma reverse, 122 Centrioles, 23 Centromere, 30 Centromere plate, 35 Ceramela, 99 Cercospora rodmanii, 395 Cerenotabdities elegans, 181 Cetus, 5

428 Chad,310 Chambon, 168 Chaperones, 138 Chargaff, 54, 56, 60 Chargaff's rules, 56 Charged tRNA, 129 Chase,155 Cheating genes, 169 Chelsea, 310 Chemical alteration, 159 Chimaera,4 Chironomous, 48 Chironomous larva, 46 Chironomous pallidivittatus, 48 Chironomous tentatus, 48 Chloe O'Brien, 319 Chloroplasts, 22 Cholera, 362 Cholesterol oxidase, 381 Chorion, 272 Chorion frondosum, 272 Chorionic villi, 272 Chorionic villus biopsy, 272 Christa, 310 Christian Anfinsen's, 137 Christian de Duve, 19 Christopher, 316 Chromatids, 34 Chromatin, 24,.30 Chromatin fibres, 28 Chromatophores, 24 Chromeoles, 35 Chromocenter, 47 Chromomeres, 35 Chromonemata, 34 Chromonematal fibrils, 34 Chromoplasts, 22 Chromosome arm, 30 Chromosome banding techniques, 40 Chromosome jumping, 259 Chromosome walking, 259 Chromosomes, 24, 28 Cilia,23 Cis-trans, 165 Cisternae, 20 Cistron, 164, 165, 167, 169, 170 Citrus,395 Cla,362 Clark Fraser, 288

Biotechnology

Claude, 20, 21 Cleft lip, 287 Clever, 48 Clonal propagation, 368 Clonal selection, 326 Clone, 4 Cloning, 210 Clostridium tetani, 260 Clostridium titani, 358 Clyde Hatchinson III, 167 Co-workers, 63 Code words, 142 Codon, 121, 131, 142, 157 Codon signal, 136 Cognate, 129 Cohen, 3 Coiled coil, 34 Colin Macleod, 152 Colinar, 144 Collar, 11 Collectotrichum gloeospriodes, 395 Common genes, 392 Complementary chains, 74 Complementary DNA, 252, 255 Complete penetrance, 200 Computer aided protein model, 7 Congenital hip defects, 287 Congenital ptosis, 286 Consanguineous matings, 287 Consensue, 112 Consensus, 112 Constitutive proteins, 173 Continuous replication, 79 Contractile vacuoles, 23 Copolymerase III, 101 Copyrights, 8 Core, 11 Core DNA, 44 Core enzyme, 124 Corepressor, 178 Corepressor-repressor complex, 174 Cornybacterium diphthriae, 357 Coupled by methionine, 213 Creutzfeldt-Jakob disease, 215 Crick, 3,54,60, 74,122,143,148,161,163 Crispina Calvert, 316 Cristae, 19 Crossing over, 162 Crotalaria juncea, 393

Index Crown gall, 205, 217 CryIA(a), 379 CryIA(b), 379 CryIA(c), 379 Cryoprecipitate, 199 Cryptic genes, 168 Crystal, 293 Cyamopsis tetragonoloba, 393 Cynthia, 293, 299 Cynthia Cutshall, 293 Cyperous rotundus, 395 Cystic fibrosis, 294, 296, 307, 319 Cytidine, 59 Cytidine monophosphate, 74 Cytidine phosphate, 59 Cytidylic acid, 59 Cytoplasm, 14 Cytosine, 56, 97, 164 Cytosol, 14 Cytotoxic T cells, 324 D &-Chain, 192 Dark bands, 46 Darwin, 282 Daucus,22 David,329 DD. Brown, 102 de Beer, 190 de novo, 199 Decidua basalis, 272 Deformylase, 132, 136 Degeneracy, 129 Dehydrouridine, 97 Demerec, 162 Deoxyinosine, 292 Deoxynucleotide monomeres, 56 Deoxyribonucleic acid, 53 Deoxyribonucleoside, 59 Deoxyribonucleoside triphosphate, 83 Deoxyribonucleotide monophosphates, 74 Developmental totipotency, 204 Diabetes mellitus, 200 Diamond v. Chakrabarty, 3, 234 Dicentric, 32 Oie, 159 Differentiation, 172 Dihydrofolate reductase, 191, 350 Dihydrofolate reductase-thymidylate synthase, 364 Dilichol phosphate, 140

429 Dimer,76 Dinucleotide, 256 Diplococcus pneumoniae, 73 Diploid number, 32 Diptera,47 Direct sequence analysis, 266 Directed mutagenesis, 335 Directive counseling, 284 Disbrey,68 Discontinously, 79 Discontinuous replication, 79 Disease, 198 Dispersed pseudogenes, 171 Dissociation, 168 Dissociation factor F3, 136 Dithiothreitol, 266 DNA coding regions, 216 DNA dependent RNA polymerase, 109 DNA fingerprint profile, 267 DNA fingerprinting, 7 DNA gyrase, 30 DNA ligase, 79, 81, 254 DNA noncoding regions, 216 DNA polymerase, 77, 83 DNA polymerase I, 255 DNA polymerase III, 78 DNA probes, 6 DNA typing, 268 DNA-directed RNA polymerase, 77 DNAase, 265 DNase 1,185 Dominant, 162 Dot blot analysis, 266 Dot blotting, 258 Dotty, 54 Double helix, 164 Double stranded DNA, 28 Double stranded RNA, 104 Downstream processing, 2 Dr. H.G. Khorana, 146 Dr. Miss Otsuka, 147 Drosophila, 31, 39, 46, 47, 51, 55, 64, 78, 163, 164,169,171,181,190,191,193,195,200 Drosophila melanogaster, 30, 39, 40, 46, 47, 161, 162, 190 Drumstick body, 40 Duchenne type, 305 Duchenne's muscular dystrophy, 279 Dujardin, 11

430 Dupraw, 43, 55 Dut,336 Dut ung, 339 dUTPase, 336 Dyson, 77 E

E. coli, 3, 11, 12, 13, 14, 29, 58, 62, 69, 70, 72, 74, 76, 78, 81, 82, 83, 98, 101, 112, 126, 130, 136,144,145,149,155,163,165,168,169,173, 174,181,183,184,186,193,197,199,212,213, 215,219,238,251,254,258,260,262,264,336, 337,338,339,340,341,345,346,347,348,356, 361,380,383,384,386,389,390,392 E. coli lac, 213 E.G. Balbiani, 48 Early gene, 119 Ecdysone, 48 Eco,392 Ecology, 211 Ectopic, 311 Ectopic pregnancy, 313 Ectoplasm, 25 Edmonds, 99 Edward Jenner, 352 EF-G,134 EF-Ts, 134 Ehinaster, 49 Eichhomia,395 Eichhornia crassipes, 395 Elaioplasts, 23 Elastase, 306 Elear,395 Electroporation, 219, 263 Elementary bodies, 16 Elementary chromosome fibril, 43 ELISA, 6 Elizabeth Blackburn, 81 Elongation, 131 Elongation factors, 134 Embryo adoption, 318 Embryo culture, 370 Embryogenic, 368 Embryoids,371 Embryonic stem (ES) cell, 223 Enables, 184 Endoduplication, 49 Endometriosis, 313 Endometrium, 182 Endomitosis, 49

Biotechnology

Endonuclease, 70 Endoplasm, 25 Endopolyploidy, 49 Endothelium, 305 Endotoxin, 356 Engineer nucleases, 349 Engineered DNA, 217 Engineered myoblasts, 305 Enhancers, 183 Enhances, 178 Enu, 300 Env,300 Enzymes, 19 Ephrussi, 166 Epithelial cells, 16 Epstein-Barr virus, 119 Ereky, 1 Erwin Chargaff, 56 Erythroid progenitor cells, 299 Erythropoietin, 3 Escherichia coli 11, 29, 155, 170, 336, 353, 356, 360,367,380,386,394 Ester bonds, 54 Estrogens, 182 Ethel, 222 Ethidium bromide staining, 266 Eubacteria, 12 Euchromatin, 39 Eugenics, 282 Eukaryotic, 11 Eukaryotic cells, 15 Ex vivo, 308 Ex vivo gene therapy, 299 Except, 182 Excision repair, 82 Exon sequences, 126 Exons, 124, 125, 192, 216 Exonuclease, 78, 79, 83 Exotoxin, 356 Explants, 225 Expression vectors, 258 Eyes, 76 F

E Jacob, 104, 193 E Ratiaman, 99 EM. Ritossa, 103 Factor dependent termination, 114 Factor IX, 198 Factor VIII. 252

Index

Faithful genes, 169 Familial hypercholesterolemia, 306 Fawcett, 21 Fedric,44 Feedback inhibition, 127 Feedback mechanism, 174 Fermentation and transformation, 2 Fibrinogen, 216 Fibroids, 313 Fingers, 117 First, 130, 266 Fischberg, 68 Fischer, 53 Flagella, 23 Flavobacterium okeanokoites, 349 Flemming, 49 F1urochrome quinacrine mustared, 42 Foetoscope, 272 Foetoscopy, 271 Foetuses, 304 Fok, 349, 350 Fold endonuclease, 349 Folded fibre model, 43 Fork,76 Forward mutation, 148 Fraenkel conrat, 160 Fraenkel-contrat, 159 Fragment, 78, 79 Frameshift mutation, 148 Frances E" Arrighi, 42 Francis Galton, 282 Frankia, 8, 385 Franklin, 60 Franklin W. Stahl, 53 Frederic Griffith, 73, 152 Freese' model, 43 Friedrich Meischer, 53 Fundulus, 238 Furberg,54 Fusing, 50 Fusion gene, 242 G G factor, 134 G-bands,42 "(-Chain 192 Gag, 300 Gain-ofcftmetion. 238, 241 GAL,341 Galactose epimerase. 341

431 Galactosemia, 199, 276 Galactoside permease, 175 Galactoside transacetylase, 175 Gall, 41, 51 Galton, 282 Gamete intrafallopian transfer, 317 Ganciclovi.", 307 Gangliosides, 197 Gay, 163 Geitter, 28 Gene, 124, 167 Gene amplification, 190 Gene as the ultimate unit of recombination, 161 Gene augmentation, 298 Gene cloning, 6, 251 Gene families, 163 Gene substitution, 298 Gene targeting, 222 Gene therapy, 7, 214 Gene-D, 168 Gene-E, 168 Gene-J, 168 Genentech, 3, 5 Genetic, 32 Genetic code, 121, 156 Genetic complementation, 386 Genetic counseling, 282 Genetic engineering, 251 Genetic screening, 270, 276 Genetic variation, 158 Genome, 29, 252 Genomic DNA, 256 Genomic library, 257, 264 Genonema, 34 Genotype, 189 George Gamow, 142 Germ-line gene therapy, 295 Giant fUSing, 50 Gieerke's disease, 197 Giles, 164 Gillian Air, 167 Glioma, 307 Glomus mosseae, 384 Glucose effect, 185 Glucose isomerase, 7 Glucose-sensitive operons, 185 Glycoprotein gene, 355 Glycoproteins, 140 Glycosidic bonds, 58, 59

Biotechnology

432 Glycosylated, 29 Glycosylation, 140 Goldstone, 109 Golgi bodies, 19 Goodenough&Le~ne, 69 Gowen, 163 Grana, 22 Granular, 20, 50 Green manuring, 392 Gregariousness in Cattle and Men, 282 Griffith, 55, 73, 152 Ground substance, 21 Growth hormone, 241, 305 Grun, 49 Guanine, 56, 97, 164 Guanosine, 59 Guanosine monophosphate, 74 Guanosine phosphate, 59 Guanylic acid, 59

H H.G. Khorana, 142, 144 Haeme, 192 Haemoglobin, 192 Haemophilia, 198, 287, 294 Haemophilia A, 216, 279 Haemophilus influenzae, 355 Haemophilus pertusis, 357 Hairy-cellleukemia, 216 Hannah,40 Haploid,32 Haplotype, 278 Hashimoto's thyroiditis, 329 Hayward, 115 Heitz, 39 Helicases, 77 Heliothix zea, 380 Helper T cells, 325 Henshaw, 98 Hepatitis B ~rus vaccine, 216 Hepatocytes; ~06 Herbert Boyer, 8 Hereditary emphyse~ 306 Hereditary genius, 282 Herman, 222 Hershey, 155 Hershey-chase experiment, 154 Hertz, 28 Hetero-chromosomes, 33 Heterocatalytlc, 68

Heterochromatic, 64 Heterochromatin, 39 Heteroduplex analysis, 260 Heteroduplex mapping, 260 Heteropycnosis, 38 Hexameric enterotoxin, 362 Hexosam inidase, 197 Highly repetitive DNA, 64 Histidine, 174, 180 Histone protein, 43 Histones, 44 HIV,330 HIV-1 envelope glycoprotein, 355 Hoffman, 280 Holocaust, 282 Homocystinuria, 276 Homologous recombination, 223 Hordeum bulbosum, 371 Host-specific genes, 392 House keeping, 172 Hsp, 343 Human growth hormone, 215, 244 Human immune deficiency ~rus, 330 Humulin, 215 Huntington's disease, 198, 278, 288 Hup, 390, 393 Huxley, 190 Hybridoma, 327 Hybritech, 5 Hydra, 16 Hydrogenase gene, 389 Hydrogenases, 389 Hydrophilic, 54 Hydrophobic, 53 Hydroxymethylcytosine, 29 Hypergamma-globulinemia, 328 Hypersensiti~ties, 331 Hyposomatotropism, 215 Hypothyroidism, 276 I Ice minus, 202 Ice plus, 202 Idiogram, 34 Imidazole ring, 57 Immobilized enzyme, 7 Immunological bank, 279 Immunotoxins, 6 In situ, 41,294,308 In situ gene therapy, 299

433

Index

In trans, 367 In utero, 270, 271 in vitro 6,39,137,144,146,147, 199,214,254, 255,256,281,296,333,370,371 In vitro fertilization, 316 In vivo, 213, 260 In vivo gene therapy, 299 Inactivated, 358 Incision enzyme, 70 Included genes, 168, 169 Inclusion body, 340 Incomplete, 200 Independence termination, 114 Inducible, 174 Inducible proteins, 173 Infertility, 311 Inherited characteristic, 189 Initiating, 144 Initiating codons, 165 Initiation, 131 Initiation end, 136 Initiation factor F1, 132 Initiation factor F2, 133 Initiation factors (IF), 131 Initiation point, 75 Initiation site, 123 Initiator proteins, 75 Initiator site, 184 Insulin, 6, 305, 351 Inter-chromomeres, 35 Interaction of genes, 162 Interbands, 46 Interferon, 6, 215, 308 Intergenic suppression, 149 Interleukin 1, 326 interleukin-2, 355 Interleukins, 308, 326 Internucleosomal DNA, 44 Internucleotide bonds, 60 Interrupted palindromes, 64 Intracytoplasmic sperm inection, 316 Intragenic suppression, 149 Introns, 124, 216, 192, 253 Intrude, 192 Invasive, 270 Isoacceptors, 129 Isochromosome, 45 Isoleucine, 180 Isozyme electrophoresis, 268

J

J. Cairns, 71, 72 J. Monod, 104, 193 J.B. Gurdon, 102 J.G. Gall, 103 J.H. Matthaei, 144 J.H. Taylor, 72 J.T. Finch, 44 Jacob,173 Jacob-Monod, 173, 174 James Phipps, 352 Jenner, 352 Johanseen, 189 Johanson, 161 Johanssen, 169, 189 John Phillips, 279 Johnson, 316 Jonathon, 310 Jonathon Loew, 310 Judge Richard N. Parslow, 316 Jumping genes, 168 K K. pneumoniae, 386, 387, 388, 389 K.L. Agarwal, 147 Kaiser, 351 Kalchar,59 Kanamycin, 377 Kaposi's sarcoma, 330 Karl Ereky, 1, 3 Karyotin, 24 Karyotypes, 34 Kaufmann, 28 Keiferia Iycopersicella, 380 Kenneth Vaux, 284 Kevin's, 310 Khorana, 3, 54, 146, 147 Khorana's direct method, 146 Killer weed, 18 Kinetochore, 36 Klebsiella ozaenae, 384 Klebsiella pneumoniae, 386 Kohler, 3 Koller, 46 Kollman, 20 Korenberg,74 Kornberg, 84 Kossel, 53 Krakow, 89 Kunkel. 280

Biotechnology

434 Kurstaki, 379 Kuwanda,28 L L. Frey, 76 L. pictus, 67 L. cella, 11 .Lac, 178, 181, 185, 186,213 Lactose, 176 LacZ,213 Lader, 146 Lagging strand, 79 Lagging strand of DNA, 78 Lamp-brush chromosomes, 35 Lampert, 44 Land marks, 49 Landgreen, 197 Lantana, 395 Last, 266 Lateral loops, 193 Laura, 292 Laura Cay Boren, 292 Lea, 162 Leader polypeptide, 179 Leader-attenuator region, 178 Leading strand, 79 Leading strand of DNA, 78 Leishmania, 364 Leishmania major, 364 Lens esculenta=L. culinaris, 393 Leonard,283 Leonard Gobea, 293 Leu, 186 Leucine, 180, 256 Leucoplasts, 22, 23 Leukemia, 293 Levine,54 Lewis, 166 Ugase, 5, 83 Ugnin,18 Ukely event, 283 Uliaceae,24 Urn, 99 Undow, 217 Unked genes, 162 Unker DNA, 44 Unus pauling, 142 Upofectins, 298 Uposomes, 219 Upsomes. 298

Lloyd,50 Locus, 162 Loew, 310, 311 Loop, 76, 96 Loss-of-function, 238, 241 Louise Joy Brown, 317 Lump, 97 Lumpy, 50 Lysine, 145 Lysosomal storage diseases, 307 Lzawa,68

M M. chase, 154 M. Masters, 76 M. tuberculosis, 358, 360 M.L. pardue, 103 MW. Nirenberg, 144 Macleod, 3, 55, 73, 164 Maclyn McCarthy, 152 Macrotyloma unifiorum=Dolichos uniflorus, 393 Maize, 35 Major area, 55 Major groove, 55 Major histocompatibility complex, 320 Males, 40 Mandel,168 Manduca sexta, 380 Maple syrup urine disease (MSUD), 276 Mark, 32, 316 Martha Chase, 154 Mary Beth Whitehead, 316 Mast cells, 332 Master, 69 Master and slave hypothesis, 50 Mauro, 310 Max Perutz, 333 Mc chelke, 47 Mc Cook, 395 Mc Devmott, 44 McCarty,3, 55,73,154,164 McClintock, 167, 168 Mediola,32 Meischer, 53 Melanoma, 308 Melilotus purvifora=M. indica, 393 Membrane protein, 137 Memory cells, 326 Mendel, 161, 169, 189 Meningomyelocele, 275

435

Index

,

Mesotoma, 32 Messenger RNA, 157 Metabolic engineering, 7 Metacentric, 35 Metacentric chromosome, 31 Metallothionine, 243 Methionine, 132, 144 Methotrexate, 191 Methylation, 100 Michele, 317 Microinjection, 209, 367 Micropropagation, 368 Microsatellites, 82 Microsome, 20, 21 Microtrabecular lattice, 17 Microtubules, 23 Miller, 68 Milstein, 3 Minor area, 55 Minor groove, 55 Mirsky,68 Mismatch repair, 82 Missense, 176 Missense mutation, 148 Mixed vaccines, 356 Modulation, 172 Moieties, 140 Molecular farming, 7 Monocistronic, 98 Monoclonal antibody, 6, 327 Monocots, 207 Monod,173 Monomer, 76 Moore, 67 Morgan, 161 Mori,59 Mouse eggs, 242 mRNA,100 MTrGH plasmid, 244 Mulder, 121 Muller, 38, 162, 164, 165 Multicellular, 11 Multigene families, 171 Multiple alleles, 162 Muscle cells, 16 Mutant selection, 226 Mutants, 166 Mutation, 158, 159, 166 Muton, 165, 167, 169

Mycobacterium tuberculosis, 56, 357, 360 Mycoherbicide, 395 Mycolasmas, 14 Mycoplasma, 13, 14 Mycorrhizae, 8 Myoblast transfer therapy, 305 Myoblasts, 305 Myrot\1ecium verrucaria, 383

N N-C. glycosidic bonds, 95 N-lmked giycoproteins, 140 N.W. Nirenberg, 142 Nancy, 220 ," Nascent polypeptide, 134 Nass,67 National Genetics Foundation, 282 Negative, 39 Neisseria meningitidis, 355 Neochetina olchhorniae, 395 Neomycin phosphotransferase gene, 380 Neonatal, 276 Network rigidity, 7 Neural tube defects, 275 Neurons, 306 Neurospora, 166, 170 Nick translation reaction, 84 Nicking closing enzyme, 90 Nicotinamide adenine dinucleotide, 81 Nif, 386, 387, 388, 389, 390 NifA, 388 Nirenberg, 142, 143, 145, 146 Nitrogenase, 386 Nod, 390, 392 NodABC, 392 Non photosynthetic, 12 Non-conservative replication, 70 Non-sense codons, 144 Nondirective counseling, 284 Nonesence mutatio~, 148 NOninvasive, 270 ~ Noqsepse, 176 No~al loops, 50 Northern blot, 367 Northern blotting, 258 Northern hybridization, 377 Nostoc, 393 Nuclear pores, 24 Nuclease Fold, 349 Nucleases, 78

436 Nucleation, 202 Nucleic acid hybridization, 264 Nucleic acid, 53 Nuclein, 53 Nucleoid, 15 Nucleolar organizer regions, 171 Nucleolar organizer, 38 Nucleoproteins, 54 Nucleosidases, 59 Nucleoside, 53, 59 Nucleosomes, 44 Nucleotide, 14, 53, 59, 164 Nucleus, 11, 24

o O-linked glycoproteins, 140 Obligate parasites, 155 Obligate photoautotrophs, 15 Oecophylla smaragdina, 395 Okazaki, 79 Okazaki fragment, 79 Okazaki pieces, 83 Okazaki segments, 78 Oligonucleotide, 256 Olistherozones, 37 One gene theory, 167 , One gene-one enzyme hypothesis, 167 One gene-one polypeptide, 167 Open complex, 112 Open reading frame, 122 Operator, 176 Operon hypothesis, 173 Ophrytrocha, 32 Organogenic, 368 Oryzias latipes, 237 Osmunda,24 Ostrini~ nubilialis, 373 Oswald Avery, 152 Othale, 115 Otsuka, 147 Oudest,44 Ovalbumin, 144, 168, 182 Ovary culture, 370 Over circular type, 24 Overlapping, 168, 169 Ovule culture, 370 p P. Broda, 76 P. fluorescens, 212

Biotechnology

P. pastoris, 342 P. syringae, 202 P. syringe, 217 PA Levine, 53 Packaging cell line, 300 Painter, 49 Painter, 48 Palade, 21 Palate, 287 Palindromic DNA, 64 Pamela, 310, 311 Panicum dicotomiflorum, 395 Paramecium, 23, 68 Pardue, 41 Particle bombardment, 219 Parvovirus B19, 299 Pasteurella pestis, 357 Paszkowski, 217 Pat, 383 Patents, 8 Pathogenesis-related (PR) proteins, 384 Pathogens, 320 Paul,116 Pauling, 192 Pauling & Corey, 54 Pavan, 47 Peacock,68 Penicillin, 3 Peptide bond, 121, 134 Peptide chain, 121 Peptide vaccines, 361 Peptidyl transferase, 134 Perfect palindromes, 64 Permease, 176 Perry & Kelly, 98 Pfitzner, 35 Phage, 251, 253, 254 Phaseolus, 46 Phenotype, 189 Phenylalanine, 145, 180 Phenylketonuria, 199 Phillip Sharp, 125 Phosphodiester, 58 Phosphodiester bond, 54, 59, 60, 83 Phosphodiesterase, 95, 185 Phosphorylase, 59, 75 Phosphorylation, 74 Photo synthetic, 12 Photolyases, 82

437

Index

,

Photoreactivation, 82 Phytophthora palmivora, 395 Pichia pastoris, 342 Piko,67 Plasma membrane, 15, 16 Plasmid, 4, 205, 217, 251, 253 Plasmodium falciparum, 214 Plastids, 21 Platt, 115 Platysome, 44 Pleiotropic, 192 Pluripotent, 304 Plusia verticillate, 395 Pneumocystic carinii, 330 Pol, 300, 331 Polar effect, 176 Polar mutations, 176 Polarity, 59, 176 Poly, 100 Poly adenylation, 100 Poly-A tail, 126 Polycentric chromosomes, 37 Polycistronic, 98 Polycistronic mRNA, 180 PolygalacturonCise, 376 Polygalacturonase mRNA, 377 Polylysine, 145 Polymerase, 79,146,176 Polymerase a-primase, 81 Polymerase chain reaction, 6, 339, 367 Polymerase I, 77, 78 Polymerase III, 78 Polynucleotide phosphorylase, 144 Polypeptide chain, 165 Polyphenylalanine, 145 Polyploid, 32 Polyproline, 145 Polyribosome, 21, 131 Polysome, 131 Polytene chromosomes, 46 Polytene theory, 48 Polyvalent vaccines, 356 Pontecorvo, 161, 162 Porter, 20 Positional effects, 374 Positive, 39 Post transcriptional controls, 126 Post translational controls, 126 Power house, 20

Predictive relationship, 245 Preimplantation genetic diagnosis, 318 Preproinsuiin, 140 Presence, 184 Prevents, 178 Pribnour Box, 112 Primary immune response, 321, 325 Primary transcript, 125 Primase,83 Primate, 77 Primer, 255 Pro-chromosomal, 2~ Probe, 257 Prochromosomes, 24 Profile, 267 Progenitors, 305 Progesterone, 182 Progesterone receptor, 182 Progesterone response element, 182 Proinsulin, 140 Prokaryotic, 11 Proline, 145 Promoter Tyr-tDNA terminator, 199 Promoters, 111 Promotor gene, 176 Proplastids, 22, 23 Proplasts, 204 Protamine, 53 Protein engineering, 7 Proteinase K, 265 Proteinases, 373 Proteolysis, 139 Protists, 23 Protoplasm, 11 Protoplast, 225 Protoplast fusion, 204 Protoxin, 373, 379 Protropin, 215 Prustner, 107 PsbA,383 Pseudo-uridine, 97 Pseudogenes, 163, 168, 171 Pseudomonas flurescens, 212 Pseudomonas putida, 383 Pseudomonas syringae, 202 Pseudomonas syringe, 217 Psychodynamics, 288 Psychosomatic, 288 Puffs, 47, 48, 193

438 Pur, 364 Purines, 56, 164 Purkinje, 11 Pyrimidines, 56, 164 Pyrophosphatase, 84 Pyrophosphate, 83

Q

Q bands, 42 R R-band,42 R. leguminosarum, 390 R leguminosarum hup, 390 R. meliloti, 390, 392 R. Okazaki, 77, 79 R.meliloti, 392 RP. Perey, 99 R W. Holleye, 144 Randall, 68 Randomly, 296 .. Rare event, 283 Rat, 242 Rate of gene transcription, 181 Ray L'Esperance, 317 Recessive, 162 Recombinant DNA, 6 Recombinant DNA molecule, 4 "". Recombinant DNA technology, 159;- ~~ "'Recombination, 158 ,~ Recombivax HB, 216 Recon, 165, 167, 169 Reconstitution experiment of H, 159 Reduced penetrance, 200 Reefs, 18 Regulator, 176, 186 Regulator gene, 176, 193 Regulatory genes, 170 Regulatory nodD gene, 392 Regulatory sequences, 243 Relational coils, 34 Release factors, 136 Reo, 164 Repair replication, 70 Repeating units, 44 Repetitive DNA, 64 Replicase, 159 Replicating units, 70 Replication, 68, 157 Replication error, 159

Biotechnology

Replication fork, 76 Replication fragments, 79 Replication point, 76 Replicons, 70 Reporter function, 238 Repressible enzymes, 174 Repressor, 184 Repressor substance, 176 Reproductive technologies, 207 Resinde,38 Ressende 37 Restriction endonucleases, 5 Restriction enzymes, 252 Restriction fragment length polymorphisms, 277 Restriction site, 252 Reticulum, 24 Reverse transcriptase, 81, 89, 253, 255 Reversion, 148 RFLPs,277 Rhinanthus, 46 Rhizobia, 393 Rhizobium, 201, 202, 385, 389, 390, 392, 393 Rhizobium leguminosarum, 390 Rho, 124 Rhoades, 167 Rhyncosciara, 46 Ribonuclease, 98 Ribonucleic acid, 53 Ribonucleoside, 59 Ribonucleotides, 59 Ribosomal gene of Drosophila, 168 Ribosomal RNA, 98 Ribosome recognition site, 97 Ribosomes, 14, 15, 21, 130 Riccardo, 310 Rich, 63 Richard Palmiter, 215 Richard Roberts, 125 Rickettssia prowazeki, 359 Ris,43 RNA particles, 20 RNA polymerase, 104, 123, 146 RNA polymerase II, 181 RNA primer, 77, 79 RNA proceSSing, 125 RNA splicing, 125 RNA,28 Robert Brown, 11 Robert Guthrie, 276

439

Index

Robert Hooke, 11 Robert M. Veatch, 285 Roberts, 115 Robosomal RNA, 101 Roger Korenberg, 44 Ron Laskey, 138 Rosanna Della Corte, 310 Rotifiers, 16 Ruckert, 49 S S. aureus, 358 S. cerevisiae, 37, 341, 342 S. Ochoa, 142 S. Pauling, 142 S. pombe, 37 S. speigelman, 103 S. viridochromogenes, 383 S.H. Kim, 98 Sl nuclease, 256 Saccharomyces cerevisiae, 3, 37, 260, 341, 346 Sagitta, 49 Salivary gland chromosomes, 35, 46 Salmonella, 212, 364 Salmonella paratyphi, 357 Salmonella typhi, 357 Salmonella typhimurium, 180, 212 Same, 122 Sarcode,ll Sat chromosomes, 38, 51 Satellite, 38, 51 Satellite DNA, 39, 64 Scattered, 168 Schenept, 394 Schizosaccharomyces pombe, 37 Schrader, 35 Second, 266 Secondary constriction I, 38 Secondary constriction II, 38 Secondary constrictions, 37 Secondary immune response, 322, 328 Selfish DNA, 168 Semiconservative model, 70 Sense RNA, 382 Sense strand, 122 Sepia, 49 Sesbania acuIeata=S. cannabina, 393 Sesbania rostrata, 393 Severe combined immune deficiency, 292 Severe combined immunity deficiency, 329

Sexchromosomes, 33 Seymour Benzer, 164 Sheath, 11 Shelton Reed, 282 Shuts down, 184 Shuttle vector, 213 Sickle celled, 192 Sickle celled anaemia, 196 Signal peptidase, 140 Signal sequence, 137, 140 Simple vaccines, 356 Singer, 160 Singh, 393 Single, 28 Single cells, 210 Single stranded model, 43 Single stranded RNA, 104 Single-copy gene, 171 Sjostrand, 20 SnowMax,217 Sol, 24 Sole, 185 Solid, 24 Solid nucleus, 24 Solmonella, 168 Soluble RNA, 96 Somaclonal variants, 225 Somatic, 32, 34 Somatic embryo, 225 Somatic hybrids, 7 Somatic-cell hybrid, 204 Somatostatin, 214 Southern blot, 367 Southern blotting, 257, 266 Southern hybridization, 377 Spacer sequences, 124 Species integrity, 236 Spectinomycin resistance (Sperl gene, 380 Spina bifida, 274, 287 Spirogyra, 38 Splicing Life, 296 Split, 169 Stable position effect, 166 Stable trait, 206 Stahl, 55, 74 Standard coils, 34 Stanley, 163 Staphylococcal nuclease, 351 Starling, 104

Biotechnology

440 Start, 122 Starting codons, 144 Steffensen, 43 Stem, 96 Stem cells, 293 Stephen Lindow, 217 Steroids, 182 Steward,38 Stizznski, 163 Stop, 122 Streptococcus pneumoniae, 355 Streptomyces, 381 Streptomyces hygroscopicus, 383 Strickberger, 165 Structural gene, 170, 193 Sub-metacentric, 31, 35 Subtilisins, 348 Suicide bags, 19 Sunbean, 217 Super imposed coils, 35 Supernatant RNA, 96 Supporting cells, 16 Suppressor genes, 149 Suppressor T cell, 325 Suppressor tRNA, 149 Surrogate mother, 315 Sutton, 166 Swanson, 161, 166 Swift, 67 Symmetrical karyotypes, 32 Synthesis, 79 Systemic lupus erythematosus (SLE), 329

T T cell receptors, 323 T cells, 322 T killer cells, 324 T lymphocytes, 322 T memory cells, 325 T-ONA,205 t-RNA,96 T. cristatus, 51 T. reesei, 342 T.H. Morgan, 162 tail, 11 tail fibers, 11 Talbot, 107 Tandem clusters, 171 Tat, 331 Tatum, 166, 167

Tau-chiuh Hsue, 42 Tay-Sachs, 277, 286 Tay-Sachs disease, 197 Taylor, 43, 67, 70, 74 TC cells, 324 TONA,217 TEL,52 Teleconemia scrupulosa, 395 Telocentric, 35 Telocentric chromosome, 32, 37 Telomerase, 81 Telomeres, 38, 81 Temin,122 Template, 68, 253 Terminal transferase enzyme, 89 Terminating codons, 136 Termination, 131 Termination factors, 136 Termination site, 123 Terminism, 122 Tertrahynema, 181 ,Tetrahymena, 38, 52, 58, 81 Tetramer, 45, 76 Tetrehymena, 58 TfdA,383 TFIIO, 182 TH cells, 325 Thalassemia, 136 Three dimensional, 98 Threonine, 180 Thromboplastin, 198 Thymidine, 59 Thymidine kinase, 307 Thymidine monophosphate, 74 Thymidylic acid, 59 Thymine, 56, 164 Thymine phosphate, 59 Ti, 205,262 Ti plasmid, 217 Time, 293 Tissue plasminogen activator, 350 TM cells, 325 Tobjorn Caspersson, 42 Topoisomerase, 90 Toxin, 356 Toxoids, 356 Trabant,38 Trade secrets, 8 Trademarks, 8

Index

Tradescantia, 31, 35, 43, 164 Trans, 229 Transacetylase, 176 Transcriptase, 38 Transcription, 109, 123, 181, 192 Transcription factor lID, 182 Transcriptional level control, 126 Transfection, 262 Transfer, 96 Transfer RNA, 128 Transferase I, 134 Transferase II, 134 Transformed, 254 Transformylase, 132 Transgenic, 262 Transgenic animals, 7, 209 Transgenic mice, 215 Transgenic plants, 7, 205, 379 Transgenic procedure, 298 Translation, 109, 124, 181 Translation factors, 126 Translation repressor proteins, 126 Translational controls, 126 Translocase, 134 Translocation, 162 Transposons, 171 Triazine, 225 Trichoderma reesei, 342 Trifolium alexandrianum, 393 Trillium, 31,32, 55 Triosephosphate isomerase, 346 Triphosphate binding site, 83 Triplets, 157 Trisomy, 13, 18, 21, 288 tRNA, 128 tRNA met, 132 Tropaeolum, 46 Trp, 178, 179, 180 TrpA,149 TrpE, 178, 179 TrpR,178 Tryptophan, 184 Tryptophan synthetase, 149 TS cells, 325 Tuberculosis, 360 Tubules, 20 Tubulin,23 Tumor necrosis factor, 308 Tumor-including, 262

441 Tumor-inducing DNA, 217 Two gene theory, 167 Tyrosinase, 195 Tyrosine, 195 Tyrosinosis, 195 Tyrosyl t-RNA synthetase, 347, 350

U U. Clever, 47 Ulrich Clever, 48 Ultimate unit of physiological function, 161 Ultra modem concept of gene, 169 Ultrasonography, 270 Umbilical cord blood, 293 Under, 275 Ung, 337 Unicellular, 11 Unidirectional, 122 Unidirectional replication, 70, 76 Unineme model, 43 Unit of biological activity, 11 Univalent vaccines, 356 Unwinders, 76 Unwinding proteins, 76 Upstream processing, 2 Uracil N-glycosylase, 337 Uridine,59 V V-bodies, 44 V. cholerae, 362, 363 V. cholera , 356 V.M. Ingram, 142 Vaccine, 354, 356, 358 VaCcinia, 354, 355 Vacuoles, 23 Valine, 144, 180 VAM fungi, 8 Variable arms, 97 Variegated position effect, 166 Veatch,285 Vector, 4, 217, 298 Vector systems, 253 Vehicle, 4 Vermillion, 195 Vesicles, 20 Vibrio cholerae, 362 Vice versa, 39 Vicia,24 Vicia faba. 72

BTotechno{ogy

442 Vigna sinensis=V. unguiculata, 393 Vir, 366 Virus chromosomes, 4 Vivo, 84 VNTR analysis, 266 W W. Waldeyer, 28 Wall,15 Walter Gilbert, 5 Watson, 3, 54, 60, 74, 161, 163 Watson & Crick, 54, 60, 63 Watson-Crick base pairing, 96 Watt-Tobin, 143 Weeds, 395 Weisis, 109 Western blot, 367 Western blotting, 258 Whiteley, 394 Wilkins, 54, 60, 161, 163 Wilson & Thomas, 64 Wobble hypothesis, 148 Woodcock, 44

x X chromosomes, 33 X-irradiation, 270 Xanthophyll, 22 Xba, 362 Xenopus, 181 Xenopus laevis, 68 Xylanase, 345 y Y chromosomes, 33 Yacca arkansana, 32 Yeast artificial chromosomes (yACs) , 261 Yeast extract, 393 Yolk sac, 304 Z Zamecnic, 121 Zea mays, 167 Zif268,349 Zinc finger proteins, 349 Zygote gene therapy, 296 Zygote intrafallopian transfer, 317 Zygotic, 32