Subcellular Biochemistry: Volume 8 [1 ed.] 978-1-4615-7953-3, 978-1-4615-7951-9

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Subcellular Biochemistry Volume

8

ADVISORY EDITORIAL BOARD

J. ANDRE Laboratoire de Biologie Cellulaire, 4 Faculte des Sciences, 91 Orsay, France D. L. ARNON Department of Cell Physiology, Hilgard Hall, University of California, Berkeley, California 94720, USA

J. BRACHET Laboratoire de Morphologic Animale, Faculte des Sciences, Universite Libre de Bruxelles, Belgium J. CHAUVEAU Institut de Recherches Scientifiques sur Ie Cancer, 16 Avenue VaillantCouturier, 94 Ville Juif, Boite Postale 8, France C. de DUVE Universite de Louvain, Louvain, Belgium and The Rockefeller University, New York, NY 10021, USA M. KLINGENBERG Institut fUr Physiologische Chemie und Physikalische Biochemie, Universitiit Miinchen, Goethestrasse 33, Miinchen 15, Germany A. LIMA-de-FARIA Institute of Molecular Cytogenetics, Tornavagen 13, University of Lund, Lund, Sweden O. LINDBERG The Wenner-Gren Institute, Norrtullsgatan 16, Stockholm, V A, Sweden V. N. LUZIKOV A. N. Belozersky Laboratory for Molecular Biology and Bioorganic Chemistry, Lomonosov State University, Building A, Moscow 117234, USSR H. R. MAHLER Chemical Laboratories, Indiana University, Bloomington, Indiana 47401, USA M. M. K. NASS Department of Therapeutic Research, University of Pennsylvania School of Medicine, Biology Service Building, 3800 Hamilton Walk, Philadelphia, Pennsylvania 19104, USA A. B. NOVIKOFF Department of Pathology, Albert Einstein College of Medicine, Yeshiva University, Eastchester Road and Morris Park Avenue, Bronx, NY 10461, USA R. N. ROBERTSON Mac1eay Building, A12, School of Biological Sciences, The University of Sydney, Sydney, N.S.W. 2006, Australia P. SIEKEVITZ The Rockefeller University, New York, NY 10021, USA F. S. SJOSTRAND Department of Zoology, University of California, Los Angeles, California 90024, USA A. S. SPIRIN A. N. Bakh Institute of Biochemistry, Academy of Sciences of the USSR, Leninsky Prospekt 33, Moscow V-71, USSR D. von WETTSTEIN Department of Physiology, Carlsberg Laboratory, GI. Carlsbergvcj 10, DK-2500, Copenhagen, Denmark V. P. WHITTAKER AbteiJung fUr Neurochemie, Max-Plantk Institut fUr Biophysikalische Chemic, D-3400 Gottingen-Nikolausberg, Postfach 968, Germany A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact the publisher.

Subcellular Biochemistry Volume 8 Edited by

Donald B. Roodyn University College London London, England

PLENUM PRESS • NEW YORK AND LONDON

The Library of Congress cataloged the first volume of this title as follows: Sub-cellular biochemistry. London, New York, Plenum Press. v. iIIus. 23 cm. quarterly. Began with Sept. 1971 issue. Cf. New serial titles. 1. Cytochemistry - Periodicals. 2. Cell organelles - Periodicals. QH61l.S84 574.8'76

73-{)43479

Library of Congress Catalog Card Num ber 73-{)434 79 ISBN-13: 978-1-4615-7953-3 e-ISBN-13: 978-1-4615-7951-9 DOl: 10.1007/978-1-4615-7951-9

This series is a continuation of the journal Sub-Cellular Biochemistry, Volumes 1 to 4 of which were published quarterly from 1972 to 1975

© 1981 Plenum Press, New York Softcover reprint of the hardcover 1st edition 1981 A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted. in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the publisher

Contributors Antony C. Bakke Department of Immunopathology, Research Institute of Scripps Clinic, La Jolla, California 92037, U.S.A. Present address: LACUSC Medical Center, Department of Medicine, Los Angeles, California 90033, U.S.A. Ambica C. Banerjee Department of Biochemistry, Bose Institute, Calcutta 700 009, India. Present address: Department of Biochemistry, Calcutta University, Calcutta 700 019, India B. Bhattacharyya Department of Biochemistry, Bose Institute, Calcutta 700 009, India B. B. Biswas Department of Biochemistry, Bose Institute, Calcutta 700 009, India Hector F. DeLuca Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706, U.S.A. Sidney W. Fox Institute for Molecular and Cellular Evolution, University of Miami, Coral Gables, Florida 33124, U.S.A. Kaoru Harada Institute for Molecular and Cellular Evolution, University of Miami, Coral Gables, Florida 33124. Present address: Department of Chemistry, University of Tsukuba, Nihari-Gun, Ibaraki-Ken, Japan P. E. Hare Geophysical Laboratory, Carnegie Institution, Washington, D.C. 20008, U.S.A. Richard A. Lerner Department of Immunopathology, Research Institute of Scripps Clinic, La Jolla, California 92037, U.S.A. Anatoly V. Lichtenstein Department of Biochemistry, Oncological Scientific Center of the Academy of Medical Sciences, Moscow 115478, U.S.S.R. S. K. Malhotra Biological Sciences Electron Microscopy Laboratory, University of Alberta, Edmonton T6G 2E9, Canada Valery L. Mojseev Department of Biochemistry, Oncological Scientific Center of the Academy of Medical Sciences, Moscow 115478, U.S.S.R. JUrgen Oelze Institute for Biology II (Microbiology), University of Freiburg, D78 Freiburg, Federal Republic of Germany

vi

Contributors

A. R. Poole Joint Diseases Laboratory, Shriners Hospital for Crippled Children, McGill University, Montreal, Quebec, Canada Vladimir S. Shapot Department of Biochemistry, Oncological Scientific Center of the Academy of Medical Sciences, Moscow 115478, U.S.S.R. Mikhail M. Zaboykin Department of Biochemistry, Oncological Scientific Center of the Academy of Medical Sciences, Moscow 115478, U.S.S.R.

Aims and Scope SUBCELLULAR BIOCHEMISTRY aims to bring together work on a wide range of topics in subcellular biology in the hope of stimulating progress towards an integrated view of the cell. In addition to dealing with conventional biochemical studies on isolated organelles, articles published so far and planned for the future consider such matters as the genetics, evolution, and biogenesis of cell structures, bioenergetics, membrane structure and functions, and interactions between cell compartments particularly between mitochondria and cytoplasm and between nucleus and cytoplasm. Articles for submission should be sent to Dr. D. B. Roodyn, Department of Biochemistry, University College London, Gower Street, London WCI E 6BT, U.K. There are no rigid constraints as to the size of the articles and in general they should be between 9,000 and 36,000 words, with an optimum size of about 20,000 words. Although articles may deal with highly specialized topics, authors should try as far as possible to avoid specialist jargon and to make the article as comprehensible as possible to the widest range of biochemists and cell biologists. Full details of the preparation of manuscripts are given in a comprehensive Guide for Contributors which is available from the Editor or Publishers on request.

vii

Preface In this volume of SUBCELLULAR BIOCHEMISTRY we cover a wide range of topics of considerable biological importance and have continued in our policy of letting authors, rather than editors, decide the "natural" length of their articles. Thus, we have some short but nevertheless significant contributions, as well as more massive chapters. We start with a detailed account by 1. Oelze of the composition and development of the bacterial photosynthetic apparatus. A number of photosynthetic bacteria are discussed, with particular emphasis on the well-studied Rhodospirillum rubrum and Rhodopseudomonas sphaeroides. The reader will no doubt be struck by the great wealth of information now available on the molecular organization of the photosynthetic and respiratory systems in these organisms. Equally important is our improved understanding of the biosynthesis and assembly of these systems. It is now generally accepted that photosynthetic bacteria are excellent model systems for the study of bioenergetic processes. It may well be that they will become equally popular as models for the study of membrane biogenesis, and it is to be hoped that Oelze's erudite and comprehensive treatment of the subject will help in this regard. The next chapter, by A. C. Bakke and R. A. Lerner, deals with another fascinating organism that is attracting a good deal of interest, namely Dictyostelium discoideum. This slime mold can exist in a vegetative form as a conventional "amoeba." However, under appropriate conditions it is transformed into a multicellular organism that shows definite morphological development and can also move as a multicellular mass. This extraordinary leap from unicellular to multicellular organism is fortunately amenable to conventional biochemical analysis. In particular, the plasma membrane can be studied at every stage by the great battery of techniques now available for the study of membranes. The results of such studies are clearly set out in Bakke and Lerner's article and show strikingly how much has been revealed so far, and also give an indication of the complex molecular mechanisms that remain to be unraveled. ix

x

Preface

The next chapter, by B. B. Biswas, A. C. Banerjee, and B. Bhattacharyya, is entitled "Tubulin and the Microtubule System in Cellular Growth and Development." This is a subject of intense current interest as the universal distribution and immense significance of microtubules have been demonstrated in the last few years. It is also now generally recognized that tubulin is an ancient protein of profound evolutionary interest. The article systematically surveys our current knowledge of the biochemistry of microtubule proteins, the mechanism of microtubule assembly, the mode of action of antimicrotubular agents, and the role of microtubules in growth and development. Significantly, one section deals with recent experiments on the cloning of the tubulin gene, and no doubt such work will lead to even greater revelations about tubulin biochemistry and biology. The chapter after this deals with a question that has intrigued biologists for at least a century, namely the relationship between nucleus and cytoplasm. A. V. Lichtenstein, M. M. Zaboykin, V. L. Mojseev, and V. S. Shapot concern themselves in particular with how the flow of genetic information from nucleus to cytoplasm is controlled. Is the information simply "pushed" out into the cytoplasm, or does the cytoplasm "pull" out from the nucleus specific information as it is required? The authors make some fascinating suggestions about the possible role of the ubiquitous nuclear pores in the process, and also discuss in a most stimulating way the many current theories on nucleocytoplasmic interaction. We then turn to the interesting question of the subcellular basis for the mode of action of vitamin D. In a short treatment of the topic, H. F. DeLuca surveys the main metabolic transformations of vitamin D and discusses the subcellular localization of various hydroxylase systems involved in these transformations. He also emphasizes the central role of 1,25-dihydroxyvitamin D3 and presents current theories on its molecular mechanism of action. The article provides a good example of the unifying trends in current research, in that investigation into the mode of action of vitamin D is becoming increasingly concerned with membrane phenomena and the role of possible receptors for its metabolites. The next chapter, by S. K. Malhotra, deals specifically with receptors of fundamental importance, namely those for acetylcholine. Again one cannot help but be struck by the magnitude of the advances that have been made. Thus, the fact that it is now possible to present models of the molecular organization of the acetylcholine receptor complex in terms of known polypeptides arranged around a central ionophoretic channel shows that the subject is now firmly established at the molecular level. An important consequence of these advances is that the borders between biophysics and biochemistry, always unnatural, have become increasingly blurred. The chapter surveys the distribution of acetylcholine receptors, their composition, structure, and morphology, and the differences between junctional and extrajunctional receptors. Again we are given the distinct impression that we are on the verge of great

Preface

xi

developments in a topic of central importance. The next article, by A. R. Poole, describes the application of modern immunological techniques to the study of tissue proteinases. The author discusses the intra- and extracellular localization of cathepsin D in particular but also deals with a range of lytic enzymes (including various other cathepsins, collagenase, elastase, serine proteinases, and acrosin). He includes a most useful section entitled "Immunological Methods for the Study of Proteinases" with helpful advice on the preparation of antibodies, immunoprecipitation, immunoinhibition, immunolocalization, etc. The article shows strikingly how current advances in immunology may be usefully applied to the study of the tissue and intracellular distribution of specific enzyme molecules, and it is hoped that some readers will be stimulated by the information supplied to apply such methods. The final chapter stands in a class of its own. It is a brief, critical, and stimulating account by S. W. Fox, K. Harada, and P. E. Hare of studies that have been made to establish the significance of the trace amounts of amino acids that have been detected in samples of lunar rock and in the core of various meteorites. The total quantity of amino acids found in various lunar rocks ranges from 5 to 45 ng/ g. The most abundant amino acids are glycine, alanine, and glutamic acid, with smaller amounts of aspartic acid, serine, and threonine. Are these amounts significant? Do they represent material of biological importance? What are the effects of rocket exhaust? Are the analytic procedures adequate at these levels? All these matters are discussed critically, and a scheme is put forward in which "core" sets of amino acids are first formed and this gives rise to proteinoids and then protocells. The significance of this contribution to the ever-increasing debate on possible molecular mechanisms for the origin of primitive life forms is that it is concerned with the interpretation of measurements on actual samples and not with hypothetical events that could have occurred so many gigayears ago. As usual we include an extensive book review section. In this volume various texts on recognition systems, methodology, cell biology, and evolution are discussed in detail, and many of the topics discussed (e.g., tubulin, immunological techniques, and the origin of life) are related to chapters in Volume 8. To conclude, both the material in the book proper and the material surveyed in the book review section attest to the dramatic and exciting advances that are currently taking place in cell biology. It is clear that we are currently observing a great increase in the unification of the subject in which techniques and approaches in one area of study are increasingly being applied to other areas. It is to be hoped that SUBCELLULAR BIOCHEMISTRY will continue to contribute to this unification process. D. B. Roodyn

London

Contents Chapter 1 Composition and Development of the Bacterial Photosynthetic Apparatus J iirgen Oelze 1. Introduction ....................... . 2. Structure and Function of Membranes .. . 2.1. Chemical Composition of Isolated Membranes ...... . 2.2. Physical Properties of Isolated Membranes ... 2.3. The Photosynthetic Apparatus 2.4. The Respiratory Electron Transport System . 2.5. Energy-Requiring Reactions Linked to Electron Transport ... 2.6. Reconstitution of Light-Dependent Reactions in Photosynthetically Incompetent Membranes 3. Development of Membranes and Its Regulation.

3.1. Bacteriochlorophyll Synthesis . . .......... . 3.2. Differentiation of the Cellular Membrane System 4. Comparative Aspects .. 5. References.

1 3

5 12 13

28

29 33 34 34

38 57 59

Chapter 2 The Cascade of Membrane Events during Development of Dictyostelium discoideum Antony C. Bakke and Richard A. Lerner 1. Introduction ............................................. . 1.1. Dictyostelium Development. . ..... . 1.2. Processes Mediated by the Plasma Membrane 2. General Composition and Structure of the Membrane ......... . 2.1. Isolation Techniques. . . ................ . xiii

75 75

78 79 79

xiv

Contents

2.2. Changes in the Phenotype of the Membrane during Development ................................ . 81 3. Functions of the Plasma Membrane during Development ....... . 93 3.1. Chemotaxis .................................... . 93 3.2. Aggregation ..................................... . 100 3.3. Cell-Cell Interaction ................................. . 106 4. Summary ............................................... . 112 5. References ............................................. . 114

Chapter 3 Tubulin and the Microtubule System in Cellular Growth and Development B. B. Biswas, Ambica C. Banerjee, and B. Bhattacharyya

1. Introduction ....................................... . 1.1. Occurrence and Function of Microtubules . 1.2. Structure of Microtubules ................... . 2. Biochemical Characterization of Microtubule Proteins ...... . 2.1. Purification of Tubulin ............................... . 2.2. Heterogeneity in Tubulin ............................. . 2.3. Carbohydrate, Lipid, and Nucleotide in Microtubules . 2.4. Enzyme Activities Associated with Microtubule Proteins. . . . . . . . . . . . . . . .................... . 2.5. Proteins Associated with Microtubules ................... . 2.6. Microheterogeneity in Tubulin ......................... . 3. Microtubule Assembly. . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . 3.1. Conditions of Assembly ............................... . 3.2. Role of Nucleotides in Assembly ....................... . 3.3. Accessory Factors for Assembly ........................ . 3.4. Mechanism of Assembly in Vitro ................. . 3.5. Regulation of Microtubule Assembly .............. . 4. Antimicrotubular Agents ........................... . 4.1. Colchicine and Its Structural Analogs ................... . 4.2. Podophyllotoxin ..................................... . 4.3. Vinblastine and Vincristine. . . . . . . . ............ . 4.4. Griseofulvin ......................................... . . ........ . 4.5. Other Microtubule Poisons .... 4.6. The Mechanism of Substoichiometric Antimitotic Drug Poisoning. . . . . . . . . . . . . . . . . . . . . . . ..... .

123 124 125 128 128 129 130

130 131 132 133 133 134 136

139 142 143 144 147 148

149 150 152

Contents

5. Microtubules in Growth and Development. . . . . . . . . . . . . . . . . . . .. 5.1. Relation of Microtubules to Morphogenesis and Maturation of Disk-Shaped Blood Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5.2. Relation of Microtubules to Morphogenesis in Other Cells . .. 5.3. Relation of Microtubules to Other Cell Organelles and Structures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5.4. Tubulin-Microtubule Association with Membrane Structures in Relation to Cell Transformation. . . . . . . . . . . . . . . . . . . . . .. 5.5. Tubulin-Microtubule Association with Plant Cell Membrane. 5.6. Biosynthesis of Tubulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5.7. Posttranslational Modification. . . . . . . . . . . . . . . . . . . . . . . . . .. 5.8. Tubulin mRNA in Developing Systems. . . . . . . . . . . . . . . . . .. 6. Cloning of the Tubulin Gene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 8. References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

xv

152 153 154 155 157 158 158 160 161 163 165 167

Chapter 4 Nucleus and Cytoplasm: Supply and Demand. What Underlies the Flow of Genetic Information? Anatoly V. Lichtenstein, Mikhail M. Zaboykin, Valery L. Mojseev, and Vladimir S. Shapot

1. Introduction.............................................. 185 2. Interdependence and Complementarity of Central and Peripheral Mechanisms in the Control of Gene Expression ................ 188 2.1. Regulation of Protein Synthesis: Control at the Transcriptional Level Is Necessary. . . . . . . . . . . . . . . . . . . . . .. 189 2.2. Regulation of Protein Synthesis: Transcription Alone Is Not Sufficient. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 195 3. Some Hypotheses on Posttranscriptional Regulation. . . . . . . . . . . .. 199 3.1. "Cascade Regulation" Model . . . . . . . . . . . . . . . . . . . . . . . . . .. 199 3.2. "Ticketing" Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 199 3.3. Model Involving Cytoplasmic Inhibitors of mRNA Function. 200 3.4. Autogenous Regulation of Gene Expression. . . . . . . . . . . . . . .. 201 3.5. Attenuation as a Mechanism for Differential Gene Activity .. 201 3.6. Hypothesis Proposing a Regulatory Role for Repetitive Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 202 3.7. Hypothesis of "Splicer" RNAs . . . . . . . . . . . . . . . . . . . . . . . . .. 203 4. The Cytoplasm as a Source of Genome-Reprogramming Activity.. 205

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Contents

5. A Model for Cytoplasm-Governed Gene Regulation ... 5.1. Qualitative Redundancy of Transcription as a Consequence of Structural Organization of the Genome ..... 5.2. Selective RNA Transport as a Mechanism for Controlling Transcription. . . . . . . . . . . . . . . . . . . . . . . ...... . 6. Regulation of Gene Expression at the Level of Nucleus-toCytoplasm Transport of RNA ............... . 6.1. Rate of RNA Transport ....... . 6.2. Comparison of Nuclear and Cytoplasmic RNAs in Various Cell Types ..................... . 6.3. Transport of Some Specific Transcripts ... 6.4. "Luxury" Functions, "Housekeeping" Functions, and Modulation of mRNA Abundance in the Cytoplasm . 6.5. Transport-Controlling Factors of the Cytosol 7. Metabolic Heterogeneity of Nuclear RNA ............. . 8. Structural Organization of Intranuclear RNA Transport ... . 9. Conclusion .................... . 10. References .......................... .

207 208 209

210 211

212 216 218 223 225 229 233 234

Chapter 5 Subcellular Mechanisms Involving Vitamin D Hector F. DeLuca 1. Introduction.... . . . . . . . . . . . . . . . . . . . . . . ...... . 2. Subcellular Aspects of Functional Vitamin D Metabolism .. . . ....... . 2.1. Vitamin D-25-Hydroxy1ase ... 2.2. 25-0H-D-1-Hydroxylase ....... . 3. Molecular Mechanism of Action of 1,25-(OH)2D3 4. Summary ................ . . .... 5. References ................ . ..................

251 256 256 257 260 264 M5

Chapter 6 Macromolecular Organization of the Nicotinic Acetylcholine Receptors S. K. Malhotra 1. Introduction................... . . . . . . . . . . . . . . . . .. 2. Distribution of Acetylcholine Receptors. .......... 2.1. Innervated Skeletal Muscle and Electroplaques ............

273 276 276

Contents

3. 4. 5. 6. 7. 8. 9.

2.2. Denervated Skeletal Muscle (Extrajunctional Ach Receptors) 2.3. Biosynthesis of Extrajunctional Ach Receptors. Composition of Acetylcholine Receptors ....... . Structure of Acetylcholine Receptors ........................ . Morphological Correlates of Acetylcholine Receptors .. Differences between Junctional and Extrajunctional Acetylcholine Receptors. ....... . ............. . Significance of Extrajunctional Acetylcholine Receptor Aggregates Conclusion. . ............. . References.

xvii

276 277 285 290 293 295 297 300 302

Chapter 7 Immunological Studies of Tissue Proteinases A. R. Poole 1. Introduction .. 2. Cathepsin D ........ . 2.1. Antiserum Production 2.2. Inhibition by Antisera ............. . 2.3. Tissue Localization: Intracellular and Extracellular .. 2.4. The Assay of Cathepsin D .. 2.5. The Structure of Cathepsin D . 2.6. The Biosynthesis of Cathepsin D. . ..... . 3. Cathepsin B and Related Thiol Proteinases . 4. Collagenase . . ..................................... . 5. Elastase and Cathepsin G 6. Serine Proteinases of Skin and Muscle. 7. Acrosin ..................... . 8. Plasminogen Activators . 9. Immunological Methods for the Study of Proteinases .. 9.1. Purification of Proteinases ..................... . 9.2. Preparation of Antisera. . ..... . . .. 9.3. Preparation of Antibodies from Antisera. 9.4. Preparation of Antibodies Using Hybridomas . 9.5. Immunoprecipitation in Gels and Solution .. 9.6. Immunoinhibition .... 9.7. Nonprecipitating Antibodies and Immunoassay 9.8. Immunolocalization ............... . 10. Conclusions 11. References.

311 312 312 313 316 324 324 326 327 330 336 338 339 340

342 342

342 343 344 345 347

347 348 350 351

Contents

xviii

Chapter 8 Amino Acids from the Moon: Notes on Meteorites Sidney W. Fox, Kaoru Harada, and P. E. Hare 1. Introduction........................ . ............ . 2. History............ .................... . ......... . 2.1. Analyses of Lunar Samples and Meteorites ... 2.2. Preparation of Samples . . . . . . . . . . . . . . . . . . . . . .. . ..... . 2.3. Method of Analysis ........... . . ........ . 2.4. Contamination in Lunar Fines .... 2.5. Sources of Amino Acids from the Moon and Meteorites ..... 2.6. The Chemical Nature of Amino Acid Precursors .......... . 3. Summary and Prospect. . . . . . ...... . 4. References .......................................... .

357 358 360 363 364 364 365 367 368 370

Recent Books in Cell Biochemistry and Biology 1. 2. 3. 4.

Recognition Systems ....... . Techniques ......... . Cell Biology and Organelles .. . Evolution of Cellular Systems ........... .

Index ............... .

376 380 382

. . . . . . . . . . . . .. 389 395

Chapter 1

Composition and Development of the Bacterial Photosynthetic Apparatus Jiirgen Oelze Institute for Biology II (Microbiology) University of Freiburg D78 Freiburg, Federal Republic of Germany

1. INTRODUCTION The order of the Rhodospirillales, commonly known as the photosynthetic or more adequately as the phototrophic bacteria, comprises prokaryotes that are able to cover their energy requirements by coupling to light-dependent electron transport processes. In contrast to the plant-type photosynthesis not only of plants but also of cyanobacteria, the photosynthesis of phototrophic bacteria does not evolve oxygen and is dependent on anaerobiosis or, at least, largely reduced oxygen partial pressures. Apart from these physiological characteristics, the phototrophic bacteria comprise organisms of rather different properties (see Table I for the classification of phototrophic bacteria referred to in this chapter). Based on the distribution of different bacteriochlorophyll derivatives contained in the photosynthetic apparatus, two suborders can be distinguished (TrOper and Pfennig, 1978). The first suborder of the Rhodospirillineae, i.e., the purple bacteria, comprises organisms that exhibit either bacteriochlorophyll a or b. This suborder contains two families, the Rhodospirillaceae (formerly Athiorhodaceae) and the Chromatiaceae (formerly Thiorhodaceae), which differ from each other by their inability and ability, respectively, to grow on sulfide (exceptions are known). The second suborder, the Chlorobiineae, comprises all of the phototrophic bacteria that produce bacteriochlorophyll c, d, or e, in addition to minor quantities of bacteriochlorophyll a. In addition to a phototrophic energy metabolism, some members of the order are also able to grow chemotrophically. Under anaerobic dark conditions, 1

2

Jllrgen Oelze

Table I Classification of Relevant Phototropic Bacteria Order: Rhodospirillales Suborder: Rhodospirillineae Family: Rhodospirillaceae Genus: Rhodospirillum Species: R. rubrum R. tenue

Genus: Rhodopseudomonas Species: R. acidophila R. R. R. R. R.

capsulata gelatinosa palustris sphaeroides viridis

Family: Chromatiaceae Genus: Chromatium Species: C. vinosum Genus: Thiocapsa Species: T. pfennigii Suborder: Chlorobiineae Family: Chlorobiaceae Genus: Chlorobium Species: C. limicola C. limicola forma sp. thiosulfatophilum Family: Chloroflexaceae

Genus: Chloroflexus Species: C. aurantiacus

chemotrophy depends on substrate level phosphorylation and, in the presence of oxygen, electron transport phosphorylation. Only the latter proceeds via membrane-bound electron transport systems. Therefore, in the context of this chapter, the term chemotrophy will be restricted to an energy metabolism coupled to a respiratory chain with oxygen as the electron acceptor. When growing chemotrophically in the presence of increased oxygen partial pressures, cultures of facultative phototrophic species, like Rhodospirillum rubrum or Rhodopseudomonas sphaeroides. exhibit the pale pinkish-beige color also typical of nonphototrophic bacteria. Electron micrographs of thinsectioned cells from such cultures exhibit the cytoplasmic membrane and the cell wall as cell envelope layers. As in other chemotrophic bacteria, the cytoplasmic membrane in phototrophic bacteria is the site of transport systems of which the respiratory electron transport chain combined with the coupling factor ATPase are of outstanding importance for cellular energy conservation. When growing anaerobically in the light, cultures of facultative phototrophic species exhibit a deep red to brownish-green color that indicates the

The Bacterial Photosynthetic Apparatus

3

presence of bacteriochlorophyll and, predominantly in the visible part of light absorption, carotenoids. Electron microscopic investigations reveal the presence of intracytoplasmic membranes in addition to the cell envelope structures (Figure 1). These intracytoplasmic membranes are known to be the preferential sites of the photosynthetic apparatus. In conclusion, chemotrophically and phototrophically grown cells represent two extreme modifications of a facultative representative of the phototrophic bacteria. The two modifications differ from each other with respect to their pigmentation, ultrastructure, composition of energy-regenerating membranes, and energy metabolism as expressions of their general physiological differences. In addition, facultatively as well as obligately phototrophic members of the order adapt structurally and functionally to appropriate changes in environmental factors when growing phototrophically. All of the processes of cellular differentiation as presented schematically in Figure 2 are reversible. It is the object of this chapter to review comprehensively our understanding of the structure, function, and development of membranes from different species of the phototrophic bacteria and thereby demonstrate the high degree of biological diversity expressed in this group of organisms.

2.

STRUCTURE AND FUNCTION OF MEMBRANES

Intracytoplasmic membranes are typically formed as tubules or lamellae. Lamellae may be arranged species specifically as single or multiple stacks, while tubules are in some species regularly bulged and therefore appear to be vesicular structures (Figure 1) (Oelze and Drews, 1972; Remsen, 1978). Irregularly shaped membrane intrusions have occasionally been observed and may represent either functionally specialized types of membranes or extensions into the cytoplasm of the cytoplasmic membrane. Some members of the phototrophic bacteria, such as Rhodospirillum tenue and Rhodopseudomonas gelatinosa, as well as members of the Chlorobiineae, exhibit only a few, if any, intracytoplasmic membranes (Cohen-Bazire et al., 1964; De Boer, 1969; Pierson and Castenholz, 1978). These organisms contain the photosynthetic apparatus in their cytoplasmic membranes (Pierson and Castenholz, 1978; Wakim et al., 1979). Upon cell homogenization, intracytoplasmic membranes, particularly of the vesicular type, break down into closed vesicles; on the basis of their pigmentation, these have been names chromatophores (Figure 1) (Schachman et al., 1952). In addition, the cytoplasmic membrane can be disrupted into vesiculating fragments of different sizes. When derived from organisms that do not produce specialized intracytoplasmic membranes, the isolated cytoplasmic membrane vesicles house all of the photopigments and as a result should also be named chromatophores. On the other hand, R. rubrum, under special con-

FIGURE 1. Electron micrographs of cells and chromatophores of phototrophically grown Rhodospirillum rubrum. (A) Thin-sectioned cell. (B) Higher magnification of a thin-sectioned spheroplast (spheroplasts allow for a better demonstration of connections between cytoplasmic

The Bacterial Photosynthetic Apparatus

5

FIGURE 2. Schematic presentation Chemotrop'hic PbototrORhic of ultrastructural and physiological (aerobic) (anaerobic) modifications of Rhodospirillum light rubrum, a typical member of the phoCWCM ICM totrophic bacteria. The organisms exhibit chemotrophic (respiratory) light flux (1) energy metabolism in the presence of sufficiently high oxygen partial presCWCM ｾ＠ sures. The respiratory chain is localized ｾｯ［＠ in the cytoplasmic membrane (CM). Upon transfer to anaerobic conditions in the light, the photosynthetic apparatus is formed concomitantly with its high light flux (2) carrier structures, the intracytoplasmic membranes (ICM). This allows for a phototrophic energy metabolism. The cellular contents of the photosynthetic apparatus and correspondingly of the ICM are regulated by external factors such as the light-energy flux. The process of adaptation from chemotrophic to phototrophic conditions is reversible. CW, cell wall.

ｾｬｏｗ＠

ｾ＠

ｾＯＮＬ＠

ｾＭ］＠

ｾ＠

ditions, forms intracytoplasmic membranes that are devoid of bacteriochlorophyll, but also forms closed vesicles when the cells are homogenized (Golecki

et al., 1980). Thus, to avoid confusion, the term chromatophore will be employed to denote vesicles derived from bacteriochlorophyll-containing intracytoplasmic membranes. All of the other types of isolated membrane vesicles will be referred to according to their respective origins. On electron micrographs, thin-sectioned specimens of phototrophic bacteria exhibit a typical gram-negative cell wall including a layer that, according to its appearance as a unit membrane, has been introduced into the literature as the outer membrane. Properties of this cell wall layer will not be discussed in this chapter; instead the reader is referred to the relevant reviews by Drews et a/. (1978) and Weckesser et a/. (1979).

2.1.

Chemical Composition of Isolated Membranes

The presentation of analytical data on the composition of membrane preparations is intended to give an impression of the various compounds involved

and intracytoplasmic membranes). (C) Freeze-fractured cell (arrowheads point to indentations on the plasmic face of cytoplasmic membranes, probably representing connections between cytoplasmic and intracytoplasmic membranes). (D) Negatively stained chromatophore preparation. CM, cytoplasmic membrane; CW, cell wall; ICM, intracytoplasmic membrane; PF, plasmic face of the cytoplasmic membrane. Bars represent 200 nm. Courtesy of Dr. 1. R. Golecki.

30 67 47 52 49

27 49 35 32

28

Total lipid

63 63

55 43

Protein

22

15.6 21.1

Phospholipid

8.3

5.9 0.02

3.2

Bacteriochlorophyll

18

12

1.8

0.3

2.3 4.2

5.6 5.6

Carbohydrates

"Cytoplasmic membranes from R. rubrum and R. spaeroides were isolated from chemotophically grown cells; all of the other preparations were from phototropically grown cells. Values represent percentages of dry weight. "Data from Oelze et al. (1975) and Collins and Niederman (1976b). 'Data from Niederman and Gibson (1978) and Parks and Niederman (1978). "Data from Cusanovich and Kamen (1968). 'Data from Takacs and Holt (1971). 'Data from Cruden and Stanier (1970).

Rhodospirillum rubrumb Chromatophores Cytoplasmic membranes Rhodopseudomonas sphaeroides' Chromatophores Cytoplasmic membranes Chromatium vinosum d Chromatophores Thiocapsa roseopersicina' Chromatophores Chlorobium thiosul/atophilum' Membranes Chlorobium limicohf Membranes

Species

Table II Chemical Composition of Membranes"

ｾ＠

f

...

'"

The Bacterial Photosynthetic Apparatus

7

in the construction of membranes. It should be kept in mind, however, that membranes in living systems change rather dynamically their functions and composition as a response not only to the cell cycle but also to changes in nutritional and other factors, such as temperature. It should also be noted that procedures employed in cell disruption as well as membrane purification may affect the structure and composition of cellular membranes in such a way that some compounds are removed while others become secondarily attached. For example, proteins categorized as peripheral membrane constituents may be detached, while polysaccharides derived from the cell wall may become attached. Table II is a compilation of analytical data on the gross chemical composition of chromatophores as well as of cytoplasmic membrane preparations from selected members of the phototrophic bacteria. Except for Chromatium vinosum and the two species of the Chlorobiineae, each species exhibits a ratio of protein to lipid that is typical of other prokaryotes (Salton, 1967; Greenawalt and Whiteside, 1975). This ratio (of protein and lipid) is also very similar to that in chromatophores and cytoplasmic membrane fragments derived from one species. Recent results, however, revealed cyclic changes of 40-50% in the protein-to-phospholipid ratio of membranes from R. sphaeroides during the cell division cycle (Figure 3) (Fraley et al., 1979a). The outstanding feature of chromatophores is, of course, their photopigment, i.e., bacteriochlorophyll, and

2.0

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....ｾ＠ ｴ［ＭＬｾ＠

1.0

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ｾ＠

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FIGURE 3. Changes in the protein-to-phos- ｾ＠ Ｎ ｾ＠ pholipid ratio (C) and in the intrinsic density ｾ＠ ... ｾＡＺｉＮ＠ () iii (8) of intracytoplasmic membranes from '\:) ｃＨｾ＠ )"'1:> t::h Rhodopseudomonas sphaeroides during syn- Ｎ ｾ＠ ｾ＠ "-b, chronous growth (A). For a detailed descripｾIt＠ "tion see Fraley et al. (i 979a). Reprinted with the kind permission of Dr. S. Kaplan.

5.0 4 .0

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0

0

0

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2

3

4

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Jurgen Oelze

8

a b c d e

A FIGURE 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of membranes from various (wild-type) phototrophic bacteria. (A) Rhodopseudomonas sphaeroides (slot a), Rhodopseudomonas palustris (slot b), Rhodopseudomonas viridis (slot c), Rhodospirillum rubrum (slot d), Rhodopseudomonas capsulata (slot e). The three polypeptides (except for R.

their carotenoid content. As will be shown later (Section 3), the amount of these particular pigments may be subject to change, depending on culture conditions. Various proteins bound to functional units have been solubilized from membranes of phototrophically grown cells as well as, to a minor extent, from

9

The Bacterial Photosynthetic Apparatus

-Start -SOH (heavy)

-d} ATPase

-p

'-H

-M -L

B viridis) from the photochemical reaction centers (RC) band within the region, as indicated. The heavily stained, fast-migrating polypeptides at the bottom can be identified in light-harvesting bacteriochlorophyll preparations. (8) Comparison of polypeptide patterns of isolated cytoplasmic membranes (left slot) and chromatophores (middle slot) from R. rubrum. The third slot presents the polypeptides patterns of chromatophores obtained after tryptic digestion of proteins exposed at the outer face (i.e., the cytoplasmic face of in situ intracytoplasmic membranes). The heavy polypeptide of succinic dehydrogenase (SDH); a and {J subunits of ATPase (FI); heavy (H), intermediate (M), and light (L) subunits of photochemical reaction centers; polypeptides associated with light-harvesting (LH) bacteriochlorophyll complexes (8875).

membranes of chemotrophically grown cells. The most prominent proteins of the chromatophores are those associated with the antenna and reaction-center pigment complexes, respectively, of the photosynthetic apparatus (Figure 4). Figure 4 reveals that antenna complexes of the Rhodopseudomonas species comprise a larger variety of polypeptides than those of R. rubrum. As calcu-

10

Jllrgen Oelze

lated on the basis of polypeptide patterns of fully developed chromatophores obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these proteins amount to more than 60% of the total (Takemoto, 1974; Nieth and Drews, 1975; Oelze and Pahlke, 1976). Considerably lower but still significant amounts of ATPase (F}) can be identified on the basis of the a and {j subunits. The heavy polypeptide of succinate dehydrogenase is also detectable in the polypeptide patterns of chromatophores from R. rubrum (Oelze, 1978) (Figure 4). In cytoplasmic membranes from chemotrophically grown R. rubrum, on the other hand, the a and (j subunits of ATPase are present as predominant polypeptides. As might be expected, polypeptides of antenna and photochemical reaction-center complexes are missing. The presence of proteins common to both chromatophores and cytoplasmic membranes was demonstrated on the basis of (1) polypeptides, (2) activities of functional units such as N AD H dehydrogenase and succinic dehydrogenase as well as ATPase (Figure 4), and recently (3) immunological methods including cross-reactivity (Collins et al., 1979; Elferink et aI., 1979; review by Drews and Oelze, 1980). A more detailed description of the composition of functional units will be given in the following sections. Membrane phospholipids of various members of the Rhodospirillineae are largely comprised of phosphatidylglycerol and phosphatidylethanolamine (see Kenyon, 1978, for a review). Some species also produce phosphatidy1choline and, in significantly lower amounts, diphosphatidylglycerol. Interestingly, R. rubrum and R. sphaeroides contain membrane-bound ornithine lipids, and only in R. sphaeroides were these lipids confined to the chromatophores (Depinto, 1967; Gorchein, 1964, 1968). To the extent that they have been investigated, it has been shown that members of the Chlorobiineae differ from members of the Rhodospirillineae in that they contain significant amounts of glycolipids as well as the phospholipids typical for Rhodospirillineae (Kenyon, 1978). The fatty acid composition of membrane lipids exhibits a remarkably high percentage, up to 90%, of unsaturated fatty acids (Table III). This does not apply to membranes of R. tenue or to the two species of the Chlorobiineae, which similarly contain "only" slightly more than 50% unsaturated acids. cisVaccenic and palmitoleic acid were the unsaturated fatty acids identified, both of which have low melting points. In general, the carbon chain length was predominantly 14 to 18 (Kenyon, 1978). The relatively high amounts of carbohydrate in membranes from Chlorobium thiosulfatophilum and C. limicola may results from the presence of glycoJipids in these organisms. However, glycolipids are not present in any of the Rhodospirillineae, as reported above. In addition, no glycoproteins have as yet been detected in membranes of the Rhodospirillineae. On the contrary, studies utilizing cytoplasmic membranes of R. rubrum have indicated that as far as can be seen, none of the membrane polypeptides are covalently bound to polysaccharide (Oelze et al., 1975). An explanation for the presence of poly-

11

The Bacterial Photosynthetic Apparatus

saccharide may be that they are similar in composition to the lipopolysaccharides of the cell wall (Hurlbert et al., 1974; Oelze et al., 1975). This suggests that they are present as contaminants rather than occurring naturally in the membranes. Functionally, membranes can be regarded as highly organized arrangements of different reaction systems. This generally includes constituents of electron transport systems, various enzyme systems such as those involved in the transport or biosynthesis of cellular and extracellular compounds, and in phototrophic bacteria pigments that collect light energy as well. The latter comprise bacteriochlorophylls as well as carotenoids. So far, five different bacteriochlorophyll derivatives have been identified, the distribution of which was described in the Introduction. More than 75 different carotenoids are synthesized by the various members of the phototrophic bacteria. But a detailed presentation of these pigments is not required in the context of this chapter (for comprehensive reviews see Liaaen-Jensen, 1978; Schmidt, 1978). A great variety of cytochromes have also been identified and characterized (Bartsch, 1978); those that are essential for an understanding of functional systems will be discussed below. Our present understanding of iron-sulfur centers has been comprehensively treated by Malkin and Bearden (1978). Electron transport systems include qui nones that are present in most of the phototrophic bacteria as ubiquinone derivatives and in some as naphthoquinones (Parson, 1978).

Table III Fatty Acid Patterns of Membranes from Rhodospirillum rubrum, Rhodospirillum tenue, Rhodopseudomonas sphaeroides, and Chromatium vinosum, and Cells of Chlorobium Iimicola and Chloroflexus aurantiacus" R. rubrurn' Fatty acid

CHR

CMc

12:0 14:0 16:0 16:1 18:0 18:1

2.6 10.4 30.8 1.4 53.3

2.9 10.4 37.8 1.6 47.4

20:1

R. tenue CMp

CMc

3.3 7.8 32.8 34.7

2.0 7.8 37.4 42.8

11.5

5.5

R. sphaeroides' CHR

CMp

1.3 3.2 4.3 89.3

2.6 3.4 2.9 88.4 2.7

C.

C.

CHR

lirnicolaf' Cells

ｶｩｮｯｳｵｭｾ＠

1.3 26.9 34.5 1.0 36.9

13 17 57

C. ｡ｵｲｮｴｩ｣ｾＧ＠

Cells

12 14 52

"CHR, chromatophores; eM, cytoplasmic membrane fragments. eM preparations were isolated from phototrophically grown (CMp) R. sphaeroides, from chemotrophically grown (CMc) R. rubrurn, and from phototrophically (CMp) and chemotrophically (CMc) grown R. lenue. 'Data from SchrOder el 01. (1969) and Oelze el 01. (1975). 'Data from Wakim el 01. (1979). "Data from Michels and Konings (1978a). 'Data from Hurlbert el 01. (1974). fData from Kenyon (1978). 'Contains 17-cyclopropane group (3%). 'contains also 17:0 (3%). 17:1 (3%), 18:2 (2%), 19:1 (3%), 20:0 (1%),20:1 (4%).

12

JUrgen Oelze

2.2. Physical Properties of Isolated Membranes Data on the physical properties of isolated membranes (like data on the chemical composition) should be viewed with caution. For example, physical properties can be subject to physiological alterations that, depending on the cell division cycle and developmental stage, are of major importance. However, alterations imposed artificially upon membranes, particularly during cell homogenization, should also be taken into consideration. For example, cytoplasmic membranes may yield fragments of different sizes; when they are derived from cells in the course of osmotic lysis, cytoplasmic membrane vesicles exhibit diameters of 100 to 500 nm. However, when they are derived from cells by sonication or by the use of a French pressure cell, their diameters are below 60 nm (Oelze et al .• 1969; Niederman et al.• 1972; Oelze et al.. 1975; Collins and Niederman, 1976a; Michels and Konings, 1978a; Parks and Niederman, 1978). Chromatophores derived from the vesicular type of intracytoplasmic membranes exhibit diameters of about 60 nm, which are similar to the diameters of the "vesicles" in situ (Worden and Sistrom, 1964; Gibson, 1965a; Cusanovich and Kamen, 1968; Collins and Niederman, 1976b). This similarity in size might be interpreted as an indication of the prior existence of closed or single vesicles in the cells. However, electron micrographs from carefully isolated intracytoplasmic membrane preparations unequivocally reveal chains of interconnected vesicular structures (Holt and Marr, 1965; Hurlbert et al.. 1974). Buoyant densities of cytoplasmic membrane vesicles and chromatophores from R. rubrum are about 1.14 and 1.16 g·cm- 3 , respectively, as determined after equilibrium sucrose density gradient centrifugation (Oeize et al.. 1975; Oelze, 1976; Collins and Niederman, 1976a,b). In R. sphaeroides. the values for both types of membranes varied between .1.14 and 1.19 g·cm- 3 (Worden and Sistrom, 1964; Gibson, 1965b; Ding and Kaplan, 1976; Michels and Konings, 1978a). Buoyant densities of 1.16 g. cm -3 were reported for cytoplasmic membrane preparations from either chemotrophically or phototrophically grown R. tenue. a species that does not form significant amounts of intracytoplasmic membranes (De Boer, 1969; Wakim et al .. 1979). In the latter case, however, the identical density of both preparations should be noted rather than the exact value, which might be influenced by the presence of cell wall contaminants. Fraley et al. (1979a), investigating membrane assembly in synchronous cultures of phototrophic R. sphaeroides. reported cyclic changes in the natural densities of chromatophore preparations ranging from 1.169 to 1.179 g. cm -3, as determined on cesium chloride gradients. Cyclic changes of the densities of isolated intracytoplasmic membranes occurred concomitantly with the earlier

The Bacterial Photosynthetic Apparatus

13

mentioned changes in the protein-to-phospholipid ratio (Figure 3) and moreover with changes in membrane fluidity (Fraley et aI., 1979b). The fluidity of bacterial membranes depends largely on the fatty acid composition of lipids (except for the Mycoplasmas, cholesterol is not generally found in prokaryotic membranes) as well as on the sizes of the polar head groups of phospholipids and on the interaction between lipid and membrane proteins (Melchior and Steim, 1976; Lenaz, 1979). Species of the Rhodospirillineae, e.g., R. capsuiata, R. rubrum, and R. sphaeroides, exhibit extremely high proportions of low-melting unsaturated fatty acids (Table III), which, within physiologically relevant temperatures, may not be expected to undergo thermotropic phase transitions from a disordered liquid-crystalline to an ordered gel state. Indeed, Fraley et al. (1978) reported thermotropic phase transition to occur below O°C in membranes of R. sphaeroides. Nevertheless, a decrease in the mobility of fatty acid side chains and a consequent decrease in membrane fluidity was observed in membranes of R. sphaeroides, which was interpreted as resulting primarily from an association between negatively charged lipids and antenna bacteriochlorophyll-protein complexes (Birrell et ai., 1978). It was also shown that immobilization by lipid-protein interactions could include up to about 70% of the total fatty acids. Interestingly, the degree of immobilization varied directly with the bacteriochlorophyll content of the chromatophores (Birrell et ai., 1978). In accordance with the dependency of lipid fluidity on the protein-to-lipid ratio, Fraley et ai. (1979b) observed the above-mentioned cyclic changes in membrane fluidity along with cyclic changes in the protein-to-phospholipid ratio during the cell division cycle (Figure 3).

2.3.

The Photosynthetic Apparatus

From its original meaning, the term photosynthesis describes the lightdependent autotrophic fixation of CO 2 into organic matter. In the phototrophic bacteria, however, photoautotrophy is one of several types of photometabolism. A great number of species are not only able to photoassimilate several simple organic compounds but are also able to cover their carbon requirements in the course of a photoheterotrophic type of metabolism (Trliper and Pfennig, 1978). Alternatively, photosynthesis may be defined as a light-dependent process leading to the production of ATP and reducing equivalents in the form of reduced pyridine nucleotides (Trebst and Avron, 1977). But since photoreduction of pyridine nucleotides is generally unlikely to occur in the phototrophic bacteria, this definition applied primarily to plant-type photosynthesis. Thus, taking into account the activities of phototrophic bacteria, photosynthesis is probably best defined as the process that transduces light energy into electronic energy and, moreover, into energy of the electrochemical proton

14

Jllrgen Oelze

gradient after charge separation followed by electron and proton transport. This in turn is assumed to be the driving force in several energy-dependent reactions of which the formation of energy-rich phosphate bonds is of central biological importance (Wraight et ai., 1978a). Accordingly, from a biological point of view, the function of ATPase may also be included in definitions of the photosynthetic apparatus. In conclusion, the bacterial photosynthetic apparatus comprises in a membrane-bound form light-harvesting antenna pigments as well as the photochemical electron transport system. Upon illumination the light-harvesting pigments become excited. When transferred to a photochemical reaction center, the excitation energy can be trapped through the primary photochemical reaction, ｾ･｡､ｩｮｧ＠ to charge separation. Electron flow from the reduced primary electron acceptor to the photooxidized primary electron donor involves special carriers that are also assumed to facilitate proton movement across the membrane. In the following subsections, our present understanding of the composition and function of the photosynthetic apparatus will be detailed. For further information the reader is referred to the comprehensive monograph on the photosynthetic bacteria (Clayton and Sistrom, 1978) as well as to reviews by Jones (1976), Gromet-Elhanan (1977), Drews (1978), Blankenship and Parson (1979), and Thornber and Barber (1979), to name a few.

2.3.1.

Light-Harvesting Units

The majority of bacteriochlorophyll molecules present in phototrophic bacteria are associated with light-harvesting antenna complexes. Accordingly, the absorption spectra of whole cells or isolated membranes are largely representative of the absorption properties of the light-harvesting pigments (Thornber et aI., 1978). So far, five different bacteriochlorophyll derivatives designated a through e have been described, of which bacteriochlorophyll a is further subdivided into two forms esterified with either phytol or geranyl-geraniol (Pfennig, 1978). Bacteriochlorophyll a is abundantly formed by members of the suborder Rhodospirillineae. Only a few strains of this suborder, such as R. viridis and Thiocapsa pfennigii, synthesize bacteriochlorophyll b. Bacteriochlorophylls c, d, and e are produced exclusively by the Chlorobiineae. While Rhodospirillineae form only one of the two derivatives a or b, Chlorobiineae contain one of the derivatives c-e as the predominant antenna pigment existing in combination with bacteriochlorophyll a, which is assumed to be involved in excitation transfer (antenna) as well as in the primary photochemical events (reaction center) (Truper and Pfennig, 1978; Pierson and Castenholz, 1978). In situ, a single bacteriochlorophyll derivative can exhibit different absorption bands in different species and under different conditions, particularly in the longer-wavelength region (Figures 5, 6, 18). The molecular basis for this is not yet fully understood, but current discussions point to possibilities

15

The Bacterial Photosynthetic Apparatus

A

QI

u

C

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o

1/1

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e. c.... := z

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188

Anatoly V. Lichtenstein et al.

would be relatively stable). The main features of the two models are shown in Figure 1. The subject of this chapter is the analysis anew of some data pertaining to the metabolism of informational RNAs and an attempt to discover whether peripheral regulation via selective nucleus-to-cytoplasm transport of RNA indeed takes place in vivo. Previously, a model for cytoplasm-governed gene regulation was suggested (Lichtenstein and Shapot, 1976) in which the idea of peripheral regulation was substantiated. Here this model is developed further in view of some recent data. It is now generally realized that the nuclear genome is not the unique genome of the cell: there are also the mitochondrial genome in all cells and the chloroplastal in plant cells, both of them being translated. There are some indications that mitochondrial products influence the expression of nuclear genes involved in mitochondrial biosynthesis. However, this chapter is not primarily concerned with the interplay of nuclear and mitochondrialjchloroplastal genetic systems; for a full discussion of such matters, the reader is referred to Gillham (1978). It should be stressed that many posttranscriptional regulatory events, such as mRNA transitions from inactive informosomes to active polyribosomes and the whole translation machinery, lie beyond the scope of this chapter.

2. INTERDEPENDENCE AND COMPLEMENTARITY OF CENTRAL AND PERIPHERAL MECHANISMS IN THE CONTROL OF GENE EXPRESSION The central dogma of molecular biology (Crick, 1970) envisages the flow of genetic information in the DNA-RNA-protein direction. Application of this dogma to eukaryotic cells implies the transfer of informational macromolecules from the nucleus to the cytoplasm, since it is the nucleus where, as a rule, RNA synthesis proceeds, while only in the cytoplasm is protein synthesized. This fact as well as a number of genetic observations have led investigators to the widely accepted view that the nucleus is not only the receptacle of the genome, but also the unique cellular organelle that controls the very realization of genetic functions (Chentsov, 1978). In accordance with this idea, most studies have focused on the chromatin structure in which one hopes to find the clue to the riddle of why some genes are expressed in the cell while others are not. The prime importance of such studies is obvious, but it is doubtful whether transcriptional regulation in itself, without the contribution of cytoplasmic factors, is able to determine the spectrum of proteins to be synthesized in the cell. It is worth stressing that the goal of such contradistinction is not to answer

Control of RNA Transport from Nucleus to Cytoplasm

189

the scholastic question of whether the nucleus or the cytoplasm is the leader in the regulation of gene expression. Indeed, both cellular compartments in vivo interact and complement each other; they find themselves separated only in the experimenter's hands. A warranted view is that interaction of the nucleus and the cytoplasm is so close that differentiation of the cytoplasm is at the same time both the result and the cause of differentiation of the nucleus (D' Amato, 1977). Our goals are to attract attention to underrated aspects of their interrelationship and to assess the contribution of each to the selection of mRNA sets. 2.1.

Regulation of Protein Synthesis: Control at the Transcriptional Level Is Necessary

There are contrasting views on the importance of transcription. The vast majority of authors hold that it is at that level that gene expression is generally regulated (see Chambon, 1978). On the other hand, there is a growing body of evidence (see below) inconsistent with the presumed predominant role of transcription. Therefore, many authors have put forward various models of posttranscriptional regulation (Scherrer and Marcaud, 1968; Tomkins et al., 1969; Harris, 1970; Sussman, 1970; Lichtenstein and Shapot, 1976; Remington, 1979; Davidson and Britten, 1979; Murray and Holliday, 1979). According to Remington's (1979) "attenuation" hypothesis, for example, there are specific factors that control a premature termination of nascent RNA chains, while the regulation of transcription, as such, does not occur. (Premature termination is the abortive arrest of transcription and the release of incomplete nascent transcripts from the template.) . Davidson and Britten (1979), summing up the results of many hybridization experiments, concluded that the nucleus of each differentiated cell contains not only all the genes ever acting in the organism but also the transcripts of all or most of them. To duly assess the hypotheses in question, it is imperative to go over the relevant data and find out whether regulation of transcription, if indeed acting, can alone ensure the cell specificity of protein synthesis. 2.1.1.

Puffs

Polytene chromosomes of Diptera exemplify differential gene activity via the formation of puffs, which are the manifestations of the activity of certain genes. According to autoradiographic observations, nucleotide precursors of RNA are rapidly incorporated into puffs, whereas DNA metabolism and DNA content in the puffs do not change (Beermann, 1964). Chromosome puffing proved to be tissue specific. Indeed, in the course of development of a given

190

Anatoly V. Lichtenstein et al.

tissue, one can see the highly ordered emergence and disappearance of puffs in certain loci, the location and timing of puffing being different in various tissues (Ashburner, 1967; Truman, 1974). The results of heat shock experiments show an obvious relation between the formation of puffs and the synthesis of certain proteins. When the temperature was elevated from 25· C to 37· C, new puffs emerged on the larval chromosomes, while some old ones disappeared. This phenomenon was accompanied by the synthesis of a new set of mRNAs coding for heat-shock-specific polypeptides and the arrest of transcription of many mRNAs synthesized at 25· C (Mirault et al., 1978). In addition, the transposition of RNA polymerase B from old puffs to new ones was demonstrated to occur as the result of heat shock (Jamrich et al., 1977). Ecdysone induces specific alterations in isolated insect tissues accompanied by the formation of specific puffs (Clever, 1968). Puromycin, an inhibitor of protein synthesis, blocks the chain of consecutive puffing, thus indicating that the proteins coded for by certain puffs serve as specific messages to induce the formation of others (see Truman, 1974).

2.1.2. Restricted Portion of the Genome Is Transcribed DNA:RNA hybridization experiments are able to give an unambiguous answer to the question of whether regulation of transcription in eukaryotic cells does in fact occur. The genome of higher organisms is characterized by a kinetic complexity of about 1-2 X 109 base pairs (Davidson and Britten, 1973). Hence, a similar kinetic complexity can be expected for cellular RNAs, if the whole genome is transcribed. In this case, about half of the DNA must be involved in hybridization assuming asymmetric transcription, or even a greater portion if both DNA strands are transcribed, which is actually found to occur in some cases (Scheller et al.. 1978; Davidson and Britten, 1979). In all experiments, however, a substantially smaller amount of DNA appears to hybridize, even under conditions of vast RNA excess. The transcription of nonreiterated DNA sequences in various human tissues has been studied by Sawada and Saunders (l974). The fraction of DNA transcribed was relatively small, amounting only to 2.5% in various types of leukemic lymphocytes and in liver and kidney. A slightly greater DNA fraction (3.2%) was transcribed in HeLa cells. Similar values for percent transcription of the genome of mouse malignant mastocytoma P8l5 cells, mouse liver, and spleen were reported by Boehm and Drahovsky (1979). Chikaraishi et al. (1978) hybridized an excess of nuclear RNAs from various rat tissues with unique DNA sequences, which are known to contain structural genes. Assuming asymmetric transcription (Le., that transcription proceeds on only one of the two complementary DNA strands), the above authors

Control of RNA Transport from Nucleus to Cytoplasm

191

have shown that in rat brain, liver, kidney, spleen, and thymus, 31.2, 21.8, 10.6, 9.6, and 9.2% of the genome are transcribed, respectively. It follows from these experiments that the proportion of the DNA transcribed in various tissues is quantitatively different, although qualitatively a significant portion of these sequences are common for many tissues studied. Although the kinetic complexity of nuclear RNA of various eukaryotic tissues, especially of brain, appears to be extremely high, being in the range of 2 X 107 to 2.6 X 108 nucleotides (Bantle and Hahn, 1976; Chikaraishi et al., 1978; Jacquet et al., 1978; Kaplan et al., 1978; Boehm and Drahovsky, 1979; Hough-Evans etal., 1979; Grouse and Grouse, 1979), in fact, it constitutes a relatively small proportion (8-26%) of the kinetic complexity of unique DNA sequences (Hahn and Laird, 1971; Turner and Laird, 1973; Holmes and Bonner, 1974; Kaplan et al., 1978; for review see also Davidson and Britten, 1973, 1974; Lewin, 1975a,b; Danehoit, 1976). Strict restriction of genome transcription is also exemplified by highly differentiated avian erythroid cells. Their nuclei contain a relatively narrow spectrum of polyadenylated RNAs, composed of about 4000 species, whereas in other avian cells it is found to be substantially broader (Tobin et al., 1978; Lasky and Tobin, 1979). From a comparison of liver and erythroid cell RNA populations, the authors infer that in avian erythroid cells gene expression is controlled strictly at the transcriptional level. Transcriptional regulation can be demonstrated not only in terminally differentiated but also in undifferentiated cells. For instance, in pluripotent embryonic carcinoma cells (PCC 3), nuclear poly(At RNAs have a complexity corresponding to only 2.5% unique sequences of the haploid genome (RNAs with or without poly(A) tracts at their 3' termini are termed poly(A)+ RNAs or poly(A)- RNAs, respectively). No globin-specific sequences, actively synthesized in erythroid cells, were found in nuclear RNA of undifferentiated PCC 3 cells (Jacquet et aI., 1978). 2.1.3.

Regulation of Transcription in Embryogenesis

There are several comprehensive reviews on the subject (Davidson, 1976; Kafiani and Kostomarova, 1978; Neyfakh and Timofeeva, 1977). Here we will only mention briefly some observations attesting to obvious reprogramming of gene activity in the course of embryogenesis. At the early stages of mouse development, the proportion of transcriptionally active reiterated DNA sequences nearly doubles (from 2.5% to 5%) and then stabilizes, amounting to 4% of the total DNA, or 13-16% of the reiterated DNA (Church and Brown, 1972). [The genome of all eukaryotes can be subdivided into three main fractions (see Britten and Davidson, 1969): unique sequences (with one or at most several copies per haploid genome), including

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most structural genes; moderately repeated sequences (with several hundred to thousands of copies per genome), which are presumably regulatory elements; and highly repeated sequences (up to a million copies per genome), comprising, as a rule, the so-called satellite DNAs.] As to the proportion of unique DNA sequences, its increase is more pronounced: from about 1% in the early stages to 10% by the moment of birth (Church and Schultz, 1974). An interesting phenomenon suggesting a reprogramming of genome activity, namely the reciprocal relationship between unique and reiterated DNA sequence activity transcribed, is revealed during the development of mammals (see Neyfakh and Timofeeva, 1977). Before any visible differentiation occurs, the ratio of transcribed reiterated sequences to unique ones is 5 (2.5-5% for reiterated and 0.5-1 % for unique sequences). By the seventh day of mouse development, this ratio drops to 1 and at the moment of birth is as low as 0.4 (4% reiterated, 10% unique sequences). As the morphological differentiation of organs proceeds, their RNAs become to some extent tissue specific, though a certain RNA fraction remains common to all cell types (Neyfakh and Timofeeva, 1977). Hybridization of cellular RNAs isolated at various stages of embryonic development with repeated DNA sequences revealed four main RNA subpopulations: (1) RNA species synthesized only during oogenesis and thereafter gradually utilized; (2) RNA species synthesized constantly in oocytes as well as in embryonic and adult tissues; (3) RNA species synthesized only after passage through a certain stage of development; and (4) RNA species synthesized transiently only at a certain stage of development and disappearing thereafter (see Kafiani and Kostomarova, 1978). Analogous RNA classes were found in Euglena gracilis chloroplasts during their development (Chelm et al., 1979). The occurrence of such RNA subpopulations is indicative of a development-dependent differential gene activity. A comparative study of structural gene sets active in embryos and adult tissues of the sea urchin showed large qualitative differences between them (Galau et al., 1976). Using sea urchin unique DNA sequences enriched in sequences transcribed in mature oocytes as molecular probes in hybridization with RNAs from different embryonic stages, Hough-Evans et al. (1977) have shown that polyribosomes of the 16-cell embryo, blastula, and gastrula contain 73, 56, and 53% of the maternal mRNA set, respectively. These data indicate a gradual narrowing of the spectrum of transcribed genes in the course of embryogenesis. Ernst et al. (1979) studied further the RNA sets of sea urchin embryos and adult tissues. Labeled DNA containing sequences absent from gastrula nuclear RNA was hybridized with nuclear RNA from the adult intestine, thereby testing the presence in the latter of sequences lacking in the gastrula. In this way, differential gene activity was shown to occur in the two tissues

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studied. About one-third of the unique DNA sequences are transcribed in both tissues (2 X 108 base pairs), while differentially transcribed unique sequences constitute about 3.5 X 107 base pairs. An interesting example of differential gene activity during sea urchin development was shown by Nemer and co-workers (Nemer, 1975; Nemer et al., 1975; Dubroff and Nemer, 1976). Poly(A)+ RNAs and poly(A)- RNAs were found to be distinct subpopulations of molecules transcribed on different genes (see also Gasaryan et aI., 1978). In the course of development, the proportions of poly(A)+ RNAs and poly(A)- RNAs substantially change. Poly(A)- RNAs are thought to code for proteins responsible for basic cell functions common to all cells, whereas poly(A)+ RNAs presumably direct the synthesis of specific proteins appearing at the late differentiation stages. Even from this very brief review one can see that the regulation of transcription contributed efficiently to the development and differentiation of animal cells.

2.1.4.

Hormone-Induced Synthesis of Specific RNAs

There is ample information pertaining to the hormonal regulation of protein synthesis, and here we will restrict ourselves to only a few examples demonstrating the hormone-induced transcription of specific RNAs. As mentioned in Section 2.1.1., molting hormone (ecdysone) has the capacity to induce in isolated insect tissues a cascade of successive puffings presumably responsible for the synthesis of specific molting-related proteins. In chicken oviduct, the transcription of ovalbumin is strongly stimulated by steroid hormones (Palmiter, 1975; Royal et al., 1979). A marked stimulation of ovalbumin gene transcription in response to estradiol treatment was revealed by quantitation of the ovalbumin mRNA content in various subcellular fractions of chick oviduct (Cox et al., 1974). Experiments with oviduct nuclei isolated at various stages of hormonal stimulation have shown that the accumulation of ovalbumin mRNA results from the activation of specific gene transcription (Schlitz et al., 1978). Similar results were obtained in experiments with isolated chromatin (O'Malley et al., 1978). Tissue-specific transcription of the ovalbumin gene was demonstrated in hybridization experiments with cloned ovalbumin cDNA (Swaneck et al., 1979). Abundant ovalbumin mRNA sequences were elicited in oviduct nuclei, but not in spleen or liver nuclei. Ovalbumin mRNA synthesis practically stopped in oviduct nuclei I hr after estrogen withdrawal but resumed upon the second induction. In an attempt to fractionate moderately hydrated chromatin from the

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chicken oviduct by means of ECTHAM-cellulose chromotography,* O'Malley and co-workers obtained two fractions that apparently corresponded to transcriptionally active and inactive chromatin (they contained 12 and 88% of the total DNA, respectively) (Strotling et al., 1976; Strotling and O'Malley, 1976). The amount of the active chromatin fraction increased from 12% in controls to 20% in animals that had undergone 9 days of estrogen induction. Hormone-induced transcription of specific genes was shown also for the synthesis of vitellogenin in the liver of chicken (Panyim et aI., 1978) and Xenopus (Tata, 1978), as well as for human placental lactogen (McWilliams et al., 1977) and rat preprolactin (Ryan et al., 1979). Also, plant hormones have been shown to affect both total and specific RNA synthesis in plants (Biswas and Roy, 1978; Varian and Sacher, 1978).

2.1.5. Transcriptionally Active Chromatin Is in an Altered Conformation Nucleosomes have been convincingly shown of late to be elementary structures of chromatin composed of a core octamer of main histones with DNA wound on its surface (see Chambon, 1978). Nucleosomal structure is broadly similar in both inactive chromatin (which makes up the predominant mass of the chromatin) and active chromatin. Nucleosomes in the latter, however, are characterized by some distinct features (presumably a more "open" conformation) that enable DNases to rapidly degrade them. Indeed, DNase I preferentially digests those nucleotide sequences that are active in a given tissue, for example, globin genes in erythroid cells (Weintraub and Groudine, 1976) and ovalbumin genes in chick oviduct cells (Garel and Axel, 1976; Axel and Garel, 1977; Garel et al., 1977). The same genes in other tissues that synthesize no such proteins are resistant to DNase I. Similarly, altered chromatin conformation was found to be associated with the actively transcribed DNA of integrated adenovirus genes (Flint and Weintraub, 1977). Transcriptionally active loci of chromatin display a high sensitivity to DNase II as well. On the basis of this peculiarity, rat liver chromatin was separated into two fractions, transcriptionally active and inactive (Gottesfeld et al., 1975). It was found that nucleosomes of active chromatin differ from those of inactive chromatin both in conformation and in protein composition. Judging by the DNase II test, globin genes of Friend leukemia cells are in active conformation both before and after the cells have been induced to differentiate with dimethylsulfoxide. However, in cells that have lost the ability to produce

*ECTHAM-cellulose is a cationic adsorbent prepared by coupling tris (hydroxymethyl) aminomethane to cellulose with epichlorhydrin.

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hemoglobin, the globin genes are no longer associated with the template active chromatin fraction (Wallace et al., 1977). In contrast to some earlier reports, there are recent data on the ability of micrococcal nuclease under certain conditions to digest active nucleosomes preferentially as well (Bellard et al., 1978; Tata and Baker, 1978; Bloom and Anderson, 1978). Since DNase I, DNase II, and micrococcal nuclease, enzymes with very different modes of action, are all able to digest preferentially template-active genes, it seems likely that these enzymes recognize only large-scale structural features manifested along the whole active regions of chromatin, rather than local and specific conformational alterations (Bloom and Anderson, 1978). A similar view has also been expressed concerning exogenous RNA polymerases, which serve apparently as nonspecific probes for the active chromatin conformation, which is presumably more easily accessible for interactions (Chambon, 1978). Electron microscopic observations clearly demonstrate substantial morphological distinctions between transcriptionally active and inactive chromatin (Franke et al., 1978). The former was found to be extended and unfolded, in contrast to condensed inactive chromatin (see also Lilley, 1978). Thus, it follows from the above review that differential gene activity is an indispensable step in the process of gene expression.

2.2.

Regulation of Protein Synthesis: Transcription Alone Is Not Sufficient

In the preceding, only observations that unequivocally attest to an important role of transcription in the regulation of protein synthesis have been discussed. This endeavor might seem unnecessary since the predominant role of transcription is often regarded as self-evident. However, the data to be reviewed below show that transcriptional regulation in itself is far from sufficient to ensure tissue-specific synthesis. There is a growing body of evidence to indicate that the extent to which the transcription of structural genes is regulated in the animal cell may be much more limited than previously thought. It has been suggested that the nucleus of all differentiated cells may contain transcripts of most, if not all, genes ever expressed in the organism (Davidson and Britten, 1979). It seems paradoxical that doubts about the predominant role of transcription have been engendered by the same approaches that first asserted this view. Hybridization studies that convincingly demonstrated that only a portion of the genome is transcribed in the eukaryotic cell have shown, at the same time, that transcription alone is insufficient for tissue-specific protein synthesis. In

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other words, far more DNA sequences are transcribed than are needed at any given moment.

2.2.1.

Molecular Probes in the Study of Chromatin Structure

In early work, it was shown that the transcription of chromatin in vitro by exogenous RNA polymerases was strongly limited as compared to the transcription of deproteinized DNA. In those experiments, a certain tissue-specificity of RNA synthesis in in vitro systems was observed, this finding being regarded as decisive evidence for cell-specific transcription in vivo. However, since the work of Zasloff and Felsenfeld (1977a), serious doubts have appeared concerning the validity of earlier results. In experiments with mercuriated nucleoside triphosphates, which permitted the extraction of in vitro synthesized transcripts by affinity chromatography on thiol-agarose, the following facts were established: (1) Contaminating preexisting RNA was present in the chromatin preparations tested; (2) E. coli RNA polymerase can synthesize RNA on a template of this contaminating RNA; (3) the tissue-specific RNA sequences belong to the preexisting RNA rather than to those synthesized de novo (the preexisting RNA sequences were carried over by the newly formed ones by molecular hybridization during the affinity chromatography). This "carry-over" phenomenon is presumably responsible for the quasi-specific in vitro transcription. When all precautions were taken, no significant specific transcription of duck reticulocyte chromatin by E. coli RNA polymerase could be observed (Zasloff and Felsenfeld, 1977a,b), although in the other system (mouse embryonic liver chromatin) it presumably did occur (Gilmour, 1979). On the other hand, no specific transcription of globin sequences could be detected when chromatin preparations from rabbit erythrocytes, reticulocytes, or brain were compared (Konkel and Ingram, 1978). In addition, it was demonstrated that E. coli RNA polymerase failed to initiate the synthesis of new globin transcripts on the template of reticulocyte chromatin but merely elongated already preexisting ones, thus revealing the specificity of an endogenous enzyme and not the specific conformation of active chromatin per se (Maryanka et al., 1979). Thus, the facts known to date make one doubt the validity of this approach as evidence for tissue-specific transcription. Many researchers share the view that exogenous RNA polymerase is nonspecific in that it interacts merely with decondensed and, as a result, more accessible regions of chromatin (Chambon, 1978). As to the regulation of transcription in vivo, it presumably involves the local site near the promoter, the specific recognition of which by an endogenous RNA polymerase is thought to play the key role in gene activation.

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Aside from exogenous RNA polymerase, a number of nucleases have been routinely used as molecular probes for chromatin structure, since they are able to digest preferentially tissue-specific genes from isolated chromatin or nuclei. An interesting fact is that DNases I and II attack not only genes with on-going transcription but also potentially active ones (Weintraub and Groudine, 1976; Garel et al., 1977; Wallace et al., 1977; D. M. Miller et al., 1978). Also, globin genes in mouse erythroleukemia cells, irrespective of whether their differentiation is or is not induced by dimethylsulfoxide, appeared equally sensitive to DNase I. It follows that globin genes in these cells are in an active conformation irrespective of the transcription rates (D. M. Miller et al., 1978). The authors infer that transcription of active genes is determined not only by their conformation in chromatin but also by some additional factors. From studies on the kinetics of DNase I digestion, Garel et al. (1977) concluded that chick oviduct ovalbumin genes transcribed at different rates are in the same conformation. Similar data were obtained using DNase II as a molecular probe (Wallace et al., 1977). The globin genes appeared to be associated with the active chromatin in Friend leukemia cells both before and after the cells were induced with dimethylsulfoxide. However, in cells that had lost the ability to produce hemoglobin, the globin gene sequences were no longer associated with the active chromatin fraction (Wallace et al., 1977). The nature of the factors that determine the rate of actual transcription of a gene remains unknown.

2.2.2. Transcription Is Not Cell Specific The availability of highly labeled complementary DNAs (cDNAs), both to total and to individual mRNAs, for hybridization studies has substantially changed the views on the regulation of transcription. The widely adopted view had been that this process was implemented just by turning genes on or off ("qualitative regulation"). Now a quantitative mode of regulation seems to be more likely, i.e., modulation of the rate of transcription on a set of permanently active genes (Lichtenstein and Shapot, 1976; Davidson and Britten, 1979; Gilmour, 1979; Remington, 1979). A further development of this idea implies that strongly overlapping or even identical gene sets function in various cells. Depending on the phenotype and physiological state of the cell, certain genes are activated to a greater extent than others. Such a view is supported by the numerous data that reveal no qualitative specificity of transcription. As already mentioned, RNAs synthesized in various cell types are similar not only in terms of kinetic complexity quantitatively but also qualitatively. For example, most of the nuclear RNA sequences of highly malignant mas-

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tocytoma P815 cells are also found in normal liver and spleen. Only a small fraction of the RNA transcribed on unique DNA was found to be specific for mastocytoma cells (Boehm and Drahovsky, 1979). A high degree of homology was found to occur between nuclear RNAs of such distant organs as rat brain, liver, spleen, and thymus. A significant portion of the nuclear RNA sequences constitutes a "core" common to various tissues (Chikaraishi et ai., 1978). Both poly(A)+ and poly(A)- RNAs isolated from various regions of rat brain (cerebellum, hypothalamus, hypocampus, and cerebral cortex) were shown to have a kinetic complexity as great as that of the whole brain (about 6 X 108 nucleotides) and a high degree of homology (over 80%) (Kaplan et ai., 1978). Supporting evidence comes also from the fact that even dramatic changes in the physiological status or phenotype of the cell are not accompanied by corresponding alterations at the transcriptional level. Indeed, no clear-cut differences were found between total nuclear RNA sets of normal and tumor cells (Drews et ai., 1968; Church et aI., 1969; Turkington and Self, 1970; Shearer and Smuckler, 1972; Garrett et ai., 1973a,b; Flickinger et ai., 1973; Sawada and Saunders, 1974; Piker and Shapot, 1976; Shearer, 1974a,b, 1977, 1979); resting and growing cells (Johnson et ai., 1974, 1976, 1977; Greene and Fausto, 1977); cells before and after hormone induction or hormone withdrawal (Church and McCarthy, 1970; Flickinger and Roche, 1972; Mizuno and Cox, 1979); and Friend erythroleukemia cells irrespective of stimulation with dimethylsulfoxide (Minty et ai., 1978). Even during embryonic development, the changes in transcriptional patterns are less pronounced that would be expected (Kleene and Humphreys, 1977). Most of the nuclear RNAs from sea urchin gastrula and from the intestine of adult animals were found to common (about 2 X 108 nucleotides), whereas the fractions of differentially transcribed sequences comprised only about 3.5 X 107 nucleotides (Ernst et ai., 1979). In some instances, template sequences not expressed in the tissue studied were nevertheless revealed among nuclear RNAs. For example, nuclear RNAs from rabbit intestine, kidney, and liver were found to be capable of directing the synthesis of globin in a cell-free system (Kruh, 1972). By the hybridization of globin cDNA with RNAs of various origin, specific globin sequences were revealed in RNAs from cells of erythroid lineage (embryonic liver and reticulocytes) as well as from nonerythroid cells (adult liver and brain, cultured lymphoma cells, transformed and nontransformed fibroblasts) (Humphries et ai., 1976). Polyribosomes of frog embryo contain distinct mRNA sets at specific stages of development (neurula and larvae), while the corresponding nuclear RNAs are very similar, if not identical (Shepherd and Flickinger, 1979). Analogous data on the presence in sea urchin embryo of template sequences in

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nuclear RNA not expressed in polyribosomes at the same stage were reported by Wold et al. (1978).

3.

SOME HYPOTHESES ON POSTTRANSCRIPTIONAL REGULATION

It is clear that the regulation of protein synthesis cannot be explained exclusively by differential gene activity, which fails to account for the broader spectrum of nuclear RNAs as compared to cytoplasmic RNAs. This situation spurred attempts to explore additional (posttranscriptionai) levels of the regulation of mRNA sets in the cytoplasm. Some of the resulting models are outlined below.

3.1. "Cascade Regulation" Model Scherrer and Marcaud (1968) advanced the "cascade regulation" model aiming at an explanation of the presence of nucleus-restricted RNAs. According to this model, transcriptional units in eukaryotes are polycistronic and, unlike those in prokaryotes, contain genes not linked to each other functionally as well as sequences that carry no structural information at all (the latter prediction proved correct when introns were discovered). The redundancy of nucleotide sequences in primary transcripts makes their processing necessary (a multistep process of extraction of the structural information needed). This process can be viewed as getting "rational kernels out of husks." The cascade regulation hypothesis introduces the important point of multistep regulation of protein synthesis as well as the notion of processing large mRNA precursors to much smaller "mature" mRNAs. This second prediction also proved correct after some 10 years of doubt and caution.

3.2. "Ticketing" Model In this hypothesis an attempt was made to link selective nucleus-to-cytoplasm transport of RNA with a special activity of ribosomes (Sussman, 1970). According to the model, there is a highly heterogeneous population of ribosomes in the cell, each ribosome being able to interact with its "own" mRNA by recognizing certain specific nucleotide sequences ("tickets"). Each ribosome shuttles between nucleus and cytoplasm transferring corresponding mRNAs. Those pre-mRNAs that find no specific ribosomes as carriers undergo a rapid degradation within the nucleus. It is suggested that each mRNA molecule has reiterated sequences at its 5' terminus that serve as ribosome recognition sites.

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As the ribosome moves along the mRNA in the process of protein synthesis, one of its enzymes cuts off the first ticket, this process being repeated until all the tickets have disappeared. Thereafter, the mRNA breaks down rapidly. Sussman's hypothesis offers an explanation for nucleus-restricted RNAs and a possible mechanism for control of mRNA stability. However, its main point, namely a high degree of heterogeneity of ribosomes, has not been confirmed experimentally. 3.3.

Model Involving Cytoplasmic Inhibitors of mRNA Function

The paradoxical phenomenon of the "superinduction" by actinomycin D of the synthesis of certain enzymes offers strong evidence for posttranscriptional regulation (Tomkins et al., 1969, 1972). Studying the induction of tyrosine aminotransferase in HTC hepatoma cells by steroid hormones, these authors observed that not only was the induction not blocked by actinomycin D-induced inhibition of RNA synthesis, but it was even more stimulated. To date many similar examples of superinduction by actinomycin Dare known, e.g., RNA synthesis in plant tissues (Sacher and Leo, 1977); ovalbumin, conalbumin, ovomucoid, and lysozyme synthesis in chick oviducts (Palmiter and Schimke,1973);production of interferon in human fibroblasts (Sehgal and Tamm, 1976; Sehgal et aI., 1977); globulin CX2u synthesis in rat liver (Chatterjee et al., 1978, 1979). An increased template activity of poly(A)+ mRNA isolated from actinomycin-D-treated cells was observed in a cell-free protein synthesis system (Kessler-Icekson and Yaffe, 1977; Kessler-Icekson et al., 1978). As an explanation, the existence in the cytoplasm of a short-lived protein repressor was suggested (Tomkins et al., 1969). The repressor prevents mRNA from functioning as a template presumably by triggering its degradation or by some other process. Induction of tyrosine aminotransferase synthesis by steroid hormones becomes possible as a result of their binding to the repressor. In cells treated with actinomycin D, the content of the short-lived repressor decreases and, as a result, synthesis of the enzyme is superinduced due to increased mRNA stability. A special class of recently discovered low-molecular-weight cytoplasmic RNA, termed translation-controlling RNA (tcRNA), may be related to the hypothetical Tomkins' repressor. tcRNAs were shown to inhibit translation of polysomal and postribosomal mRNAs in a rather specific way (Kennedy et al., 1974; Fuhr and Overton, 1975; Bester et al., 1975). The presence of long poly(U) tracts in some tcRNAs led the authors to suggest that inhibition of mRNA template activity occurs as a result of hybridization of poly(U) of

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tcRNA with the poly(A) tract of mRNA, thus bringing the latter into an inactive conformation (Bester et al., 1975).

3.4.

Autogenous Regulation of Gene Expression

This hypothesis postulates a universal principle, according to which a protein directly controls expression of its own structural gene (Goldberger, 1974). Apart from this, the protein carries out special functions acting as enzyme, structural protein, antibody, etc. This principle is illustrated by the mouse myeloma protein that specifically binds to mRNA, thus preventing it from participation in translation. In this way, autogenous regulation could operate at the translational level.

3.5. Attenuation as a Mechanism for Differential Gene Activity This model proposed by Remington (1979) suggests that regulatory molecules interact not with DNA but with nascent pre-mRNAs, and that it is this process that underlies the control of differential transcription. Based on the model of Lee and Yanofsky (1977) for the trp operon of E. coli, this hypothesis ascribes a special role to the noncoding "leader" sequences at the 5' termini of eukaryotic pre-mRNAs. It is suggested that a specific conformation of the 5' terminus of pre-mRNA determines the extent of premature termination of transcription (attenuation). In one of the two alternative conformations, abortive synthesis occurs, i.e., the release of incomplete nascent transcripts from the template. A modulation of the secondary and/or tertiary structure at the 5' terminus mediated by the regulatory molecules (antiattenuators) prevents premature termination, resulting in the production of complete transcripts. The premise for this idea is that the differences in patterns of transcription in cells of various origin and physiological state are quantitative rather than qualitative. Therefore, all or most structural genes are thought to be in an "open" conformation and potentially active. As to their actual expression, this is determined by the contribution of regulatory molecules ensuring synthesis of full-length transcripts. According to Remington, a certain probability, inequal for different genes, of transcription "read through" exists even in the absence of regulatory molecules whereby complete, though not functionally needed, transcripts are formed. (The readthrough phenomenon is random movement of the transcription complex beyond the site of early termination under conditions that would normally be expected to result in termination of transcription.) This read through may account for, on the one hand, a high degree of overlapping of nuclear pre-mRNAs from various tissues, many of which are not expressed

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in polyribosomes at any given time, and, on the other hand, an extremely high degree of heterogeneity of nuclear RNAs and the low frequency of most of them. Besides this, premature termination of transcription is expected to result in the accumulation of short abortive transcripts associated with chromatin, which might constitute the so-called chromosomal RNAs (Holmes and Bonner, 1974), an enigmatic fraction with as yet unknown functions.

3.6. Hypothesis Proposing a Regulatory Role for Repetitive Sequences Proceeding from the already mentioned data on the absence of tissue specificity in the pattern of transcription of most structural genes and the much greater diversity of nuclear RNAs compared to cytoplasmic RNAs, Davidson and Britten (1979) have distinguished two main DNA fractions. The first fraction includes the so-called constitutive transcriptional units, which represent structural genes transcribed continuously in all cell types, at more or less similar rates. The production of constitutive transcriptional units forms a highly heterogeneous spectrum of RNAs common to various tissues. The second DNA fraction includes integrating regulatory transcription units, which carry no structural information and are transcribed in a cell-specific fashion. This latter DNA is made up of interspersed repetitive and single-copy sequences or of clusters of repetitive sequences. The sorting out of cell-specific structural transcripts is suggested to occur at the posttranscriptionallevel via intranuclear molecular hybridization between structural transcripts and cell-specific sets of integrating regulatory transcripts. Due to the specific arrangement of repeated sequences within structural transcripts and complementary sequences on appropriate regulatory transcripts, the hybrids resulting from sequence-dependent base pairing acquire a specific conformation. This conformation is an obligatory requirement for further processing of mRNA and its transport to the cytoplasm. As a result, only hybridized structural transcripts survive and pass through processing, the other undergoing rapid degradation. The point is that the intranuclear concentration sequences of a given family of repeats on regulatory transcripts may vary in a wide range depending on the number of family members transcribed. Accordingly, the fraction of "salvaged" pre-mRNAs from each gene will vary from 0 to 100% for it is the intranuclear concentration of appropriate repeats on regulatory RNAs that determines the rate and extent of completeness of RNA:RNA hybridization. Thus, according to Davidson and Britten, "both the quantitative and qualitative structure of cytoplasmic mRNA populations are controlled posttranscriptionally. " A limited set of genes coding for the so-called superprevalent mRNAs

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(with more than 104 copies per cell), such as ovalbumin mRNA in the oviduct of the laying hen and globin mRNA in mouse or chick reticulocytes, are transcribed nonconstitutively. The synthesis of these mRNAs is undoubtedly regulated at the transcriptional level, and they are outside the scheme proposed. The authors assume that the genes coding for superprevalent mRNAs constitute a special class characterized by a high tissue specificity.

3.7. Hypothesis of "Splicer" RNAs This hypothesis (Murray and Holliday, 1979) is akin to the DavidsonBritten model, as both regard specific intermolecular RNA:RNA interactions as a general mechanism for posttranscriptional regulation. It has been shown recently that many structural genes in eukaryotes consist of expressed structural sequences (exons) and intervening sequences (introns) that carry no structural information (Flavell et al., 1978; Tilghman et al., 1978a; Breathnach et al., 1978; Darnell, 1978; Tonegawa et al., 1978; Leder et al., 1978; Catterall et al., 1978; Smith and Lingrel, 1978; Ghosh et al., 1978; Konkel et al.,1978 ; Rabbitts, 1978). The latter are thought to be transcribed but subsequently excised by a process of RNA splicing (Tilghman et al., 1978b; Gilbert, 1978). The authors hypothesize that a special class of so-called splicer RNAs of low molecular weight is synthesized in the cell on certain genetic loci. The role of splicer RNAs would be to establish a precise localization of the breakagejoining sites in different pre-mRNAs by a common enzymatic mechanism. This is carried out by hybridization of splicer RNA with pre-mRNA in such a way that adjacent exons are brought together, whereas introns form out-of-hybrid loops that are the targets for cleavage enzymes. Following ligase action would ensure end-to-end splicing of adjacent exons (see Figure 2). Strong evidence for this mechanism comes from analysis of the structure formed from adenovirus type 2 virus associated RNA and the third splice point of adenovirus 2 exon pre-mRNA. The authors suggest that small nuclear RNAs (150-250 nucleotides in length)(Weinberg and Penman, 1968; Brown and Marzluff, 1978) found in pre-mRNA-containing ribonucleoproteins are suitable candidates for the role of splicer RNAs, especially since they appear in some cases to be base-paired to pre-mRNAs (Deimel et aI., 1977; Guimont-Ducamp et al., 1977; Flytzanis et al., 1978; Seifert et al., 1979; Lerner and Steitz, 1979). This prediction may be correct as extensive complementarity has been revealed between some small nuclear RNA sequences and sequences present around the splice junctions in heterogeneous nuclear RNAs (Lerner et al., 1980; see also Roberts, 1980). It is hypothesized further that if intron excision is a necessary prerequisite

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for transportation of mRNA to the cytoplasm, then the mechanism for RNA splicing may be a means of regulating gene expression. Indeed, the set of splicer RNAs in this case would determine the spectrum of pre-mRNAs properly processed and transported to the cytoplasm, this process being in principle similar to the "salvage" of structural transcripts in the Davidson-Britten model.

4.

THE CYTOPLASM AS A SOURCE OF GENOMEREPROGRAMMING ACTIVITY

In the Davidson-Britten model as well as in some others, the supply of cell-specific messengers is regarded as a purely nuclear process. Weare of the opinion, however, that the participation in this process of the other major cellular compartment, the cytoplasm, should not be ignored. The possible mechanism by which the cytoplasm may influence gene activity was formulated in our model of cytoplasm-governed gene regulation (Lichtenstein and Shapot, 1976; for details see below). The prerequisite for such a model would be that gene activity in the nucleus is forced to exhibit a mode of action dictated by its cytoplasmic environment. In this section we will outline some cytological, embryological, and biochemical observations that attest to the ability of some cytoplasmic factors to exert powerful and, in certain instances, highly specific effects on the nuclear genetic apparatus. Our emphasis will first be on the cytoplasmic localization of morphogenetic determinants in the early development of a number of multicellular organisms. This topic has been expertly covered by Davidson (1976). He has gathered numerous data revealing that specification of the fate of the embryonic cell is determined by the sector of egg cytoplasm inherited by embryonic blastomeres and that each blastomere lineage then gives rise only to specific differentiated cell types, although each blastomere has a nucleus that contains all the genetic information inherent in the organism. Eight-cell embryos of ctenophores are composed of "E" and "M" cells. The descendants of E cells form several structures, in particular swimming prominent cilia, whereas those of the M cells differentiate to other structures, including highly specialized light-producing photocytes. Cilia and photocyte determinants were shown to be localized in different regions of the egg cytoplasm, reaching final positions at different times (Freeman, 1976). Commenting on Hegner's (1911) experiments with Coleoptera, Davidson (1968) came to the conclusion that the egg cytoplasm contained molecules capable of specific and selective activation of certain genes. Indeed, when the egg cytoplasm, which determined the development of primary germ cells, was destroyed by a hot needle, the embryos lost their capacity to extract corre-

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sponding information from the genome, and the resulting adult insects, unlike the normal ones, had no gametes, i.e., were sterile. The cytoplasm was shown to be able to reverse the direction of cell differentiation. Grasshopper neuroblasts divide asymmetrically: one of the daughter cells is preserved as a neuroblast ready for the next division, whereas another differentiates to form the gangliocyte. The cytoplasm of the dividing neuroblast at the edge of the potential gangliar cell differs morphologically from that at the opposite pole where the neuroblast is to be formed. Carlson (1952) used a microneedle to rotate the spindle of the dividing neuroblast during anaphase in such a way that the chromosomes, destined to migrate to the gang liar pole, were directed to the opposite side and vice versa. In spite of the abnormal position of the chromosomes, the cytoplasm of the gang liar pole continued to form gangliocytes with either chromosome set. In this connection the transplantation experiments of McKinnell et al. (1976) are worth mentioning. These authors demonstrated that the amphibian oocyte cytoplasm can direct the normal development of the embryo by changing a settled program of the differentiated cell genome. Nuclei isolated from mitotically active cells of Lucke carcinoma, characterized by a triploid chromosome set, were implanted into the enucleated and activated oocytes of healthy frogs with a diploid karyotype. The organism resulting from such a nuclear-cytoplasmic hybrid developed normally and reached the stage of swimming tadpole, all the cells of its tissues containing nuclei of a triploid karyotype, i.e., of kidney carcinoma origin. Doubts raised at the time pertained to the possible contamination of malignant cell nuclei with normal stromal cells, but the very effect of the oocyte cytoplasm on the functioning of the differentiated cell genome was never questioned. A series of well-known studies by Gurdon are in line with the above results (see Gurdon, 1970, 1974). For example, nuclei isolated from adult frog brain and then implanted into frog oocytes of various developmental stages as well as into unfertilized eggs behaved differently in terms of DNA and RNA synthesis depending on the type of cell cytoplasm in which they found themselves (Gurdon, 1974). Especially impressive are recent experiments of Gurdon's group (De Robertis and Gurdon, 1977). Nuclei isolated from cloned Xenopus laevis kidney cells were injected into oocytes of the newt Pleurodeles waltlii. A high-resolution electrophoretic technique was used to fractionate proteins synthesized by injected and intact Pleurodeles oocytes, Xenopus oocytes, and somatic donor cells. As a result, protein sets specific for each cell type mentioned were revealed. They differed both in electrophoretic mobility and in fingerprint patterns. Nonexpressed "early" genes of Xenopus somatic cells were found to be expressed anew in oocytes, whereas the genes expressed in Xenopus somatic cells were turned off after their nuclei were injected into Pleurodeles oocyte:>.

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Very interesting results were obtained by Lucas's group (Lipsich et al.• 1979) that attest to the capacity of the cytoplasm to activate an individual "silent" gene. The experiments were carried out on karyoplasts and cytoplasts (purified nuclei and enucleated cells, respectively) in a short form culture. A culture of pure cytoplasts was obtained from rat HTC hepatoma cells containing the tissue-specific enzyme tyrosine aminotransferase, the synthesis of which is stimulated by glucocorticoids. A culture of pure nuclei (uncontaminated with cytoplasm) isolated from mouse A9 fibroblasts, unable to synthesize tyrosine aminotransferase, served as the other half of the fusion experiments. By fusion of rat cytoplasts with mouse karyoplasts, viable nuclear-cytoplasmic hybrids were formed that preserved the genetic marker of the nuclear donor (resistance to 8-azaguanine). The hybrid cells synthesized thermostable mouse tyrosine aminotransferase, inducible by dexamethasone, over a period of 75-100 generations. It is worth emphasizing that reprogramming of the fibroblast genome by the hepatoma cytoplasm turned out to be strictly selective; indeed, the genes coding for other liver-specific proteins, e.g., albumin, were not activated. Summing up this section, one has to bear in mind that the cytoplasmic factors affecting gene expression represent, at the same time, the end products of the genome activity. Here we face the two counterflows of information, centrifugal and centripetal, complementing each other.

5. A MODEL FOR CYTOPLASM-GOVERNED GENE REGULATION Numerous data indicate that the spectrum of RNAs synthesized in the nucleus is far broader than that required for protein synthesis in the cytoplasm (see Section 2.2 and below). Various models attempt to explain how this surplus of nuclear information might be eliminated at the posttranscriptionallevel. The question that is not answered, however, relates to the origin of the phenomenon itself, i.e., why are more DNA sequences transcribed than needed? The reason for this surplus transcription would be the occurrence of "split genes" or "genes in pieces" recently discovered (see Gilbert, 1978; Darnell, 1978; Flavell et al., 1978). Thus, the primary transcript of a given gene contains not only structural information, but also nontemplate intervening sequences, introns, which are eliminated during processing of pre-mRNA. Therefore, one could suggest that it is exactly the intron sequences, present in nuclear RNAs and absent from cytoplasmic RNAs, that make the former much more diverse than the latter. However, the phenomenon of split genes alone may only partially solve this problem. The point is that the qualitative surplus of nuclear sequences, as compared to cytoplasmic sequences, arises not only because of nontemplate intron sequences but also because of sequences of potential nuclear messengers

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(see below). The mechanism that presumably underlies the qualitative redundancy of transcription is considered in the following section.

5.1. Qualitative Redundancy of Transcription as a Consequence of Structural Organization of the Genome The term "leaky genes" was coined some time ago (Gal au et al., 1976) to designate genes transcribed rarely and not expressed in the given tissue. Such "leakage," which results, for example, from the fortuitous readthrough transcription of functionally unneeded genes (Remington, 1979), may give rise to qualitative redundancy of transcription. In our model this fact is accounted for in another way. It is suggested that in the very organization of the genome, as it appears at present in the model of Britten and Davidson (1969), there is some redundancy in effect in comparison with what is needed (Lichtenstein and Shapot, 1976). It is likely that the genome organization is suitable for large-scale rearrangements of cell protein synthesis during embryonic development. However, it seems to be too coarse to adjust itself to the requirements of already differentiated cells, varying constantly but with a small amplitude. According to the Britten-Davidson model (1969), structural genes physically not linked to each other may nevertheless form functionally related "batteries." Activation of all members of a battery in concert is implemented by means of reiterated regulatory sequences adjoining each of them. However, the regulatory sequences scattered throughout the genome are highly reiterated and may easily activate some structural genes in excess of what an inducing stimulus was intended to do. Actually, any structural gene may be a constituent of various functional batteries. Thus, the latter can be expected to overlap extensively, thereby forming functional networks. Analogous networks presumably exist in another informational machine, the animal brain, in which every neuron may belong to various functional and dynamic networks. If true, such overlapping must also occur in the case of regulatory sequences. As a result, activation of a certain gene battery is not an indifferent event for others; some "related" batteries concomitantly affected may give rise to a generalization of the effect. As a result, the wider the set of structural genes needed to be activated, the more diverse and redundant the set of structural genes actually transcribed. Thus, the inevitable consequence of a very complex organization of the genome would be that it is impossible to induce only those genes that are needed to be transcribed at a given moment. We believe that precisely this "parasitic" activation of unneeded genes is the cause of redundant transcription, which has a pattern that is not cell specific and which entails the necessity of additional posttranscriptional selection.

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Selective RNA Transport as a Mechanism for Controlling Transcription

In our model the cell nucleus, apart from its function of synthesizing RNA, may be regarded, at the same time, as a siore of molecules produced but not yet utilized. As for the cytoplasm, it is endowed with the capacity to extract selectively a relatively small portion of mRNA species from the pool of potential nuclear messengers to be utilized in the protein-synthesizing machinery. The spectra of mRNAs extracted would vary both qualitatively and quantitatively, depending on the functional state of the cell. It seems likely that the nuclear envelope is a part of the mechanism controlling the transport of mRNA. It has been suggested that the nuclear pore complex alters its permeability to specific messengers under the control of cytoplasmic factors. Such a possibility may be true if only one mRNA species were to pass through a given nuclear pore. Indeed, it seems most unlikely that a nuclear pore complex would be able to select many thousands of specific mRNAs destined to be transported to the cytoplasm out of the many times larger amount of RNAs present in the nucleus. However, the informational burden on each pore complex would be drastically relieved if the flow of RNA molecules could somehow be ordered. Proceeding from this hypothesis, a prediction could be made that RNAs are transported within the nucleus not as free molecules and not stochastically but in association with intranuclear structures and in such a way that they inevitably pass only through certain pores, depending on the RNA. Some data (see below) on the association of posttranscriptional RNAs with chromatin and the association of the latter, in turn, with the nuclear envelope prompted us to advance the idea that chromatin and the nuclear envelope are a functional entity. By this we meant that chromatin fibrils possibly serve both as templates for RNA synthesis and as "tracks" for RNA molecules en route to those nuclear pores that the chromatin fibrils are linked (or adjoined) to. In this case, a single pore complex would deal with only one type of RNA molecule, i.e., a molecule synthesized on one gene. The pore complex could then operate in a simple "yes" or "no" mode of action. The idea of "one active operon - one nuclear pore" suggests that the control of gene expression at the level of the exit of transcripts to the cytoplasm may work in such a way that cytoplasmic transport-controlling proteins could specifically block certain pores either completely or partially, thus achieving qualitative and quantitative regulation of the cytoplasmic mRNA set. The functional activity of transport-controlling, presumably allosteric, proteins could be altered by effectors (metabolites, hormones, cyclic nucleotides, etc.). In the case of negative control, transport-modulating proteins are

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thought of as being able to block specific nuclear pores and, as a result, corresponding pre-mRNAs would remain within the nucleus. However, the block of specific pores could be relieved or abolished in the presence of certain effectors that are able to interact with transport-controlling proteins, thus restoring the transport of specific messengers. This model is consistent with the hypothesis of Tomkins et al. (1969) on the occurrence of cytoplasmic short-lived specific repressors of mRNA template function (see above), as well as with the data on the effect of cyclic nucleotides on RNA transport in a cell-free system (Schumm and Webb, 1978). Thus, the transcriptional activity of chromatin is believed to be under dual control. First, there is central regulation ensuring large-scale alterations of gene activity in response to hormones or embryonic inducers. Second, there is peripheral regulation issued from the cytoplasm and affecting the transcriptional activity of chromatin through selective extraction of specific mRNAs from the nucleus. Pre-mRNA molecules not in demand by the cytoplasm suppress their own synthesis by end-product inhibition. The possible mechanism of end-product inhibition would be either the direct inhibition of RNA polymerase activity by accumulating RNA transcripts, as was shown to be the case in the experiments in vitro of Sasaki et al. (1974), or the blockage of structure-oriented movement of potential mRNAs en route to the cytoplasm at the nuclear pores (see below). With the release of certain pre-mRNAs from the nucleus, chromatin fills the deficit caused by increased synthesis. Thus, the rate of pre-mRNA synthesis would depend to a certain extent on the rate of its utilization in the cytoplasm. We believe that such dual control of transcriptional activity would endow the protein-synthesizing machinery with a higher flexibility of reaction to varying cell requirements as compared to the powerful but apparently too coarse control at the transcriptional level alone. True, no conclusive evidence as to the validity of the main point of the model (the effect of cytoplasmic factors on the transcriptional activity through selective RNA transport) has been obtained so far, but a number of biochemical observations give support to it as well as to some predictions that ensued.

6.

REGULATION OF GENE EXPRESSION AT THE LEVEL OF NUCLEUS-TO-CYTOPLASM TRANSPORT OF RNA

With the evolutionary origin of the nuclear envelope in eukaryotic cells, transcription and translation became spatially uncoupled, resulting in the appearance of a novel stage in the multistep process of gene expression, namely nucleus-to-cytoplasm transport of RNA. The very significant question arises as

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to whether this stage is merely an automatic translocation of RNA molecules or whether it is involved in the control of the quantitative and qualitative structure of the cytoplasmic mRNA population. The data to be described below indicate that nucleus-to-cytoplasm transport of RNA is indeed regulated, at least in some cases. 6.1.

Rate of RNA Transport

In the early development of the sea urchin, the rate of RNA transfer is found to be relatively low and constitutes about 6% of the total RNA per hour (Aronson and Wilt, 1969), the labeled RNA being conserved within the nucleus for a long time with no sign of degradation. At later stages, after gastrulation, the rate of nucleus-to-cytoplasm transfer greatly increases (Kijima and Wilt, 1969). On the whole, during development the rate of RNA transfer changes drastically, 10- to IS-fold (Neyfakh, 1974; Kostomarova, 1977). The rates of transport of polyadenylated RNAs from the nucleus to the cytoplasm were shown to change significantly in different rat brain regions in accordance with the stages of maturation of the neural tissue (Hall and Lim, 1978). It is widely accepted that macromolecules pass to the cytoplasm through pores in the nuclear envelope (Franke, 1970; Zbarsky, 1972; Franke and Scheer, 1974a,b; Maul, 1977a,b). Attempts have been made to estimate the rate of RNA flow through nuclear pores (Franke, 1970). In exponentially growing HeLa cells, an estimated rate was about 1 molecule (molecular weight 1.15 X 106)/min per pore. A relatively close value (0.79 molecule/min per pore) was obtained for Xenopus laevis oocytes at the lampbrush stage. On the other hand, in exponentially growing Tetrahymena pyriformis cells, this parameter was found to be 20 to 25 times higher. It is possible that RNA transport is regulated to some extent by the number of pores in the nuclear envelope. Indeed, their frequency per unit area and the total amount per nucleus vary significantly and correlate with the functional state of the cell and with transcriptional activity (Franke and Scheer, 1970; Zbarsky, 1972; Maul, 1977a). The data mentioned above characterized the rate of RNA passage through the nuclear envelope. Another point is whether the delay known to exist between RNA synthesis and its appearance in the cytoplasm is regulated. It is interesting that in embryonic development the transport of RNA is often postponed significantly after the time of RNA synthesis. For example, in embryos of Misgurnus fossilis, RNAs synthesized at the late blastula stage begin to pass to the cytoplasm much later, at the stage of gastrula formation (Spirin et al., 1964; Neyfakh and Kostomarova, 1971; Neyfakh et al., 1973; Neyfakh, 1974). Similar results on Xenopus laevis embryos were obtained by

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Crippa and Gross (1969). Some authors (Neyfakh and Kostomarova, 1971; Neyfakh, 1974) proposed that the varying rate of RNA transport may control the successive changes of morphogenetic stages. A prolonged intranuclear preservation of RNAs is also characteristic of embryonic development. Mouse embryo RNAs labeled for 3 hr at the eightblastomere stage and the morula stage were found to be localized exclusively in the nuclear fraction and started entering the cytoplasm far later (Hillman and Tasca, 1969). Similar results using radioautography were obtained by Knowland and Graham (1972). The phenomenon of a prolonged RNA existence in the nucleus was also observed in avian erythroid cells in which globin genes were known to be transcribed. Under some conditions (virus transformation, certain stages of differentiation), globin pre-mRNAs synthesized are blocked within the nucleus for many hours (Chan, 1976; Therwath and Scherrer, 1978). In the search for the mechanism of adenovirus-induced blockage of host protein synthesis in HeLa cells, Beltz and Flint (1979) have found that synthesis of nuclear RNAs proceeds unchanged after infection. However, the RNAs were restricted to the nucleus, while viral RNAs entered the cytoplasm without hindrance. Ordered timing of the synthesis and transport to the cytoplasm of various RNA classes (4, 5,28, and 18 S rRNA, and poly(A)+ RNA) was found to occur in Xenopus laevis embryos according to the stage of development (Shiokawa et al., 1979). Thus, more or less prolonged storage of RNAs in the nucleus depending on their functional role seems to be characteristic of the eukaryotic cell.

6.2.

Comparison of Nuclear and Cytoplasmic RNAs in Various Cell Types

The paradox was mentioned earlier that the spectra of heterogeneous nuclear RNA species from tissues differing from one another phenotypically or functionally have much less qualitative distinctions than would be expected. The similarity of nuclear RNAs, on the one hand, and differences in sets of proteins synthesized, on the other hand, in functionally distinct cells, e.g., in resting and growing cells, prompted many investigators to search for some posttranscriptional events as the basis for the cell specificity of protein synthesis. In this connection, it is worth comparing nuclear and cytoplasmic RNAs from various tissues with regard to their kinetic complexity and cell specificity. As far back as 1959, a special, rapidly turning over fraction of RNA never leaving the nucleus was revealed by autoradiographic studies (Harris, 1959; Watts and Harris, 1959). Thorough investigations that followed substantiated the notion of "nucleus-restricted" RNAs (Attardi et al., 1966; Soeiro et al., 1966; Weinberg and Penman, 1968; Penman et al., 1968). There is ample evidence that heterogeneous nuclear RNAs of nearly all eukaryotic cells studied exceed

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cytoplasmic mRNAs by many times with respect to size and kinetic complexity (Getz et aI., 1975; Ryffel, 1976; Hames and Perry, 1977; Kleiman et al., 1977; Minty et al., 1977; Rolton et al., 1977; Sippel et al., 1977a,b; Grady et aI., 1978; Jacquet et al., 1978; Lasky et al., 1978; Tedeschi et al., 1978; Samal and Bekhor, 1979). Cytoplasmic poly(A)+ RNAs represent less than 1% of the nuclear poly(A)+ RNA complexity in rat liver (Sippel et aI., 1977b) and about 2% in Ehrlich ascites carcinoma (Samal and Bekhor, 1979) and avian erythroid cells (Lasky et al., 1978), as judged by hybridization studies employing cDNA. Proceeding from the kinetic approach, Brandhorst and McConkey (1974) found that only 2% of the RNA synthesized in the nucleus of mouse L cells was transported to the cytoplasm. It has been suggested that during nuclear processing, large primary transcripts undergo cleavage and excision of noninformational sequences, resulting in the formation of relatively small mature mRNAs (Scherrer and Marcaud, 1968; Georgiev, 1969; for review see also Lewin, 1975a,b; Daneholt, 1976; Molloy and Puckett, 1976; Perry, 1976). In reality, recent studies prove the existence of large nuclear precursors to cytoplasmic mRNAs (Hames and Perry, 1977; Goldberg et al., 1977; Sippel et al., 1977a; Nordstrom et aI., 1979; Harpold et al., 1979). Decisive evidence for such a scheme came with the recent discovery of split genes (see Section 3.7). Indeed, transcripts of noninformational intervening sequences eliminated during intranuclear processing may account for the higher diversity of nuclear RNA sequences as compared with cytoplasmic ones. The question arises, however, as to whether noninformational intron sequences are responsible for all the redundancy of nucleusrestricted RNAs. This question seems to be answered in the negative, since the fraction of nucleus-restricted RNAs includes, as will be shown below, not only noninformational but also template sequences. The notion of "potential nuclear messengers" resulted from the early work of Church and McCarthy (1967, 1970), Shearer and McCarthy (1967, 1970), Drews et al. (1968), and Flickinger and Roche (1972), in which some RNAs usually restricted to the nucleus were shown to be present in the cytoplasm when the functional state of the cell changed (during neoplastic transformation, regeneration of liver, or hormonal stimulation). Analyzing the results of numerous comparative studies of nuclear and cytoplasmic RNAs, one gains the impression that the nuclear RNAs display substantially less tissue and cell specificity than the cytoplasmic RNAs. This point may be exemplified by the observation that no differences in nuclear poly(A)+ RNAs of normal and transformed mouse 3T3 fibroblasts could be revealed in hybridization experiments. They differed neither in kinetic complexity nor in frequency distribution. As for polyribosomal mRNAs, they did differ, although not very much, in the frequency of certain RNA sequences (Rolton et al., 1977). Rat kidney, brain, and liver nuclear RNAs transcribed on reiterated

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DNA sequences were shown to be very similar, whereas mRNAs from microsomes of the same cells proved qualitatively distinct (Garrett et al., 1973a). Tedeschi et al. (1978) studied liver RNAs at the early stages (6-12 hr) of liver regeneration, when a considerable reprogramming of the genetic apparatus, preceding DNA replication, could be expected to occur. However, neither qualitative nor quantitative changes in nuclear RNAs were detected. RNA transport reacted to partial hepatectomy more promptly: within the first hours a substantial broadening of the mRNA set entering the cytoplasm was noted (Greene and Fausto, 1977). In numerous studies dealing with RNA transport from the nucleus to the cytoplasm, a decrease in its selectivity in tumor cells has been reported (Drews et al., 1968; Shearer and Smuckler, 1972; Smuckler and Koplitz, 1973; Shearer and Mayer, 1974; Shearer, 1974a,b; Lichtenstein et al., 1978; Austin et al., 1978; Shearer, 1979). In the latter paper, competitive hybridization of RNAs with repeated DNA revealed neither genome repression nor its derepression in dimethylnitrosamine-induced kidney tumor as compared with normal kidney. On the contrary, the set of RNAs transferred to the cytoplasm of tumor cells was far richer than that found in normal cells, the first changes in RNA transport appearing as early as within 2 days of carcinogen feeding. On the other hand, the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene, which is not carcinogenic for kidney, caused no changes in the transport of RNA from the nucleus to the cytoplasm in this tissue even after 9 days of feeding (Shearer, 1979). In rat hepatoma induced by N-2-acetylaminoftuorene, the amount of cytoplasmic mRNAs hybridized with unique DNA sequences was found to increase by 20% as compared with that of normal rat liver, whereas the extent of hybridization of nuclear RNAs did not change (Austin et al., 1978). The set of mRNA transported to the cytoplasm in frog embryos changes in response to the action of triiodothyronine (Flickinger and Roche, 1972). The same effect was observed in rat liver and uterus as a result of treatment with estradiol-17 {3 (Church and McCarthy, 1970) as well as in rat liver in the course of embryonic development or regeneration (Church and McCarthy, 1967, 1970; Greene and Fausto, 1974). The ability of some RNAs stored in the nucleus for a long time to enter the cytoplasm as soon as the conditions changed was revealed by Hammett and Katterman (1975) and Chan (1976). I t is widely held that the poly(A) segment attached to the 3' end of RNA is characteristic of molecules with template function. It is therefore of great interest to compare nuclear and cytoplasmic poly(A)+ RNA sets to answer the question as to how many of the pre-mRNAs synthesized in the nucleus are utilized in the cytoplasm. According to Ryffel (1976) and Sippel et al. (1977b), a significant portion of nuclear polyadenylated RNAs are not represented in the cytoplasm. Thus, the presence of the poly(A) segment in the RNA molecule is insufficient in itself to ensure RNA transport.

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The view that mRNA transport is a controlled process is supported also by the finding that mRNA sequences revealed in polyribosomes of sea urchin blastulae are also present in nuclear RNAs of various adult tissues but not in their polyribosomes (Wold et ai., 1978; see also Kleene and Humphreys, 1977). Analogous evidence for the posttranscriptional control of mRNA diversity in the frog embryo cytoplasm was presented by Shepherd and Flickinger (1979). Nuclear RNA and mRNA populations from the early neurula and larval stages of Rana pipiens were compared by hybridization with labeled singlecopy DNA. A great number of sequences were common to the two nuclear RNA populations, and mRNA complexity almost doubled during the developmental period studied (from 4.7% of the DNA complexity at the early neurula to 8.7% at the larval stage). Moreover, mRNA sequences detected on neurula polyribosomes, but absent from larval polyribosomes, were found in larval nuclei. Also, mRNAs detected on larval polyribosomes, but absent from neurula polyribosomes, were found in the neurula nuclei. These data seem to indicate that varying structural information is extracted from the same nuclear store according to the developmental stage. In addition to what was mentioned earlier on changes in the nucleus-tocytoplasm transport of RNA in tumor cells, some recent data indicate that this level of regulation may be involved also in other pathological phenomena. Morphogenesis of the sea urchin embryo, for instance, is restrained considerably as a result of sulfate deficiency, which does not affect the synthesis of RNA but blocks its transport to the cytoplasm (Gezelius, 1976). Moreover, Beltz and Flint (1979) have described a surprising phenomenon in which cellular mRNAs are restricted to the nucleus after adenovirus type 2 infection of HeLa cells. It was found that 16 hr following infection, almost all the newly formed mRNAs reaching the cytoplasm were of viral origin. However, normal patterns of host nuclear RNA synthesis, polyadenylation, and breakdown were preserved throughout the course of adenovirus type 2 infection. Thus, the gradual shift from the synthesis of cellular to viral proteins at the late phase of virus infection can result from the prevention of host mRNAs from entering the cytoplasm. The authors infer that the regulation of gene expression by retention within the nucleus of a specific set of mRNA molecules may have a more general significance rather than simply constituting a response to adenovirus infection, and the above situation possibly represents an extreme example of a regulatory mechanism that is characteristic of eukaryotic cells. Summing up this section, one can infer that while some of the nuclear RNAs are transported to the cytoplasm shortly after their synthesis and processing is completed ("real mRNAs"), others ("potential mRNAs") are preserved within the nucleus for some time. The synthesis of the latter RNAs is apparently not necessary to meet the immediate requirements of the cell, but may be a consequence of the structural organization of the cell genome as well as of a certain inflexibility in transcriptional regulation.

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It is possible that apart from the two RNA subpopulations mentioned, the nuclei contain still more RNA molecules that are functionally distinct and have no template functions at all. Some recent data are in favor of this suggestion (Berger and Cooper, 1978; Harpold et aI., 1979).

6.3. Transport of Some Specific Transcripts All the examples of regulated RNA transport discussed above concerned the flow of total nuclear RNAs and may be vulnerable to the criticism that they give no information on individual transcripts. Rapid progress in DNA cloning and preparation of DNA probes complementary to purified mRNAs makes it possible, however, to investigate the transport of specific mRNAs. Most papers concerned with this deal with globin mRNAs. Avian erythroblasts transformed in culture by erythroblastosis virus lose, in a few days the ability to synthesize hemoglobin and to be induced by dimethylsulfoxide to differentiate. Analysis of the phenomenon by using globin cDNA has shown that globin genes in these cells are transcribed, thus giving rise to globin pre-mRNAs; these, however, do not appear in the cytoplasm (Therwath and Scherrer, 1978). The authors believe that virus transformation affects posttranscriptionallevels of regulation, preventing the transport of host transcripts from the nucleus to the cytoplasm. Such a conclusion is consistent with the data of Beltz and Flint (1979) described above. In embryonic differentiating erythroid cells, synthesized globin RNAs are kept within the nucleus for as long as 24 hr and only thereafter appear in the cytoplasm (Chan, 1976). From the data of Nienhuis et al. (I977), impairment of the transport of ｾＭｧｬｯ｢ｩｮ＠ mRNA may be suggested as a cause of homozygous ｾＫ＠ thalassemia. The observations on the presence of globin mRNA sequences in the nuclei or nonerythroid tissues deserve special attention. In the early work of Kruh (1972), nuclear RNAs of rabbit liver, kidney, and intestine were shown to stimulate globin synthesis in a reticulocyte cell-free system. Cytoplasmic sequences RNAs of the same tissues display no such activity. (X- and ｾＭｧｬｯ｢ｩｮ＠ were found in the nuclear RNA of leukocytes, separated from erythroid cells, obtained from patients with chronic myeloleukemia (Gianni et al., 1978). It is interesting that globin mRNAs encoding the proteins that seem to appear only in fully differentiated cells are nevertheless revealed in Xenopus laevis oocytes (Perlman et al., 1977). Humphries et al. (1976) reported that globin mRN A sequences were present in low amounts in many non erythroid cells (such as adult brain and liver, lymphoma cells in culture, normal and transformed fibroblasts). The content of globin mRNA sequences in nuclear RNA did not change significantly from one tissue to another, while in the cytoplasm of erythroid cells globin RNA sequences accumulated in much higher quantities than in the cytoplasm of nonerythroid cells. The authors sug-

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gested that some type of posttranscriptional mechanism operates that allows the cytoplasmic accumulation of globin mRNAs only in erythroid tissues. All these observations demonstrate the transcription of "alien" genes, not expressed in a given tissue. It is worth mentioning, however, that the problem of the presence of globin RNA sequences in non erythroid cells has not been unequivocally resolved, since these sequences are found neither in pluripotent embryonic carcinoma cells (Jacquet et al.. 1978) nor in chicken embryo brain (Knochel and KohnertStavenhagen, 1977). At the same time, globin mRNAs were detected in various lymphoid cells (Storb et al.. 1977). Another example of posttranscriptional regulation is provided by the synthesis of histones which proceeds in general during the S phase of the cell cycle. Accordingly, histone mRNAs are found in the cytoplasm of only those cells that synthesize DNA (Wilkers et al.. 1978). Inhibition of DNA synthesis by cytosine arabinoside or hydroxyurea results in the destruction of preexisting histone mRNAs. The results of experiments with native and reconstructed chromatin suggested that histone synthesis is regulated at the transcriptional level depending on the phase of the cell cycle (Stein et al.. 1975). Melli et al. (1977) studied histone mRNA synthesis in HeLa cells at various cell-cycle stages as well as after the suppression of DNA synthesis by cytosine arabinoside. Histone mRNA sequences were detected by hybridization with cloned sea urchin histone DNA. Unexpectedly, they were found to be synthesized throughout the entire cell cycle and even after cytosine arabinoside-induced inhibition of DNA synthesis, while their content in the cytoplasm beyond the S phase, e.g., in the G 2 phase, was very small. However, it was augmented drastically (about 14-fold) during the S phase. In the control of histone mRNA entering the cytoplasm, a highly unstable protein factor seems to be involved, as blockage of protein synthesis by cycloheximide results in an increase in the histone mRNA content in the cytoplasm. The authors emphasize that, contrary to widely accepted views, there are very few sufficiently documented examples of purely transcriptional control, and in the case of histone mRNAs of HeLa cells the control seems to be posttranscriptional (and may even be extranuclear) rather than transcriptional. This observation may suggest that the regulation of histone gene expression occurs at the level of RNA transport from the nucleus to the cytoplasm, although selective and rapid degradation of histone mRNAs in the cytoplasm of cells beyond the S phase as an alternative mechanism cannot be ruled out at present. Analogous data obtained by Stein et al. (1977) show that the inhibition of DNA synthesis in HeLa cells by cytosine arabinoside or hydroxyurea abruptly decreases (by more than 99%) the content of histone mRNA sequences in polyribosomes but not in the nuclei and chromatin. Under these conditions, histone templates accumulate in the postpolyribosomal cytoplasmic fraction. The idea that the regulation of histone gene expression may occur at the

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posttranscriptional level was questioned in recent reports from Stein's group (Detke et al., 1978, 1979). In contrast to the results of Melli et al. (1977) and their own earlier evidence (Stein et al., 1977) indicating that the coupling of histone gene expression and DNA synthesis is not mediated at the transcriptionallevel, these authors maintain, first, that histone mRNA synthesis in isolated nuclei of HeLa S3 cells proceeds in the S phase, but not in the G 1 phase (Detke et al., 1978), and, second, that the data of Melli et al. (1977) can be explained by an artifact of the synchronization procedure, i.e., by the presence of S-phase cells in a G] population. This argument is not convincing evidence against posttranscriptional regulation per se, since drastic differences between nuclear and cytoplasmic histone mRNA content found in cells either beyond the DNA duplication phase or treated with cytosine arabinoside or hydroxyurea (Melli et al., 1977; Stein et al., 1977) cannot be the result of the lack of synchronization. It seems likely, therefore, that control at the transcriptional and posttranscriptional levels are not mutually exclusive but rather complement each other in the regulation of histone gene expression. An abnormal subcellular distribution of mRNAs coding for the K chain of immunoglobulins was revealed in mastocytoma and leukemic cells, the specific mRNA sequences being present in the nuclei but not in the cytoplasm (Storb et al., 1977). One more example comes from the study of hereditary hypothyroid goat goiter related to thyroglobulin deficiency. Nuclear RNA fractions of the goiter tissue (characterized by negligible amounts of thyroglobulin) contained almost normal quantities of 33 S thyroglobulin mRNA sequences, whereas the cytoplasm and microsomes respectively had no more than 6.6 and 2% of the mRNA sequences found in the control (Van Voorthuizen et al., 1978). Nuclei of CVl cells 8 hr after SV40 infection contained both early and late virus-specific RNA sequences. However, no late virus-specific transcripts were found on the polyribosomes, indicating the involvement of posttranscriptiona I control in the regulation of viral gene expression (Chumakov, 1979). 6.4.

"Luxury" Functions, "Housekeeping" Functions, and Modulation of mRNA Abundance in the Cytoplasm

Ephrussi (1972) distinguished "housekeeping" functions, which are inherent to all cells without exception (i.e.,metabolic processes necessary for the very existence and growth of all cells), and differentiated("luxury")functions, which ensure the survival of the multicellular organism but not of the cell. The proteins implementing luxury functions are synthesized when the cell differentiates in a certain direction, and they are therefore specific for a given cell type. The synthesis of proteins involved in luxury functions is likely controlled by a mechanism different from that involved in housekeeping functions. Such

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an unexpected conclusion was drawn from studies on somatic hybrids. The results of numerous experiments reviewed by Ephrussi (1972) indicate that after fusion of phenotypically distinct cells, luxury functions in the resulting hybrids are, as a rule, turned off, while housekeeping functions are preserved. Bromodeoxyuridine (BUdR) is also known to suppress luxury functions in various differentiated cells, not affecting cell viability significantly (Silagi and Bruce, 1970; Rutter et aI., 1973; Githens et al., 1976; Colbert and Coleman, 1977). Some of the tumor cell lines after BUdR treatment continue to proliferate but lose their tumorigenicity. However, this capacity is restored after the release of BUdR from the cellular DNA (Price, 1976). The similar suppression of malignancy and differentiation both by BUdR treatment and by cell fusion suggests that the mechanisms underlying differentiation and malignancy may have something in common (Silagi and Bruce, 1970) and are distinct from the mechanisms controlling housekeeping functions. The idea of cellular housekeeping functions was revived on biochemical grounds by Britten and Davidson's group (Galau et al., 1976; Davidson and Britten, 1979). Evaluation of the structural genes expressed at various stages of sea urchin development has shown that the tissues studied contained distinct but partially overlapping RNA sequences, the mRNA set common to all of them comprising about 1500 species (Galau et al., 1976). These molecules are likely to represent housekeeping gene transcripts. The extent of overlapping of cytoplasmic mRNA sets appeared to be extremely high for various cell types that were sometimes phenotypically very distant. For example, 85% homology was found between chick liver and oviduct mRNAs (Axel et al., 1976); 80% between human liver and leukemic lymphocytes (Ostrow et aI., 1979); 90% between mouse embryo ,fibroblasts,and Friend erythroleukemia cells (Minty et al., 1978); and at least 55% between mouse embryo, brain, and liver (Young et al., 1976). Similar results were obtained for various eukaryotic cells (Getz et al., 1976; Affara et al., 1977; Wilkers et al., 1979). These findings have led many investigators to the conclusion that the predominant part of the genetic information expressed as polyribosomal mRNAs is used for the fulfillment of functions common to phenotypically distinct cells, i.e., housekeeping functions (Getz et al., 1976; Axel et al., 1976; Ostrow et al., 1979). As for the mRNAs involved in the realization of luxury functions, they are likely to comprise qualitatively a small proportion of the total mRNAs. Another facet of the problem is the quantitative aspect of the mRNA population, i.e., the intracellular amounts (the number of copies) of the different individual mRNAs. From the pioneering work of Bishop et al. (1974), Birnie et al. (1974), and Levy and McCarthy (1975), it is known that there are several, usually three to four, abundance classes in cytoplasmic mRNA populations from various eukaryotic cells. Analysis of the hybridization kinet-

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ics, for example, of HeLa cell cytoplasmic poly(A)+ RNAs with their complementary DNAs, prepared by reverse transcriptase, has shown that the approximately 35,000 different mRNAs present in HeLa cell cytoplasm can be grouped in three distinct abundance classes: highly repetitive (with 15 individual mRNA sequences each present in 8000 copies per cell); moderately repetitive (330 mRNA sequences, 440 copies per cell); and rarely repetitive (36,000 mRNA sequences present in only 8 copies per cell) (Bishop et al., 1974). Numerous studies have shown that a frequency distribution of cytoplasmic mRNAs that is similar in principle occurs in many other eukaryotic cells; i.e., Drosophila cells (Levy and McCarthy, 1975); Friend erythroleukemia cells (Birnie et al., 1974; Affara et al., 1977; Mauron and Spohr, 1978; Minty et al., 1978); mouse embryo, brain, and liver (Young et al., 1976); pluripotent embryonic carcinoma cells (Affara et al., 1977; Jacquet et al., 1978); mouse myoblasts (Affara et al., 1977); Ehrlich ascites carcinoma cells (Samal and Bekhor, 1979); embryonic AKR cells (Siegal et al., 1979); differentiated and undifferentiated neuroblastoma cells (Felsani et aI., 1978); rat normal (Sippel et al., 1977b; Savage et al., 1978), regenerating (Wilkers et al., 1979), and hypothyroid liver (Towle et al., 1979); rat prostate (Parker and Mainwaring, 1977) and pancreas (Harding et al., 1977); human liver and leukemic cells (Ostrow et al., 1979); chick oviduct (Cox, 1977), liver (Axel et al., 1976), and myoblasts (Paterson and Bishop, 1977); rooster liver (Grouse and Grouse, 1979); bovine hypophysis (Meuli et al., 1979); and avian erythroid cells (Lasky et aI., 1978). The question arises as to whether there is a correlation between the involvement of a given mRNA in the realization of luxury or housekeeping functions and its belonging to a definite abundance class. This question is far from fully answered, but some available data favor the suggestion that at least some mRNAs associated with luxury functions belong to the class of highly repetitive mRNA sequences (which have many thousands of copies per cell). This seems to be true of globin mRNA in erythroid cells and ovalbumin mRNA in the oviduct of the laying hen (for review see Davidson and Britten, 1979); myosin, actin, and tropomyosin mRNAs in chicken myoblasts (Paterson and Bishop, 1977); casein mRNA in the mammary gland of the pregnant rabbit (Shuster et al., 1976); and pancreatic secretory protein mRNAs in the rat pancreas (Harding et al., 1977). The frequency distribution of different mRNA species in the population is a rather dynamic parameter, apparently linked to the functional state of the cell. For example, a transient hormone-induced appearance of the highest frequency class comprised of specific mRNAs was shown to occur in rat prostate (Parker and Mainwaring, 1977), the mammary gland of the pregnant rabbit (Shuster et al., 1976), and chicken oviduct (Cox, 1977). Analogous shifts in the frequency distribution of specific mRNAs apparently associated with the

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fulfillment of luxury functions also take place upon cellular differentiation of chicken myoblasts (Paterson and Bishop, 1977) and dimethylsulfoxide-induced Friend erythroleukemia cells (Minty et al., 1978). Treatment of chicken myoblasts with BUdR, an inhibitor of cellular differentiation, prevents the appearance of the high-frequency class of mRNAs coding for muscle proteins (Paterson and Bishop, 1977). A number of comparative reports demonstrate a high qualitative homology between mRNAs from various tissues studied, concomitant with a clearcut tissue specificity of the highest frequency class, i.e., mRNAs present at high frequency in one tissue were also found in the other, though at sharply reduced concentrations (Ryffel and McCarthy, 1975; Axel et al., 1976; Young et aI., 1976; Hastie and Bishop, 1976). The data supporting the great significance of quantitative regulation of the mRNA population may suggest that the cellular phenotype is determined to a greater extent by the relative intracellular concentrations of certain mRNA species rather than by their mere presence or absence. In this connection, it is very important to establish the mechanisms determining the quantitative composition of the cytoplasmic mRNA population. Conceivably, the mechanisms would be, first, differential RNA synthesis, then differential nucleus-to-cytoplasm RNA transport, and, finally, differential mRNA stability. As to control at the transcriptional level, it seems to be most important for the formation of the highest frequency mRNA class involved in luxury functions. A thorough analysis of the data available has led Davidson and Britten (1979) to the conclusion that the genes coding for superprevalent mRNAs are under strict transcriptional control (see Section 3.6). The observations of an increased globin mRNA synthesis in dimethylsulfoxide-induced Friend erythroleukemia cells (Orkin and Swerdlow, 1977) as well as of hormone-stimulated synthesis of specific mRNAs (see Section 2.l.4) corroborate this idea. It is clear, however, that certain posttranscriptional events must also contribute to the formation of mRNA abundance classes. As a matter of fact, the frequency patterns of RNA sequences in the nucleus and the cytoplasm appeared drastically different. Most nuclear RNA species are present in amounts of one to several copies per nucleus and even the highest frequency class (relatively small) includes molecules reiterated only 10 to 80 times per nucleus, i.e., present at concentrations several orders of magnitude lower than those of the highest frequency class in the cytoplasm (Herman et al., 1976; Sippel et al., 1977b; Mauron and Spohr, 1978; Lasky et al., 1978; Jacquet et al., 1978; Siegal et al., 1979; Mansson and Harris, 1979; Samal and Bekhor, 1979). Thus, the nuclear RNA sequences appear to be much more diversified qualitatively and many times narrower in their frequency distribution than the cytoplasmic ones. Most authors agree that such a drastic difference in both the

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qualitative and the quantitative composition of nuclear and cytoplasmic RNA populations implies the involvement of posttranscriptional selection. One of the posttranscriptional mechanisms proposed is that various cytoplasmic mRNAs differ in their stability. This mechanism proved to operate in Friend erythroleukemia and spleen cultured cells induced to differentiate. The accumulation of nothing but globin mRNA (about 98% of the total mRNA population), which takes place at the terminal differentiation stage, can only be accounted for by a selective destabilization of all the nonglobin mRNAs (Aviv et al., 1976; Bastos et aI., 1977; Bastos and Aviv, 1977). Moreover, a positive correlation between mRNA stability and its intracellular concentration has been shown to exist in mouse L cells (Meyuhas and Perry, 1979). The data available indicate that apart from RNA synthesis and stabilization, RNA transport also significantly contributes to the formation of the quantitative composition of the mRNA population. As a matter of fact, the metabolic heterogeneity of cytoplasmic mRNAs does not seem to be, in general, high enough to ensure great differences in the relative intracellular concentrations of various mRNA species. In most eukaryotic cells studied, only two metabolic mRNA classes (with half-lives of 1.5-7 and 18-24 hr) are usually observed (Singer and Penman, 1973; Spradling et al., 1975; Puckett et al., 1975; Perry, 1976; Perry et al., 1976). The half-life of the most stable mRNAs is only 2.5 to 16 times more prolonged than that of the rapidly turning over fraction, these differences being significantly lower than those needed for the mRNA intracellular frequency distribution usually observed. Besides, the positive correlation between mRNA stability and its abundance shown for L cells (Meyuhas and Perry, 1979) and Drosophila cells has not been observed in HeLa cells (Lenk et aI., 1978). On the other hand, the rate of RNA transport to the cytoplasm can vary by extremely wide limits. Thus, newly formed mRNA may appear on polyribosomes within 15-20 min. On the contrary, there can be complete arrest of RNAs within the nucleus upon, for example, virus infection (Beltz and Flint, 1979), i.e., the rate of transport may be zero. A number of other examples of prolonged conservation of template RNAs in the nucleus are reviewed above (see Section 6.2 and 6.3). Direct evidence for differential rates of RNA transport was obtained for avian erythroid cells by Tobin et al. (1978), who estimated that globin mRNAs appear in the cytoplasm at a rate of approximately 1 molecule per minute, while other mRNAs do so at a rate 50 times lower. Thus, certain quantitative features of the cytoplasmic mRNA population seem to be the result of the activity of both transcriptional and posttranscriptional mechanisms operating in concert. However, it seems possible that these mechanisms contribute differently to the formation of various abundance classes. The highest frequency class comprising tissue-specific mRNAs, for example, apparently arises as a result of enhanced transcription on correspond-

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ing genes, while lower frequency classes are regulated posttranscriptionally (see Davidson and Britten, 1979). It is interesting to note that Ephrussi (1972), proceeding from quite different approaches (see above), came to the conclusion that the luxury and housekeeping functions are regulated by different mechanisms. This is consistent with the idea that the luxury functions performed by the highest frequency mRNAs are under transcriptional control, while the housekeeping functions carried out by lower frequency mRNAs would be controlled posttranscriptionally, their genes being constantly switched on.

6.5. Transport-Controlling Factors of the Cytosol In an attempt to clarify the nature of factors controlling RNA transport, several cell-free systems have been elaborated permitting the study of RNA release from isolated nuclei to the incubation medium as a model of the process proceeding in vivo. Incubation under appropriate conditions of isolated nuclei, containing RNAs labeled in vivo, results in the release of ribosomal and nonribosomal RNAs (Samarina and Zbarsky, 1964; Ishikawa et al., 1969, 1972; Schumm and Webb, 1972; Schumm et al., 1973a,b; Chatterjee and Weissbach, 1973; Raskas and Rho, 1973; Schumm and Webb, 1974a). The RNAs released from the nuclei are in the form of ribonucleoprotein particles and are able to associate with postmitochondrial polyribosomes (Ishikawa et al., 1970, 1972). The release of RNAs is blocked by cordicepin, an inhibitor of RNA polyadenylation and transport in vivo (Schumm and Webb, 197 4b). The release of RNAs from the nuclei is ATP dependent (Ishikawa et al., 1969, 1970, 1972; Raskas and Rho, 1973; Schumm and Webb, 1975a; Toyoji et al., 1977), except for some tumor cells (see below). RNA transport in vitro seems to be energy dependent and connected with various enzymatic events. A nonspecific nucleoside triphosphatase was revealed in the vicinity of nuclear pores, its activity correlating with the rate of RNA transport. Inhibitors of the enzyme suppress the release of RNA (Agutter et al., 1976). Clawson and coworkers (Clawson et al., 1978; Clawson and Smuckler, 1978) have demonstrated a linear dependence of RNA transport on the energy of exogenous nucleoside triphosphates and estimated an activation energy of RNA translocation of 12.5-13 kcaljmol. They came to the conclusion that RNA transport is catalyzed by specific enzymes and is not just a simple diffusion. All the data described along with the results of comparative studies of RNAs released from nuclei in vivo and in vitro by competitive hybridization indicate the adequacy of the cell-free systems elaborated for the exploration of nucleus-to-cytoplasm transport of RNA in vivo (Racevskis and Webb, 1974; Schumm and Webb, 1974a,b). The adequacy of the cell-free systems is supported by the well-known in

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vivo peculiarities of RNA transport in various tumor cells, which can also be manifested in tumor-derived cell-free systems. Comparison of nuclear and cytoplasmic RNAs in many tumors has demonstrated a reduction in the high selectivity of RNA transport from the nucleus to the cytoplasm, which is characteristic of normal cells. The impairment of this selectivity is observed at the early stages of carcinogenesis and has proved to be a stable feature of at least some tumor cells (Drews et al., 1968; Shearer and Smuckler, 1971, 1972; Shearer and Mayer, 1974; Shearer, 1977; Austin et aI., 1978). An analogous situation was observed in a cell-free system. First, some chemical carcinogens, but not their noncarcinogenic analogs, caused early, stable, and significant changes in RNA transport in a cell-free system derived from the target tissue. There was a sharply increased ability of the cytosol to extract RNAs from isolated nuclei (Schumm et al., 1977). This ability is preserved by the cytosol from transplantable tumor cells (Schumm et al., 1973b). Second, some tumor nuclei, as opposed to normal ones, partially or completely fail to show ATP dependence of in vitro RNA transport in accordance with the degree of malignancy of the corresponding tumor cells (Stuart et al., 1975; Schumm and Webb, 1975b). Thus, the cell-free system proved to be suitable as model system for the study of transport of RNA from the nucleus to the cytoplasm in vivo. It provides the opportunity to evaluate the role of various factors in this process. The data of Webb and Schumm's group attest to the fact that certain cytosolic factors regulate both the quantitative release of RNAs from isolated nuclei and the qualitative composition of the RNAs released (Schumm and Webb, 1972; Schumm et al., 1973a,b; Schumm and Webb, 1974a,b; Yannarell et al., 1976; Schumm et al., 1977). These authors have demonstrated, for example, that qualitatively different sets of nonribosomal RNAs are extracted from isolated rat liver nuclei incubated in the cytosol of either normal rat liver or rat hepatoma (Schumm et al., 1973b). The phenomenon of active extraction by the cytoplasm of specific mRNAs from the nucleus is obviously manifested by these observations, in line with the model of cytoplasm-governed gene regulation (see Section 5). Cytosolic factors controlling the release of nuclear RNAs are a heterogeneous population of negatively charged proteins (in rat liver their molecular weights range from 10,000 to 30,000) capable of affecting the release of RNA from nuclei of rat liver (Schumm et al., 1973a; Yannarell et al., 1976) and of mouse brain (Weck and Johnson, 1978) both by positive and by negative regulation. The turnover rate of these proteins seems to be rapid, since RNA transport in a cell-free system was significantly enhanced a short time after the administration to the animals of the protein synthesis inhibitor cycloheximide or phenobarbitone (Ch'ih et al., 1979; Hazan and McCauley, 1976).

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Fractionation of mouse brain cytosol revealed a very heterogeneous spectrum of factors that function at both the transcriptional and posttranscriptional levels. In particular, some of these factors stimulate RNA transport in a cellfree system, while others have the reverse effect (Weck and Johnson, 1978). Apparently, it is the balance between transport-promoting and transport-inhibiting factors that determines the actual rate of RNA translocation. There are some indications that cyclic nucleotides, such as cAMP and cGMP, are also involved in this process. Addition of cAMP or cGMP at physiological concentrations to a cell-free system derived from rat liver, Novikoff hepatoma, and Morris 5123 D hepatoma stimulated the release of poly(A)+ RNA but not of rRNA. The extent of stimulation is dependent on the in vivo level of endogenous cyclic nucleotides in the tissue studied (Schumm and Webb, 1978). It is worth emphasizing that the cytosolic factors affect not only RNA transport, but also RNA synthesis in isolated nuclei, both quantitatively and qualitatively (McNamara et al., 1975; Weck and Johnson, 1976; Morry and Gefter, 1977; Bastian, 1980). It remains to be seen, however, whether a causal relationship between the two processes-stimulation of RNA transport and stimulation of RNA synthesis-does exist, as we proposed earlier. In other words, does the augmented release of RNA transcripts from the nucleus serve as an inducer of accelerated RNA synthesis?

7. METABOLIC HETEROGENEITY OF NUCLEAR RNA The result of regulated RNA transport must be either the accumulation within the nudeus of those RNA molecules that have been overproduced but not called for by the cytoplasm, or their rapid intranuclear breakdown. The solution to this problem, which is very important for the choice of alternative hypotheses, may be achieved by the study of turnover rates of nuclear RNAs. If the preservation of nuclear RNAs does in fact occur, then these molecules would constitute a pool of stable nuclear RNAs in addition to the rapidly turning over RNA fraction most intensively studied so far. It should be stressed here that the well-known phenomenon of a rapid intranuclear breakdown of the most rapidly labeled RNAs in all probability has nothing to do with the problem in question, since it is a manifestation of the processing of large mRNA precursors coupled with the elimination of noncoding RNA sequences, introns included (Sherrer and Marcaud, 1968; Georgiev, 1969; Lewin, 1975a,b; Perry, 1976; Chambon, 1978), rather than the degradation of potential mRNAs. As to the latter, their intranuclear fate is not well understood, probably because of the short duration of RNA labeling experiments, which is usually suitable for detection of short-lived, but not stable, molecules. Nevertheless, there are some data, both electron microscopic

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and biochemical, demonstrating that nuclear RNAs contain not only molecules with a high turnover rate, but also a large (by mass) sub population of metabolically stable molecules. First, several specific ribonucleoprotein (RNP) structures have been revealed in nuclei of eukaryotic cells by electron microscopy using selective staining techniques (Monneron and Bernhard, 1969). Perichromatin fibrils, perichromatin granules, and interchromatin granules were shown to be constant nuclear components. Evidence for the ribonucleoprotein nature of these structures was obtained by demonstrating their breakdown after treatment with RNases and proteases. The RNPs of the perichromatin fibrils and granules incorporate radioactive label actively, are increased in number as a result of stimulation of RNA synthesis, and seem to be structurally and functionally interrelated. By these criteria, the rapidly labeled nuclear structures may be identified with nuclear RNP particles thoroughly studied by biochemical methods (Samarina et al., 1968; Parsons and McCarty, 1968; Faiferman et al., 1970; Pederson, 1974a,b). As for the third type of structure (interchromatin granules), they are more resistant to RNases than the others. This is presumably due to distinctive structural features; they also incorporate radioactive label very poorly (Monneron and Bernhard, 1969; Fakan and Bernhard, 1971; Chentsov and Polyakov, 1974; Bouteille et al., 1974; Bachellerie et al., 1975; Puvion and Bernhard, 1975; Beljaeva, 1977). These authors maintain that interchromatin granules, which are common to all the nuclei studied, represent stable structures not migrating to the cytoplasm. Second, some biochemical observations accumulating of late indicate that the heterogeneous nuclear RNA population includes stable molecules (with half-lives of several hours or more) that are preserved in the nucleus (Lichtenstein et al., 1974; Tarantul et al., 1974; Spohr et al., 1974; Lichtenstein et al., 1976a; Gasaryan et al., 1977 a, b; Lewis and Penman, 1977; Gasaryan et al., 1979a,b). In our earlier experiments we studied the turnover rates of nuclear high-molecular-weight DNA-like RNAs of rat liver purified by ion-exchange chromatography on methylated albumin-kieselguhr columns. Unexpectedly, it was found that actinomycin D-induced blockage of RNA synthesis after a short (40 min) labeling of nuclear RNAs resulted in the almost complete removal of radioactive label from the DNA-like RNA fraction without any apparent reduction in its total amount as measured by optical density (Lichtenstein et aI., 1974). These data were interpreted to mean that in rat liver nuclei there was a large subpopulation of RNA molecules that remained practically unlabeled during the entire experiment. This conclusion was confirmed further by employing a kinetic approach. The specific radioactivities of free UTP and of UMP residues in rat liver nuclear DNA-like RNA were compared after a single injection of [14C]orotic

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acid into the animals (Lichtenstein et al., 1976a; Lichtenstein and Shapot, 1976). This experiment was expected to answer the question as to whether there are in fact long-lived molecules among the nuclear RNAs, and if so, what their proportion of the nuclear RNA population is. If there were no long-lived molecules at all or their proportion were negligible, then the specific radioactivity of the VTP pool would become identical to that of the VMP residues of nuclear RNA shortly after reaching its maximum. Assuming that the half-life of nuclear rapidly labeled RNA is about 15-30 min, the leveling of specific radioactivities of the precursor (VTP) and product (VMP in RNA) would be expected to occur after 3-4 half-life periods (i.e., within 1-2 hr), when practically all (90-95%) of the "cold" VMP residues in RNA have been replaced by the radioactive ones. If observed, this leveling of specific radioactivities of VTP and VMP in RNA would attest to the absence of poorly labeled molecules in the nuclear RNA population; however, if such molecules were present, they would significantly reduce the overall specific radioactivity of VMP in RNA as a result of dilution of highly labeled VMP-RNA in the rapidly turning over fraction with poorly labeled VMPs of metabolically inert RNAs. The latter situation was found to be the case. The specific radioactivity of VTP exceeded that of VMP in RNA by a factor of 2.5-3.5 during all the experiments that lasted for 6-16 hr (the specific radioactivity in VTP reached a maximum 1.5-2.5 hr after [14C]orotic acid injection) (Lichtenstein et al., 1976a; Lichtenstein and Shapot, 1976). Assuming that intracellular VTP in rat liver is not compartmentalized, we estimated that the half-life of rapidly labeled nuclear RNAs was 30 min and that the stable nuclear RNAs made up 7580% of the total nuclear RNA. In further investigations we attempted to evaluate the half-life of stable rat liver nuclear RNAs by employing daily injections of ['4C]orotic acid over a period of 7-10 days. Prior to isotope injection the animals were subjected to partial hepatectomy in order to ensure that there would be exhaustive labeling of all RNAs (including the most stable ones) which must proceed rapidly in growing cells. The specific radioactivity of nuclear RNAs and of intracellular VTP were measured for a period of over 2 months. Vnder these nonconventional conditions, a fraction of nuclear RNA with a half-life of 5-7 days was revealed. Its stability was comparable to or even exceeded that of cytoplasmic rRNAs used as an internal control. Thus, special conditions were required to detect this extremely stable nuclear RNA fraction (Mojseev, Lichtenstein, and Shapot, in preparation). Stable nuclear RNA has also been demonstrated in avian erythroid cells (Tarantul et al., 1974). A significant portion of it is not transported to the cytoplasm, thus preserving its association with chromatin. This stable RNA was found to be rather homogeneous with a sedimentation coefficient of about 28 S, but by various criteria (nucleotide composition, melting curves, hybridization with reiterated and unique DNA sequences) it was shown to be different

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from rRNA (Gasaryan et al., 1977a,b, 1979a,b). In the nucleus this RNA is a constituent of RNP complexes firmly bound to chromatin and is characterized, like informosomes, by a buoyant density in CsCI gradients of 1.41 g/ cm 3 (Gas aryan et al., 1977b, 1979a). A homology observed between stable nuclear RNAs, on the one hand, and giant nuclear nonpolyadenylated RNAs as well as cytoplasmic polyadenylated RNAs, on the other hand, has led these authors to the conclusion that a portion of the nuclear nonpolyadenylated RNAs undergoes incomplete processing with the formation of 28 S RNA that is stored within the nucleus for a long time (up to 30 hr). Later on, cytoplasmic polyadenylated mRNAs are formed from this stable precursor (Gasaryan et al., 1979b). Analogous data indicating a significant metabolic heterogeneity of nuclear RNAs in cultured Drosophila cells were obtained by Lewis and Penman (1977). Third, some data indicate that at least some stable nuclear RNAs are of the messenger type. 1. A portion of the stable nuclear molecules was reported to by poly adenylated (Hammett and Katterman, 1975; Herman and Penman, 1977; Hendrickson and Johnson, 1978), this characteristic being typical of the mRNA class of molecules. For instance, the bulk of the mRNAs in germinating cotton seeds is polyadenylated and preserved within the nucleus for a long time (Hammett and Katterman, 1975). Hendrickson and Johnson (1978), studying HeLa and 3T6 cells, came to the conclusion that conventional pulse labeling cannot reveal the presence of stable nuclear RNAs. In their experiments labeled RNAs were chased under different conditions. Normally, when no metabolic inhibitors were used, stable nuclear RNAs with half-lives of 8-12 hr were revealed, the latter being comparable with the generation time of these cells. A significant portion of the nuclear polyadenylated RNAs, probably more than half, belongs to the stable type. The authors discussed several possibilities: (a) these RNAs may be stored in the nucleus to be exported to the cytoplasm later on; (b) they may be mRNA precursors undergoing very slow processing; and (c) they may have no bearing on cytoplasmic mRNAs at all, in spite of their being polyadenylated. 2. More direct evidence for the template function of at least some stable nuclear RNAs comes from the work of Chan (1976). Studies on the kinetics of globin mRNA synthesis and transport in cultured erythroid cells of chick embryos showed that within the first 24 hr in culture the cells synthesize globin mRNAs that could be detected in nuclei but not in cytoplasm. Only on the second day did globin mRNAs start to appear in the cytoplasm, so that by 36 hr half of them had left the nucleus. In HeLa cells and avian erythroblasts, three subpopulations of nuclear RNAs of various half-lives were observed, the most stable having a half-life of about 15 hr. In erythroid cells the most stable fraction of nuclear RNAs con-

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tained globin mRNA sequences as well as many other nonglobin pre-mRNAs. Spohr et af. (1974) infer that intermediate products of pre-mRNA processing may be preserved within the nucleus over a long period of time. It is appropriate to recall here the data of Shepherd and Flickinger (1979) and Wold et al. (1978) already cited. They attest to the fact that in the course of embryonic development, diverse mRNA sets appear in polyribosomes in succession, whereas in the nuclei no tangible changes could be observed, nuclear RNAs comprising both pre-mRNAs currently being expressed in polyribosomes and pre-mRNAs that are due to be expressed in the future.

8.

STRUCTURAL ORGANIZATION OF INTRANUCLEAR RNA TRANSPORT

The regulation of gene expression at the level of RNA transport (the evidence for which is given in the preceding sections) envisages the necessity of certain mechanisms of RNA selection. It is conceivable that the nuclear envelope and nuclear pores greatly contribute to this process (Franke and Scheer, 1974a,b; Chentsov and Polyakov, 1974; Lichtenstein and Shapot, 1976; Maul, 1977a; Zbarsky, 1978). RNA transport through nuclear pores may, in principle, proceed in two ways. 1. Nuclear pores may not be specific for the nucleotide sequences of RNAs passing through them, but may recognize certain features ("passwords") that permit the pores to discriminate between molecules destined to leave the nucleus and those remaining it it. Certain modified bases in RNA, specific constituents of the protein moiety of RNP particles, or special conformations of RNA termini may serve as passwords. In that event, regulation of RNA transport would occur during RNA processing and/or RNP assembly. This model is consistent with Spirin's (1978) hypothesis "omnia mea mecum porto,"* according to which RNP particles carrying mRNAs contain at the time of their appearance all the components required for subsequent maturation and functioning. In this case, the nuclear pore apparatus would fulfil merely the executive function of an "admission post," whereas the regulation itself would be implemented at higher levels. This model does not require ordered transport of RNA from the nucleus to the cytoplasm; in other words, any of the RNP particles moving freely in the nuclear sap would be able to pass at random through any of the nuclear pores in the nuclear envelope. 2. An alternative mechanism of RNA transport postulating a highly ordered process was suggested in a model for cytoplasm-governed gene regu-

*"All that I have, I carry with me."

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lation (Lichtenstein and Shapot, 1976; see also Section 5). In this model, the regulation of RNA transport is ascribed directly to the nuclear pore apparatus, which by interacting with cytoplasmic transport-controlling proteins (see Section 6.5) either permits the passage of a given RNA species to the cytoplasm or prohibits it. The principal difficulty that arises here is the visualization of a way by which specific selection of the required RNAs is achieved. If one admits that RNA particles are able to pass through any of the nuclear pores at random then the specific interaction between RNA and the pore apparatus, which is obligatory for RNA selection, would seem to be impossible. The situation would be simplified if RNA transport were not just simple diffusion in the liquid phase of the nucleus but instead a strictly ordered process. The idea of "one active gene, one nuclear pore" advanced in our hypothesis allows us to consider intranuclear transport of the transcripts of one gene as an ordered movement of molecules in a queue only through their individual exit, which is a particular pore (the "conveyor model"). A role for chromatin fibrils as tracks for RNA molecules en route to the nuclear pores was discussed earlier (Section 5.2). Nonrandom distribution of nuclear pores in the envelope, on the one hand, and an ordered orientation and association of chromatin fibrils with the nuclear envelope, on the other hand (Comings, 1968; Comings and Okada, 1970; Engelhardt and Pusa, 1972; Zbarsky, 1972; Franke and Scheer, 1974a,b; Chentsov and Polyakov, 1974; Dye and Toliver, 1975; Maul, 1977a,b; Chentsov, 1978; Zbarsky, 1978), are consistent with this assumption. A partial or complete block of certain pores by specific cytoplasmic proteins (see Section 6.5) would modulate the delivery of mRNAs to the cytoplasm both qualitatively and quantitatively. The expression of a gene, in this case, would proceed provided two obligatory conditions were met: first, gene activation, and second, free exit to the cytoplasm for gene products. Although the question of whether intranuclear RNA movement is indeed oriented toward its "own" nuclear pore cannot be answered in the affirmative so far, the model enables one to consider much hitherto unexplained experimental data from a new viewpoint. It is generally held that primary transcripts, after their synthesis is completed, are immediately released into the nuclear sap and remain there as free RNP particles before entering the cytoplasm. In accordance with this view, many authors who have revealed the presence of a high-molecular-weight RNA in purified chromatin preparations have regarded it only as a nascent RNA (see, e.g., Bhorjee and Pederson, 1973; Pederson, 1974a,b; Monahan and Hall, 1975). This conclusion was apparently supported by the occurrence in the nuclear sap of RNP particles containing heterogeneous nuclear RNA (Samarina et al., 1968; Parsons and McCarthy, 1968; Faiferman et al., 1970; Lukanidin et al., 1972; Pederson, 1974a,b; Stevenin et al., 1977). However, a growing body of evidence indicates that during the posttran-

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scriptional period pre-mRNAs are not present in the liquid nuclear phase but are associated with some intranuclear structures. Gasaryan et al. (1971) have demonstrated that a significant portion of rapidly labeled nuclear RNAs is firmly bound to chromatin even posttranscriptionally. In nuclear RNP particles, small quantities of DNA were revealed (Naora, 1969). The occurrence of RNP-chromatin complexes was uncovered by both electron microscopic and biochemical methods (Price et al.. 1974; Tata and Baker, 1975; Bachellerie et al.. 1975; Puvion and Bernhard, 1975). The relationship between nuclear RNP particles and chromatin in Zajdela ascites hepatoma cells was examined in detail in this laboratory (Lichtenstein et al.. 1976b; Zaboykin et al.. 1978). To study interactions between nuclear components that had been doubly labeled with [14C]uridine and pH]thymidine, three independent methods were used, namely sucrose density centrifugation, CsC! density centrifugation and nucleoprotein-Celite chromatography. [The latter is a new technique elaborated in this laboratory that permits the fractionation of nucleic acids according to the tightness of their bonds with proteins in native nucleoproteins (Lichtenstein et al .. 1975, 1976c, 1979).] We established the fact that in Zajdela hepatoma cells, nearly all of the nuclear RNP particles are connected with chromatin, some of their RNA constituents being polyadenylated. The latter observation proves the posttranscriptional association of RNA with chromatin, since addition of a poly(A) tract to the 3' end of RNA takes place after completion of RNA transcription. An assumption was made that newly formed nuclear RNAs within the RNP complexes may undergo successive processing steps while preserving their association with chromatin. Virtually identical results were obtained simultaneously by Kimmel et al. (1976), who found that in mouse myeloma cells the bulk of the rapidly labeled nuclear RNAs, including polyadenylated ones, is firmly associated with chromatin. Similar observations on artichoke nuclei were reported by Chapman and Ingle (1976). In line with the above data is the well-known fact that the isolation of nuclear RNAs, including polyadenylated ones (see Hemminki, 1974), is associated, as a rule, with considerable difficulties and requires rather drastic treatments (e.g., phenol, SDS, heating up to 70-80 C). Firm association of heterogenious nuclear RNAs with some intranuclear structures becomes obvious from this observation since the protein components of the RNP particle per se can be separated from RNA relatively easily (Lukanidin et al .. 1973). A constant and firm association of nuclear RNP particles with chromatin accounts for the necessity to fragment chromatin, mechanically or enzymatically, prior to extraction of RNPs from nuclei. Extraction of RNPs from nuclei of tissues with low nuclease activity proceeds with difficulty but is facilitated by the preincubation of nuclei at 37 C, i.e., under conditions favoring the action of 0

0

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endogenous nucleases (Lukanidin et al., 1973; Pederson, 1974a,b). It seems likely that it is precisely these endogenous nucleases that are responsible for the presence in the nuclear sap of "free" RNP particles arising as a result of the degradation of structures that had originally been associated with chromatin. Indeed, the data presented by Tata and Baker (1975) make it possible to regard the so-called "free nucleoplasmic particles" as products of the hydrolysis of high-molecular-weight RNAs previously bound to chromatin. Feldherr (1980), studying transport of rRNA in oocytes, punctured the nuclear envelope in many sites by a fine needle. The damage to the nuclear envelope did not affect either synthesis or transport of labeled rRNA from the nucleus. The author suggested that rRNAs are bound within the nucleus until their efflux to the cytoplasm and presumably are transported directly to the pores along some structural elements. While the notion of "structure-oriented" rather than "diffusible," intranuclear transport of RNA is shared at present by many investigators, there is not universal consent as to the nature of the intranuclear component to which RNA is bound posttranscriptionally. The data on the association of nuclear RNAs with chromatin have been mentioned. At the same time, there are indications that all nuclear RNAs are constituents of an intranuclear ribonucleoprotein network that links chromatin with the nuclear envelope and, eventually, with the cytoplasm (Faiferman and Pogo, 1975; T. E. Miller et al., 1978). Association of RNAs with the nuclear protein matrix has also been reported (T. E. Miller et al., 1978; Herman et al., 1978; Herlan et al., 1979; Berezney et al., 1979). The occurrence in eukaryotic nuclei of special components in the form of a bundle of aggregated fibril-coil structures has been shown by electron microscopy. The structures containing RNP complexes and traversing the nuclear interior are connected with the nuclear pores, presumably taking part in RNA translocation (Franke and Scheer, 1974a). For the purposes of this discussion, the precise intranuclear component to which the RNAs are bound is of little significance, particularly since chromatin, the matrix, and the ribonucleoprotein network all seem to be complexed within the nucleus into an integrated superstructure. The important fact actually is that RNA translocation is structure-oriented, Le., proceeds in association with the solid phase of the nucleus. If this is so, one may hypothesize that the translocation of RNA is coupled with its processing. On the one hand, built-in processing enzymes in the nuclear matrix, located along the passage taken by the RNA, may exert their effects according to a strict schedule. On the other hand, successive interactions of an RNA molecule on its way to the cytoplasm with different sets of "solid-phase" proteins may endow it with a specific conformation needed for structural modifications of RNA by specific enzymes. From this point of view, the heterogeneous population of RNP par-

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ticles that is usually extracted from cell nuclei (the heterogeneity being in the protein moiety) may represent RNA molecules at different stages of processing, associated with the different sets of proteins that are specific for each processing stage. Finally, association with specific intranuclear structures may direct RNA movement to certain nuclear pores as we have proposed above. A similar view was expressed by Herlan et al. (1979), who suggested that at least in Tetrahymena the coupling of transcription, processing, and translocation of ribosomal RNAs occurs along elements of the nuclear matrix. Thus, if regulation at the level of the nuclear pore does exist, then the latter may be regarded as a kind of receptor for certain chromatin regions that detect specific protein signals from the cytoplasm and organize the chromatin activity in accordance with the requirements of the cytoplasmic environment.

9. CONCLUSION Recent years have been marked by profound changes in our ideas on the structural organization of the genome and of chromatin, as well as of the successive steps of gene expression. It was previously thought to be quite obvious that the eukaryotic phenotype, like that of prokaryotes, would be determined almost entirely by differential gene activity, i.e., various cell types would have qualitatively different sets of functioning genes. However, the true position turned out to be far more intricate than such a clear-cut picture. The paradox is that the differences between the ｾ･ｴｳ＠ of nuclear RNAs in cells that differ either phenotypically or functionally are significantly less pronounced that would be expected. Moreover, even the sets of cytoplasmic mRNAs, although substantially more tissue specific than the nuclear RNAs, have a great deal in common with each other. Proceeding from the above findings, many authors have expressed the view that protein synthesis in eukaryotes is regulated not only by qualitative but also by quantitative properties of the mRNA population. In other words, the sets of mRNAs in phenotypically different cells may be very close qualitatively but certain individual messengers may be present in them in markedly different concentrations. One of the conclusions to be drawn from recent data is the recognition of the important role of posttranscriptional regulation in protein synthesis. Indeed, the fact that cytoplasmic mRNAs have a higher tissue specificity than nuclear RNAs, on the one hand, and the drastic differences in quantitative composition of nuclear and cytoplasmic RNA populations, on the other hand, imply the existence of mechanisms of selection operating at the boundary of the nucleus and cytoplasm. It seems likely that it is precisely the nuclear enve-

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lope and its specialized structures (the nuclear pores) that serve as a system coupling specific nuclear and cytoplasmic processes and mediating their interactions. In the study of transcriptional and posttranscriptionallevels of regulation, it is necessary to bear in mind probable changes in their interrelationships that may occur in the course of cell development and differentiation. It is quite conceivable that in embryogenesis, when abrupt and large-scale alterations in cell structure and function occur, strictly programmed gene regulation predominates, whereas in already differentiated cells the reverse is more plausible, namely the pattern of active genes remains constant and the delivery of specific mRNAs to the cytoplasm is under peripheral regulation. This idea is in line with that of Tomkins et al. (1969), who suggested that transcriptional control is most important for successive gene activation during development as well as for the maintenance of the differentiated state. Meanwhile, posttranscriptional regulation is involved in more subtle adjustments of protein synthesis. Thus, the question in the title of this chapter may be answered by saying that both nuclear "supply" and cytoplasmic "demand" determine the flow of genetic information, complementing each other, although their relative contributions to the whole process may vary at different stages in the life of the cell.

10. REFERENCES Affara, N. A., Jacquet, M., Jakob, H., Jacob, F., and Gros, F., 1977, Comparison of po1ysomal polyadenylated RNA from embryonal carcinoma and committed and erythropoietic cell lines, Cell 12:509-520. Agutter, P. S., McArdle, H. J., and McCaldin, B., 1976, Evidence for involvement of nuclear envelope nucleoside triphosphatase in nucleocytoplasmic translocation of ribonucleoprotein, Nature (London) 263:165-167. Aronson, A. I., and Wilt, F. H., 1969, Properties of nuclear RNA in sea urchin embryos, Proc. Natl. Acad. Sci. USA 62:186-193. Ashburner, M., 1967, Patterns of puffing activity in the salivary gland chromosomes of Drosophila, Chromosoma 21:398-428. Attardi, G., Parnas, H., Hwang, M.-1. H., and Attardi, B., 1966, Giant size rapidly labeled nuclear ribonucleic acid and cytoplasmic messenger ribonucleic acid in immature duck erythrocytes, J. Mol. Bioi. 20:145-182. Austin, G. E., Russo, R. J., and Moyer, G. H., 1978, Comparison of transcripts of unique DNA sequences in hepatomas induced by N-2-acetylaminoftuorene and in rat liver, Fed. Proc. 37:N 728. Aviv, H., Voloch, Z., Bastos, R., and Levy, S., 1976, Biosynthesis and stability of globin mRNA in cultured erythroleukemic Friend cells, Cell 8:495-503. Axel, R., and Garel, A., 1977, Structure of the ovalbumin gene in chromatin, Ann. N. Y. Acad. Sci. 286:135-146. Axel, R., Feigelson, P., and Schutz, G., 1976, Analysis of the complexity and diversity of mRNA from chicken liver and oviduct, Cell 7:247-254. Bachellerie, J.-P., Puvion, E., and Zalta, J.-P., 1975, Ultrastructural organization and biochem-

Control of RNA Transport from Nucleus to Cytoplasm

235

ical characterization of chromatin-RNA-protein complexes isolated from mammalian cell nuclei, Eur. J. Biochem 58:327-337. Bantle, J. A., and Hahn, W. E., 1976, Complexity and characterization of polyadenylated RNA in the mouse brain, Cell 8: 139-150. Bastian, C., 1980, The effect of cytosol from regenerating rat liver on the in vitro RNA synthesis of isolated cell nuclei from a Morris hepatoma; comparative studies on molecular hybridization of nuclear RNA, Biochem. Biophys. Res. Commun. 92:80-88. Bastos, R. N., and Aviv, H., 1977, Theoretical analysis of a model for globin messenger RNA accumulation during erythropoiesis, J. Mol. Bioi. 110:205-218. Bastos, R. N., Volloch, Z., and Aviv, H., 1977, Messenger RNA population analysis during erythroid differentiation: A kinetical approach, J. Mol. Bioi. 110:t91-203. Beermann, W., 1964, Control of differentiation at the chromosomal level, J. Exp. Zool. 157:4961. Beljaeva, E. M., 1977, Enzymatic and cytological aspects of processing and nucleus-to-cytoplasm transport of RNA, Usp. Sovrem. Bioi. 83:198-212. Bellard, M., Gannon, F., and Chambon, P., 1978, The structure and transcriptional activity of the chromatin containing the ovalbumin and globin genes in chick oviduct nuclei, Cold Spring Harbor Symp. Quant. Bioi. 42(2):779-791. Beltz, G. A., and Flint, S. J., 1979, Inhibition of HeLa cell protein synthesis during adenovirus injection. Restriction of cellular messenger RNA sequences to the nucleus, J. Mol. Bioi. 131:353-373. Berezney, R., Basler, J., Hughes, B. B., and Caplan, S. c., 1979, Isolation and characterization of the nuclear matrix from Zajdela ascites hepatoma cells, Cancer Res. 39:3031-3039. Berger, S. L., and Cooper, H. L., 1978, The relationship between hnRNA +-poly(A) and mRNA +-poly(A) in non-dividing human lymphocytes. Evidence for distinct synthetic pathways for mRNA-precursor and non-precursor hnRNA, Biochim. Biophys. Acta 517:84-98. Bester, A. J., Kennedy, D. S., and Heywood, S. M., 1975, Two classes of transcriptional control RNA: Their role in the regulation, Proc. Natl. Acad. Sci. USA 72:1523-1527. Bhorjee, J. S., and Pederson, T., 1973, Chromatin: Its isolation from cultured mammalian cells with particular reference to contamination by nuclear ribonucleoprotein particles, Biochemistry 12:2766-2772. Birnie, G. D., MacPhail, E., Young, B. D., Getz, M. J., and Paul, J., 1974, The diversity of the messenger RNA population in growing Friend cells, Cell Differ. 3:221-232. Bishop, J. 0., Morton, J. G., Rosbash, M., and Richardson, M., 1974, Three abundance classes in HeLa cell messenger RNA, Nature (London) 250:199-204. Biswas, B. B., and Roy, P., 1978, Plant growth substances as modulators of transcription, Subcell. Biochem. 5:187-219. Bloom, K. S., and Anderson, J. N., 1978, Fractionation of hen oviduct chromatin into transcriptionally active and inactive regions after selective micrococcal nuclease digestion, Cell 15:141-150. Boehm, T. L. J., and Drahovsky, D., 1979, Sequence complexity of transcribed unique DNA sequences in genome of mouse P815 mastocytoma cells, Z. Naturforsch. 34:436-441. Bouteille, M., Laval, M., and Dupuy-Coin, A. M., 1974, Localization of nuclear functions as revealed by ultrastructural autoradiography and cytochemistry, in: The Cell Nucleus (H. Busch, ed.), pp. 5-71, Academic Press, New York. Brandhorst, B. P., and McConkey, E. H., 1974, Stability of nuclear RNA in mammalian cells, J. Mol. Bioi. 85:451-463. Breathnach, R., Benoist, C., O'Hara, K., Gannon, F., and Chambon, P., 1978, Ovalbumin gene: Evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries, Proc. Natl. Acad. Sci. USA 75:4853-4857.

Anatoly V. Lichtenstein et al.

236

Britten, R. J., and Davidson, R. H., 1969, Gene regulation for higher cells: A theory, Science 165:349-357. Brown, A., and Marzluff, W., 1978, Biosynthesis of low molecular weight RNA in mouse myeloma cells, Biochim. Biophys. Acta 521:662-676. Carlson, J. G., 1952, Microdissection studies of the dividing neuroblast of the grasshopper Chortophaga viridifasciata (De Geer), Chromosoma 5:199-220. Catterall, J. F., O'Malley, B. W., Robertson, M. A., Staden, R., Tanaka, J., and Brownlee, G. G., 1978, Nucleotide sequence homology at 12 intron-exon junctions in the chick ovalbumin gene, Nature (London) 275:510-5\3. Chambon, P., 1978, Summary: Molecular biology of eukaryotic genome is coming of age, Cold

Spring Harbor Symp. Quant. Bioi. 42(2):1211-1236. Chan, L.-N. L., 1976, Transport of globin mRNA from nucleus into cytoplasm in differentiating embryonic red blood cells, Nature (London) 261:157-159. Chapman, K S. R., and Ingle, J., 1976, The stability, polyadenylic acid content and ribonucleoprotein form of nuclear ribonucleic acid in artichoke, Biochem. J. 159:585-600. Chatterjee, B., Hopkins, J., and Roy, A. K, 1978, Increased hepatic messenger RNA for lX'u globulin after superinduction with actinomycin D, Fed. Proc. 38:399. Chatterjee, 8., Hopkins, J., Dutchak, D., and Roy, A. K, 1979, Superinduction of lX'u globulin by actinomycin D: Evidence for drug-mediated increased in lX'u mRNA, Proc. Natl. Acad.

Sci. USA 76:1833-1837. Chatterjee, N. K, and Weissbach, H., 1973, Release of RNA from HeLa cell nuclei, Arch. Biochem. Biophys. 157: 160-166. Chelm, 8. K, Hallick, R. 8., and Gray, P. W., 1979, Transcriptional program of the chloroplast genome of Euglene gracilis during chloroplast development, Proc. Natl. Acad. Sci. USA 76:2258-2262. Chentsov, Ju. S., 1978, General Cytology, Moscow State University Press, Moscow. Chentsov, Ju. S., and Polyakov, V. lu., 1974, Ultrastructure of Cell Nucleus, Nauka, Moscow. Ch'ih, J. J., Duhl, D. M., Faulkner, L. S., and Devlin, T. M., 1979, Regulation of mammalian protein synthesis in vivo. Stimulated transport of nuclear ribonucleoprotein complexes to the cytoplasm after cycloheximide treatment, Biochem. J. 178:643-649. Chikaraishi, D. M., Deeb, S. S., and Sueoka, N., 1978, Sequence complexity of nuclear RNAs in adult rat tissues, Cell 13: 111-120. Chumakdv, P. M., 1979, Symmetric transcription of simian virus 40 DNA at the early phase of productive infection, Biochem. Biophys. Res. Commun. 89:1035-1040. Church, R. B., and Brown, I. R., 1972, Tissue specificity of genetic transcription, in: Results and Problems in Differentiation (H. Ursprung, ed.), Vol. 3, pp. 11-24, Springer-Verlag, New York. Church, R., and McCarthy, 8. J., 1967, Changes in nuclear and cytoplasmic RNA in regenerating mouse liver, Proc. Natl. Acad. Sci. USA 58: 1548-1553. Church, R. 8., and McCarthy, B. J., 1970, Unstable nuclear RNA synthesis following estrogen stimulation, Biochim. Biophys. Acta 199:103-114. Church, R. 8., and Schultz, G. A., 1974, Differential gene activity in the pre- and postimplantation mammalian embryo, Curro Top. Dev. Bioi. 8: 179-202. Church, R. 8., Luther, S. W., and McCarthy, 8. J., 1969, RNA synthesis in Taper hepatoma and mouse liver cells, Biochim. Biophys. Acta 190:30-37. Clawson, G. A., and Smuckler, E. A., 1978, Activation energy for RNA transport from isolated rat liver nuclei, Proc. Natl. Acad. Sci. USA 75:5400-5404. Clawson, G. A., Koplitz, M., Castler-Schechter, B., and Smuckler, E. A., 1978, Energy utilization and RNA transport: Their interdependence, Biochemistry 17:3747-3752. Clever, u., 1968, Regulation of chromosome function, Annu. Rev. Genet. 2: 11-30.

Control of RNA Transport from Nucleus to Cytoplasm

237

Colbert, D. A., and Coleman, J. R., 1977, Transcription expression of non-repetitive DNA during normal and BUdR-mediated inhibition of myogenesis in culture, Exp. Cell Res. 109:3142. Comings, D. E., 1968, The rationale for an ordered arrangement of chromatin in the interphase nucleus, Am. J. Hum. Genet. 207:440-460. Comings, D. E., and Okada, T. A., 1970, Association of chromatin fibers with the annuli of the nuclear membrane, Exp. Cell Res. 62:293-302. Cox, R. F., 1977, Estrogen withdrawal in chick oviduct. Selective loss of high abundance classes of polyadenylated messenger RNA, Biochemistry 16:3433-3443. Cox, R. F., Haines, M. E., and Emtage, J. S., 1974, Quantitation of ovalbumin mRNA in hen and chick oviduct by hybridization to complementary DNA. Accumulation of specific mRNA in response to estradiol, Eur. J. Biochem. 49:225-236. Crick, F., 1970, Central dogma of molecular biology, Nature (London) 227:561-563. Crippa, M., and Gross, P. R., 1969, Maternal and embryonic contributions to the functional messenger RNA of early development. Proc. Natl. Acad. Sci. USA 62:120-127. D'Amato, F., 1977, Nuclear cytology in relation to development, in: Developmental and Cell Biology Series (M. Abercrombie, D. R. Newth, and J. G. Torrey, eds.), pp. 6-52, Cambridge University Press, London. Daneholt, B., 1976, Nuclear RNA, in: Handbook of Genetics (R. E. King, ed.), Vol. 5, pp. 189217, Plenum Press, New York. Darnell, J. E., 1978, Implications of RNA· RNA splicing in evolution of eukaryotic cells, Science 202:1257-1260. Davidson, E. H., 1968, Gene Activity in Early Development, 1st ed., Academic Press, New York. Davidson, E. H., 1976, Gene Activity in Early Development, 2nd ed., Academic Press, New York. Davidson, E. H., and Britten, R. J., 1973, Organization, transcription, and regulation in the animal genome, Q. Rev. Bioi. 48:565-614. Davidson, E. H., and Britten, R. J., 1974, Molecular aspects of gene regulation in animal cells, Cancer Res. 34:2034-2043. Davidson, E. H., and Britten, R. J., 1979, Regulation of gene expression: Possible role of repetitive sequences, Science 204:1052-1059. Deimei, B., Louis, Ch., and Sekeris, C. E., 1977, The presence of small molecular weight RNAs in nuclear ribonucleoprotein particles carrying HnRNA, FEBS Lett. 73:80-84. De Robertis, E. M., and Gurdon, J. B., 1977, Gene activation in somatic nuclei after injection into amphibian oocytes, Proc. Natl. Acad. Sci. USA 74:2470-2474. Detke, S., Stein, J. L., and Stein, G. S., 1978, Synthesis of histone messenger RNAs by RNA polymerase II in nuclei from S phase, HeLa S, cells, Nucleic Acids Res. 5:1515-1528. Detke, S., Lichtler, A., Phillips, I., Stein J., and Stein, G., 1979, Reassessment of histone gene expression during cell cycle in human cells by using homologous H. histone cDNA, Proc. Natl. Acad. Sci. USA 76:4995-4999. Drews, J., Brawerman, G., and Morris, H. P., 1968, Nucleotide sequence homologies in nuclear and cytoplasmic ribonucleic acid from rat liver and hepatomas, Eur. J. Biochem. 3:284292. Dubroff, L. M., and Nemer, M., 1976, Developmental shifts in the synthesis of heterogeneous nuclear RNA classes in the sea urchin embryo, Nature (London) 260:120-124. Dye, D. M., and Toliver, A. P., 1975, Evidence for the existence of a stable association between nascent DNA and the nuclear membrane of HeLa cells, Biochim. Biophys. Acta 414:173184. Engelhardt, P., and Pusa, K., 1972, Nuclear pore complexes: "Press-Stud" elements of chromosomes in pairing and control, Nature New Bioi. 240:163-166. Ephrussi, B., 1972, Hybridization of Somatic Cells, Oxford University Press, London.

238

Anatoly V. Lichtenstein et al.

Ernst, S. G., Britten, R. J., and Davidson, E. H., 1979, Distinct single-copy sequence sets in sea urchin nuclear RNA's, Proc. Natl. Acad. Sci. USA 76:2209-2212. Faiferman, I., and Pogo, A. 0., 1975, Isolation of a nuclear ribonucleoprotein network that contains heterogeneous RNA and is bound to the nuclear envelope, Biochemistry 14:38083816. Faiferman, I., Hamilton, M. G., and Pogo, A. 0., 1970, Nucleoplasmic ribonucleoprotein particles of rat liver. I. Selective degradation by nuclear nucleases, Biochim. Biophys. Acta 204:550-563. Fakan, S., and Bernhard, W., 1971, Localization of rapidly and slowly labelled nuclear RNA as visualised by high resolution autoradiography, Exp. Cell Res. 67:129-141. Feldherr, C. M., 1980, Ribosomal RNA synthesis and transport following disruption of the nuclear envelope, Cell Tissue Res. 205:157-162. Felsani, A., Berthelot, F., Gros, F., and Groizat, 8., 1978, Complexity of polysomal polyadenylated RNA in undifferentiated and differentiated neuroblastoma cells, Eur. J. Biochem. 92:569-577. Flavell, R. A., Glover, D. M., and Jeffreys, A. J., 1978, Discontinuous genes, Trends Biochem. Sci. 3:241-244. Flickinger, R. A., and Roche, F. M., 1972, Effect of tri-iodothyronine on the transcription of deoxyribonucleic acids of different degrees of redundancy in frog larvae, Biochem. J. 130:319-320. Flickinger, R. A., Roche, F. M., and Rodriguez, L. V., 1973, Qualitative restriction of transcription in human leukemic lymphocytes, Eur. J. Biochem. 9:363-366. Flint, S. J., and Weintraub, H. M., 1977, An altered subunit configuration associated with the actively transcribed DNA of integrated adenovirus genes, Cell 12:783-794. Flytzanis, c., Alonso, A., Louis, C., Krieg, L., and Sekeris, C. E., 1978, Association of small nuclear RNA with hnRNA isolated from nuclear RNP complexes carrying hnRNA, FEBS Lett. 96:201-206. Franke, W. W., 1970, Nuclear pore flow rate, Naturwissenschaften 57:44-45. Franke, W. W., and Scheer, u., 1970, The ultrastructure of the nuclear envelope of amphibian oocytes: A re-investigation. II. The immature oocyte and dynamic aspects, J. Ultrastruct. Res. 30:317-327. Franke, W. W., and Scheer, U., 1974a, Structures and functions of the nuclear envelope, in: The Cell Nucleus (H. Busch, ed.), Vol. I, pp. 219-347, Academic Press, New York. Franke, W. W., and Scheer, U., 1974b, Pathways of nucleocytoplasmic translocation of ribonucleoproteins, in: Transport at the Cellular Level (M. A. Sleigh and D. H. Jennings, eds.), pp. 249-282, Cambridge University Press, London. Franke, W. W., Scheer, U., Trendelenburg, M., Zentgraf, H., and Spring, H., 1978, Morphology of transcriptionally active chromatin, Cold Spring Harbor Symp. Quant. Bioi. 42(2):755772. Freeman, G., 1976, The role of cleavage in the localization of development potential in ctenophore Mnemiopsis leidyc, Dev. Bioi. 49:143-154. Fuhr, J. E., and Overton, M., 1975, Translational control of globin synthesis by low molecular weight RNA, Biochem. Biophys. Res. Commun. 63:742-747. Galau, G. A., Klein, W. H., Davis, M. M., Wold, B. J., Britten, R. J., and Davidson, E. H., 1976, Structural gene sets active in embryos and adult tissues of the sea urchin, Cell 7:487505. GareI, A., and Axel, R., 1976, Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei, Proc. Natl. Acad. Sci. USA 73:3966-3970. Garel, A., Zolan, M., and Axel, R., 1977, Genes transcribed at diverse rates have a similar conformation in chromatin, Proc. Natl. Acad. Sci. USA 74:4867-4871.

Control of RNA Transport from Nucleus to Cytoplasm

239

Garrett, C. T., Moore, R. E., Katz, C., and Pitot, H. C., 1973a, Competitive DNA-RNA hybridization of microsomal and nuclear RNA in normal tissues of the rat, Cancer Res. 33:24642468. Garrett, C. T., Moore, R. E., Katz, c., and Pitot, H. c., 1973b, Competitive DNA-RNA hybridization of nuclear and microsomal RNA in normal, neoplastic, and neonatal liver tissues, Cancer Res. 33:2469-2475. Gasaryan, K. G., Lipasova, V. A., Kiraynov, G. I., Ananyantz, T. G., and Ermakova, N. G., 1971, Study on rapidly labelled nuclear RNA of animal cells, Mol. Bioi. (Moscow) 5:680689. Gasaryan, K. G., Kuznetsova, E. D., Fetisova, I. V., and Tarantul, V. Z., 1977a, Metabolically stable messenger-like 28S RNA fraction in the nuclei of pigeon-bone marrow cells, FEBS Lett. 77:251-254. Gasaryan, K. G., Nikolaev, A. I., and Tarantul, V. Z., 1977b, The comparison of melting properties of the 28S nuclear messenger-like RNA, 28S ribosomal RNA and 9S globin mRNA from pigeon erythroid cells, FEBS Lett. 84:320-322. Gasaryan, K. G., Kuznetsova, E. D., and Tarantul, V. Z., 1978, Are polyadenylated and nonpolyadenylated giant hnRNA molecules transcribed from different sites in the genome?, FEBS Lett. 94: 136-138. Gasaryan, K. G., Tarantul, V. Z., Kuznetsova, E. D., Stvolinskaya, N. S., Nikolaev, A. I., Fetisova, I. V., Lipasova, V. A., and Popov, L. S., 1979a, Metabolically stable classes of nuclear informosomal RNA. I. Detection and characteristics, Mol. Bioi. (Moscow) 13:1629. Gasaryan, K. G., Kuznetsova, E. D., Fetisova, I. V., and Tarantul, V. Z., 1979b, Metabolically stable classes of nuclear informosomal RNA. II. Homology between 28S nuclear RNA, giant poly(A)- hnRNA and poly(A)+ mRNA, Mol. Bioi. (Moscow) 13:761-768. Georgiev, G. P., 1969, On the structural organization of operon and the regulation of RNA synthesis in animal cells, J. Theor. Bioi. 25:473-490. Getz, M. J., Birnie, G. D., Young, B. D., MacPhail, E., and Paul, J., 1975, A kinetic estimation of base sequence complexity of nuclear poly(A)-containing RNA in mouse Friend cells, Cell 4:121-129. Getz, M. J., Elder, P. K., Benz, E. W., Stephens, R. E., and Moses, H. L., 1976, Effect of cell proliferation on levels and diversity of poly(A)-containing mRNA, Cell 7:255-265. Gezelius, G., 1976, Further aspects on the effect of sulphate deficiency on the RNA synthesis in sea urchin embryos, Zoon 4:43-46. Ghosh, P. K., Reddy, V. 8., Swinscoe, J., Lebowitz, P., and Weissman, S. M., 1978, Heterogeneity and 5'-terminal structures of the late RNAs of simian virus 40, J. Mol. Bioi. 126:813846. Gianni, A. M., Dalla, F. R., Polli, E., Merisio, I., Giglioni, 8., Comi, P., and Ottolenghi, S., 1978, Globin RNA sequences in human leukemia peripheral blood, Nature (London) 274:610-612. Gilbert, W., 1978, Why genes in pieces?, Nature (London) 271:501. Gillham, N. W., 1978, Organelle Heredity, Raven Press, New York. Gilmour, R. S., 1979, Transcriptional control of native chromatin, in: Chromatin Structure and Function (C. A. Nicolini, ed.), Part A, pp. 41-64, Academic Press, New York. Githens, S., Pictet, R., Phelps, P., and Rutter, W. J., 1976, 5-Bromodeoxyuridine may alter the differentiative program of the embryonic pancreas, J. Cell Bioi. 71:341-356. Goldberg, S., Schwartz, H., and Darnell, J. E., 1977, Evidence from UV transcription mapping in HeLa cells that heterogeneous nuclear RNA is the messenger RNA precursor, Proc. Natl. Acad. Sci. USA 74:4520-4523. Goldberger, R. F., 1974, Autogenous regulation of gene expression, Science 183:810-817.

240

Anatoly V. Lichtenstein et al.

Gottesfeld, J. M., Murphy, R. F., and Bonner, J., 1975, Structure of transcriptionally active chromatin, Proc. Natl. Acad. Sci. USA 72:4404-4408. Grady, L. J., North, A. B., and Campbell, W. P., 1978, Complexity of poly(A)+ and poly(A)polysomal RNA in mouse liver and cultured mouse fibroblasts, Nucleic Acids Res. 5:697712. Greene, R. F., and Fausto, N., 1974, Studies on poly(A) sequences associated with regenerating liver RNA, Biochim. Biophys. Acta 366:23-34. Greene, R. F., and Fausto, N., 1977, Analysis of gene expression in regenerating rat liver by hybridization of nuclear and cytoplasmic RNA with DNA, Cancer Res. 37:118-127. Grouse, J. M., and Grouse, L. D., 1979, Effect of estrogen on gene expression in rooster liver, Biochem. Biophys. Res. Commun. 86:545-551. Guimont-Ducamp, C., Sri-Widada, J., and Jeanteur, P., 1977, Occurrence of small molecular weight RNAs in HeLa nuclear ribonucleoprotein particles containing HnRNA (HnRNP), Biochimie 59:755-758. Gurdon, J. B., 1970, Nuclear transplantation and the control of gene activity in animal development, Proc. R. Soc. London Ser. B 176:303-314. Gurdon, J. B., 1974, The Control o/Gene Expression in Animal Development. Oxford University Press (Clarendon), London. Hahn, W. E., and Laird, C. D., 1971, Transcription of nonrepeated DNA in mouse brain, Science 173: 158-160. Hall, C., and Lim, L., 1978, The metabolism of high-molecular-weight ribonucleic acid in hypothalamic and cortical regions of the developing female rat brain, Biochem. J. 176:511521. Hames, B. D., and Perry, R. P., 1977, Homology relationship between the messenger RNA and heterogeneous nuclear RNA of mouse L cells. A DNA excess hybridization study, J. Mol. Bioi. 109:437-453. Hammett, J. R., and Katterman, F. R., 1975, Storage and metabolism of poly(adenylic acid)mRNA in germinating cotton seeds, Biochemistry 14:4375-4379. Harding, J. D., MacDonald, R. J., Przybyla, A. E., Chirgwin, J. M., Pictet, R. L., and Rutter, W. J., 1977, Changes in the frequency of specific transcripts during development of the pancreas, J. Bioi. Chern. 252:7391-7397. Harpold, M. M., Evans, R. M., Salditt-Georgieff, M., and Darnell, J. E., 1979, Production of mRNA in Chinese hamster cells: Relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences, Cell 17:1025-1035. Harris, H., 1959, Turnover of nuclear and cytoplasmic ribonucleic acid in two types of animal cells with some further observations on the nucleolus, Biochem. J. 73:362-370. Harris, H. 1970, Nucleus and Cytoplasm. 2nd ed., Oxford University Press (Clarendon), London. Hastie, N. D., and Bishop, J. 0., 1976, The expression of three abundance classes of messenger RNA in mouse tissues, Cell 9:761-774. Hazan, N., and McCauley, R., 1976, Effect of phenobarbitone on the nucleocytoplasmic transport of ribonucleic acid in vitro. Biochem. J. 156:665-670. Hegner, R. W., 1911, Experiments with chrysomelid beetles. III. The effects of killing parts of the eggs of Leptinotarsa decemlineata. Bioi. Bull. (Woods Hole) 20:237-246. Hemminki, K., 1974, Polyadenylic acid in RNA extracted by thermal phenol fractionation from chick embryo brain and liver, Biochim. Biophys. Acta 340:262-268. Hendrickson, S., and Johnson, L. F., 1978, Evidence for highly stable nuclear poly(A) in cultured mammalian cells, Biochim. Biophys. Acta 517:287-295. Herlan, G., Eckert, W. A., Kaffenberger, W., and Wunderlich, F., 1979, Isolation and characterization of an RNA-containing nuclear matrix from Tetrahymena macronuclei, Biochemistry 18: 1782-1788.

Control of RNA Transport from Nucleus to Cytoplasm

241

Herman, R. c., and Penman, S., 1977, Multiple decay rates of heterogeneous nuclear RNA in HeLa cells, Biochemistry 16:3460-3465. Herman, R. c., Williams, J. G., and Penman, S., 1976, Messenger and non-messenger sequences adjacent to poly(A) in steady-state heterogeneous nuclear RNA in HeLa cells, Cell 7:429437. Herman, R., Weymouth, L., and Penman, S., 1978, Heterogeneous nuclear RNA-protein fibers in chromatin-depleted nuclei, J. Cell Bioi. 78:663-674. Hillman, N., and Tasca, R. J., 1969, Ultrastructural and autoradiographic studies of mouse cleavage states, Am. J. Anat. 126: 151-174. Holmes, D. S., and Bonner, J., 1974, Sequence composition of rat nuclear deoxyribonucleic acid and high molecular weight nuclear ribonucleic acid, Biochemistry 13:841-849. Hough-Evans, B. R., Wold, B. J., Ernst, S. G., Britten, R. J., and Davidson, E. H., 1977, Appearance and persistence of maternal RNA sequences in sea urchin development, Dev. Bioi. 60:258-277. Hough-Evans, B. R., Ernst, S. G., Britten, R. J., and Davidson, E. H., 1979, RNA complexity in developing sea urchin oocytes, Dev. Bioi. 69:258-269. Humphries, S., Windass, J., and Williamson, R., 1976, Mouse globin gene expression in erythroid and non-erythroid tissues, Cell 7:267-277. Ishikawa, K., Kuroda, c., and Ogata, K., 1969, Release of ribonucleoprotein particles containing rapidly labeled ribonucleic acid from rat liver nuclei. Effect of adenosine-5'-triphosphate and some properties of the particles, Biochim. Biophys. Acta 179:316-331. Ishikawa, K., Ueki, M., Nagai, K., and Ogata, K., 1970, Incorporation of nuclear rapidly labeled RNA into polysomes in the reconstructed system of rat liver, Biochim. Biophys. Acta 213:542-544. Ishikawa, K., Ueki, M., Nagai, K., and Ogato, K., 1972, Transportation of messenger RNA from nuclei to polysomes in rat liver cells, Biochim. Biophys. Acta 259: 138-154. Jacquet, M., Affara, N. A., Robert, B., Jakob, H., Jacob, F., and Gros, F., 1978, Complexity of nuclear and polysomal polyadenylated RNA in a pluripotent embryonal carcinoma cell line, Biochemistry 17:69-79. Jamrich, M., Greenleaf, A. L., and Bautz, E. K. F., 1977, Localization of RNA polymerase in polytene chromosomes of Drosophila melanogaster, Proc. Natl. Acad. Sci. USA 74:20792083. Johnson, L. F., Abelson, H. T., Green, H., and Penman, S., 1974, Changes in RNA in relation to growth of the fibroblasts. I. Amounts of mRNA, rRNA and tRNA in resting and growing cells, Cell 1:95- 100. Johnson, L. F., Lewis, R., Abelson, H. T., Green, H., and Penman, S., 1976, Changes in RNA in relation to growth of the fibroblasts. IV. Alterations in the production and processing of mRNA and rRNA in resting and growing cells, J. Cell Bioi. 71:933-938. Johnson, L. F., Abelson, H. T., Penman, S., and Green, H., 1977, The relative amounts of the cytoplasmic RNA species in normal, transformed and senescent culture cell lines, J. Cell. Physiol. 90:465-470. Kafiani, K. A., and Kostomarova, A. A., 1978, Informational Macromolecules in Early Animal Development, Nauka, Moscow. Kaplan, B. 8., Schachter, 8. S., Osterburg, H. H., Vellis, J. S., and Finch, C. E., 1978, Sequence complexity of polyadenylated RNA obtained from rat brain regions and cultured rat cells of neural origin, Biochemistry 17:5516-5524. Kennedy, D. S., Bester, A. J., and Heywood, S. M., 1974, The regulation of protein synthesis by translational control RNA, Biochem. Biophys. Res. Commun. 61:365-373. Kessler-Icekson, G., and Yaffe, D., 1977, Increased translatability in a cell-free system of RNA extracted from actinomycin D-treated cultures, Biochem. Biophys. Res. Commun. 75:6268.

242

Anatoly V. Lichtenstein et al.

Kessler-Icekson, G., Singer, R. H., and Yaffe, D., 1978, The capacity of polyadenylated RNA from myogenic cells treated with actinomycin D to direct protein synthesis in a cell-free system, Eur. J. Biochem. 88:403-410. Kijima, S., and Wilt, F. H., 1969, Rate of nuclear ribonucleic acid turnover in sea urchin embryos, J. Mol. BioI. 40:235-246. Kimmel, C. B., Sessions, S. K., and MacLeod, M. C., 1976, Evidence for an association of most nuclear RNA with chromatin, J. Mol. BioI. 102:177-191. Kleene, K. C., and Humphreys, T., 1977, Similarity of hnRNA sequences in blastula and pluteus stage in urchin embryos, Cell 12: 143-155. Kleiman, L., Birnie, G. D., Young, B. D., and Paul, J., 1977, Comparison of the base-sequence complexities of polysomal and nuclear RNAs in growing Friend erythroleukemia cells, Biochemistry 16: 1218-1223. Knochel, W., and Kohnert-Stavenhagen, E., 1977, Are globin coding sequences expressed in nuclear RNA of chick embryo brain?, Hoppe-Seyler's Z. Physiol. Chern. 358:835-842. Knowland, J., and Graham, C., 1972, RNA synthesis at the two-cell stage of mouse development, J. Embryol. Exp. Morphol. 27:167-176. Konkel, D. A., and Ingram, V. M., 1978, Is there specific transcription from isolated chromatin?,

Nucleic Acids Res. 5:1237-1252. Konkel, D. A., Tilghman, S. M., and Leder, P., 1978, The sequence of the chromosomal mouse (3-globin major gene: Homologies in capping, splicing, and poly(A) sites, Cell 15:11251132. Kostomarova, A. A., 1977, RNA transport in embryo cells, Ontogenesis 8:630-648. Kruh, J., 1972, RNA et biosynthese de l'hemoglobine, Biochimie 54:589-595. Lasky, L., and Tobin, A. J., 1979, Transcriptional regulation in avian erythroid cells, Biochemistry 18: 1594-1598. Lasky, L., Nozick, N. D., and Tobin, A. J., 1978, Few transcribed RNAs are translated in avian erythroid cells, Dev. Bioi. 67:23-39. Leder, A., Miller, H. I., Hamer, D. H., Seidman, J. G., Norman, 8., Sullivan, M., and Leder, P., 1978, Comparison of cloned mouse a- and (3-globin genes: Conservation of intervening sequence locations and extragenic homology, Proc. Natl. Acad. Sci. USA 75:6187-6191. Lee, F., and Yanofsky, Y., 1977, Transcription termination at the trp operon attenuators of Escherichia coli and Salmonella typhimurium: RNA secondary structure and regulation of termination, Proc. Natl. Acad. Sci. USA 74:4365-4369. Lenk, R., Herman, R., and Penman, S., 1978, Messenger RNA abundance and lifetime: A correlation in Drosophila cells but not in HeLa, Nucleic Acids Res. 5:3057-3070. Lerner, M. R., and Steitz, J. A., 1979, Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus, Proc. Natl. Acad. Sci.

USA 76:5495-5499. Lerner, M. R., Boyle, J. A., Mount, S. M., Wolin, S. L., and Steitz, J. A., 1980, Are snRNPs involved in splicing?, Nature (London) 283:220-224. Levy, W. 8., and McCarthy, 8. J., 1975, Messenger RNA complexity in Drosophila melano-

gaster, Biochemistry 14:2440-2446. Lewin, B., 1975a, Units of transcription and translation: The relationship between heterogeneous nuclear RNA and messenger RNA, Cell 4:11-20. Lewin, 8., 1975b, Units of transcription and translation: Sequence components of heterogeneous nuclear RNA and messenger RNA, Cell 4:77-93. Lewis, R., and Penman, S., 1977, The metabolism ofpoly(A)+ and poly(A)- hnRNA in cultured Drosophila cells studied with a rapid uridine pulse-chase, Cell 11 : I 05-113. Lichtenstein, A. V., and Shapot, V. S., 1976, A model for cytoplasm-governed gene regulation, Biochem. J. 159:783-794.

Control of RNA Transport from Nucleus to Cytoplasm

243

Lichtenstein, A. Y., Kartoshkin, Y. M., Piker, E. G., and Shapot, Y. S., 1974, Study on nuclear DNA-like RNA by the ion-exchange chromatography on methylated albumin-kieselguhr columns, Mol. Bioi. (Moscow) 8:703-710. Lichtenstein, A. Y., Alechina, R. P., and Shapot, Y. S., 1975, Ribosomal and informational ribonucleoprotein complexes of animal cells. Study on rat liver ribonucleic acids as constituents of ribonucleoprotein complexes by chromatography on nucleoprotein-Celite columns, Biochem. J. 147:447-456. Lichtenstein, A. Y., Zaboykin, M. M., Alechina, R. P., and Shapot, Y. S., 1976a, Demonstration of fraction of stable nuclear DNA-like RNA in rat liver cells, Dokl. Akad. Nauk SSSR 227:240-243. Lichtenstein, A. Y., Zaboykin, M. M., and Shapot, Y. S., 1976b, Association of nuclear RNP particles with chromatin in Zajdela ascites hepatoma cells, Dokl. Akad. Nauk SSSR 227:252-255. Lichtenstein, A. Y., Alechina, R. P., and Shapot, Y. S., 1976c, Study of RNP particles by the method of RNA chromatography on nucleoprotein-Celite columns, Mol. Bioi. (Moscow) 10:55-63. Lichtenstein, A. Y., Alechina, R. P., and Shapot, Y. S., 1978, Studies on informational RNAs and ribonucleoproteins of normal rat liver, Zajde\a hepatoma and liver of hepatoma-bearing rats, Eur. J. Cancer 14:939-947. Lichtenstein, A. Y., Zaboykin, M. M., Mojseev, Y. L., and Shapot, Y. S., 1979, Gradient dissociation of total cellular nucleoproteins as a principle for separation and analysis of functionally different nucleic acids, Dokl. Akad. Nauk SSSR 245:1005-1009. Lilley, D. M. 1.,1978, Active chromatin structure: Mini-review, Cell Bioi. Int. Rep. 2:1-10. Lipsich, L. A., Kates, 1. R., and Lucas, 1. 1., 1979, Expression of a liver-specific function by mouse fibroblast nuclei transplanted into rat hepatoma cytoplasts, Nature (London) 281:7476. Lukanidin, E. M., Zalmanson, E. S., Komaromi, L., Samarina, O. P., and Georgiev, G. P., 1972, Structure and function of informoferes, Nature New Bioi. 238: 193-197. Lukanidin, E. M., Kulguskin, Y. Y., Ajtchozhina, A. M., Komaromi, L., Tichonenko, A. S., and Georgiev, G. P., 1973, Nuclear ribonucleoproteins containing informational RNA. 10. Isolation and characteristics of free informoferes, Mol. Bioi. (Moscow) 7:360-367. McKinnell, R. G., Steven, L. M., and Labat, D. D., 1976, Frog renal tumors are composed of stroma, vascular elements, and epithelial cells: What type nucleus programs for tadpoles with the cloning procedure?, in: Progress in Differentiation Research (N. MUller-Berat, ed.), pp. 319-330, North-Holland, Amsterdam. McNamara, D. 1., Racevskis, 1., Schumm, D. E., and Webb, T. E., 1975, Ribonucleic acid synthesis in isolated rat liver nuclei under conditions of ribonucleic acid processing and transport, Biochem. J. 147:193-197. McWilliams, D., Callahan, R. c., and Boime, 1.,1977, Human placental lactogen mRNA and its structural genes during pregnancy: Quantitation with a complementary DNA, Proc.

Natl. Acad. Sci. USA 74:1024-1027. Mansson, P.-E., and Harris, S. E., 1979, Complexity of poly(A)-containing heterogeneous nuclear ribonucleic acid from mouse embryoid bodies (OTT6050), Biochemistry 18:20732078. Maryanka, D., Cowling, G. 1., Allan, 1., Fey, S. 1., Huvos, P., and Gould, H. S., 1979, Transcription of globin genes in reticulocyte chromatin, FEBS Lett. 105: 131-136. Maul, G. G., 1977a, The nuclear and the cytoplasmic pore complex: Structure, dynamics, distribution, and evolution, Int. Rev. Cytol. Suppl. 6:75-186. Maul, G. G., 1977b, Nuclear pore complexes. Estimation and reconstruction during mitosis, J.

Cell Bioi. 74:492-500.

244

Anatoly V. Lichtenstein et al.

Mauron, A., and Spohr, G., 1978, Frequence distribution of mRNA and pre-mRNA in growing and differentiated Friend Cells, Nucleic Acids Res. 5:3013-3032. Melli, M., Spinelli, G., and Arnold, E., 1977, Synthesis of histone messenger RNA of HeLa cells during the cell cycle, Cell 12:167-174. Meuli, C., Ryan, R., and Gorski, J., 1979, Sequence complexity of bovine pituitary poly(A) RNA, Mol. Cell. Endocrinol. 16:71-80. Meyuhas, 0., and Perry, R. P., 1979, Relationship between size, stability and abundance of the messenger RNA of mouse L cells, Cell 16:139-148. Miller, D. M., Turner, P., Nienhius, A. W., Axelrod, D. E., and Gopalakrishnan, T. V., 1978, Active conformation of the globin genes in uninduced and induced mouse erythroleukemia cells, Cell 14:511-522. Miller, T. E., Huang, C.-y', and Pogo, A. 0., 1978, Rat liver nuclear skeleton and ribonucleoprotein complexes containing hnRNA, J. Cell Bioi. 76:675-691. Minty, A., Kleiman, L., Birnie, G., and Paul, J., 1977, Nucleus-restricted polyadenylated ribonucleic acids species in mouse Friend cells, Biochem. Soc. Trans. 5:679-681. Minty, A. J., Birnie, G. D., and Paul, J., 1978, Gene expression in Friend erythroleukemia cells following the induction of hemoglobin synthesis, Exp. Cell Res. 115:1-14. Mirault, M.-E., Goldschmidt-Clermont, M., Moran, L, Arrigo, A. P., and Tissieres, A., 1978, The effect of heat shock on gene expression in Drosophila melanogaster, Cold Spring Harbor Symp. Quant. Bioi. 42(2):819-827. Mizuno, S., and Cox, R. F., 1979, Estrogen withdrawal in chick oviduct. Evidence for continued expression of active unique genes using an "expressed" DNA probe, Biochemistry 18:20492056. Molloy, G., and Puckett, L., 1976, The metabolism of heterogeneous nuclear RNA and the formation of cytoplasmic messenger RNA in animal cells, Prog. Biophys. Mol. Bioi. 31:1-38. Monahan, J. J., and Hall, R. H., 1975, A high molecular weight RNA fraction in chromatin, Biochim. Biophys. Acta 383:40-55. Monneron, A., and Bernhard, W., 1969, Fine structural organization of the interphase nucleus in some mammalian cells, J. Ultrastruct. Res. 27:266-279. Morry, Y. Y., and Gefter, M. L., 1977, Synthesis of messenger RNA-like molecules in isolated myeloma nuclei, Nucleic Acids Res. 4:1739-1757. Murray, V., and Holliday, R., 1979, Mechanism for RNA splicing of gene transcripts, FEBS Lett. 106:5-7. Naora, H., 1969, Nuclear ribonucleoprotein particles associated with DNA, Biochim. Biophys. Acta 174:137-146. Nemer, M., 1975, Developmental changes in the synthesis of sea urchin embryo messenger RNA containing and lacking polyadenylic acid, Cell 6:559-570. Nemer, M., Dubroff, L. M., and Graham, M., 1975, Properties of sea urchin embryo messenger RNA containing and lacking poly(A), Cell 6:171-178. Neyfakh, A. A., 1974, Molecular aspects of nucleo-cytoplasmic relationship in embryonic development, in: Neoplasia and Cell Differentiation (G. V. Sherbet, ed.), pp. 27-59, Karger, Basel. Neyfakh, A. A., and Kostomarova, A. A., 1971, Migration of newly synthesized RNA during mitosis. I. Misgurnus fossilis embryo cells, Cytologia (Moscow) 13:231-236. Neyfakh, A. A., and Timofeeva, M. Ja., 1977, Molecular Biology of Developmental Processes, Nauka, Moscow. Neyfakh, A. A., Kostomarova, A. A., and Burakova, T. A., 1973, A study of RNA transfer from nucleus to cytoplasm in early embryos of the loach (Misgurnus fossilis) and its hybrids (Misgurnus fossilis I' X Carassius auratus ｾＩＬ＠ Ontogenesis 4:331-339. Nienhuis, A. W., Turner, P., and Benz, E. J., 1977, Relative stability of (X- and /3-globin messenger RNAS in homozygous /3+ thalassemia, Proc. Natl. Acad. Sci. USA 74:3960-3964.

Control of RNA Transport from Nucleus to Cytoplasm

245

Nordstrom, J. J., Roop, D. R., Tsai, M.-J., and O'Malley, B. W., 1979, Identification of potential ovumucoid mRNA precursors in chick oviduct nuclei, Nature (London) 278:328-331. O'Malley, B. W., Tsai, M.-J., Tsai, S. V., and Towle, H. C., 1978, Regulation of gene expression in chick oviduct. Cold Spring Harbor Symp. Quant. Bioi. 42(2):605-613. Orkin, S. H., and Swerdlow, P. S., 1977, Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: Direct evidence for increased transcription during erythroid differentiation, Proc. Natl. Acad. Sci. USA 74:2475-2479. Ostrow, R. S., Woods, W. G., Vosika, G. J., and Faras, A. J., 1979, Analysis of the genetic complexity and abundance classes of messenger RNA in human liver and leukemic cells, Biochim. Biophys. Acta 562:92-102. Palmiter, R. D., 1975, Quantitative parameters that determine the role of ovalbumin synthesis,

Cell 4:189-197. Palmiter, R. D., and Schimke, R. T., 1973, Regulation of protein synthesis in chick oviduct. III. Mechanism of ovalbumin "superinduction" by actinomycin D, J. Bioi. Chem. 248:15021512. Panyim, S., Ohno, T., and Jost, J.-P., 1978, In vitro RNA synthesis and expression of vitellogenin gene in isolated chicken liver nuclei, Nucleic Acids Res. 5: 1353-1370. Parker, M. G., and Mainwaring, W. I. P., 1977, Effects of androgens on the complexity of poly(A) RNA from rat prostate, Cell 12:401-407. Parsons, J. T., and McCarthy, K. S., 1968, Rapidly labelled messenger ribonucleic acid-protein complex of rat liver nuclei, J. Bioi. Chem. 243:5377-5384. Paterson, B. M., and Bishop, J. 0., 1977, Changes in the mRNA population of chick myoblasts during myogenesis in vitro, Cell 12:751-765. Pederson, T., 1974a, Proteins associated with heterogeneous nuclear RNA in eukaryotic cells, J.

Mol. Bioi. 83:168-183. Pederson, T., 1974b, Gene activation in eukaryotes: Are nuclear acidic proteins the cause or the effect?, Proc. Natl. Acad. Sci. USA 71:617-621. Penman, S., Vesco, c., and Penman, M., 1968, Localization and kinetics of formation of nuclear heterodisperse RNA, cytoplasmic heterodisperse RNA and polyribosome-associated messenger RNA in HeLa cells, J. Mol. Bioi. 34:49-69. Perlman, S. M., Ford, P. J., and Rosbash, M. M., 1977, Presence of tadpole and adult globin RNA sequences in oocytes of Xenopus laevis, Proc. Natl. Acad. Sci. USA 74:3835-3839. Perry, R. P., 1976, Processing of RNA, Annu. Rev. Biochem. 45:605-629. Perry, R. P., Bard, E., Hames, B. D., Kelley, D. E., and Schibler, U., 1976, The relationship between hnRNA and mRNA, Prog. Nucleic Acid Res. Mol. Bioi. 19:275-292. Piker, E. G., and Shapot, V. S., 1976, A comparative study on nuclear RNAs of liver and Zajdela ascites hepatoma cells by hybridization DNA-RNA, Biokhimiya 41:807-813. Price, P. M., 1976, The effect of 5-bromodeoxyuridine on messenger RNA production in cultured cells, Biochim. Biophys. Acta 447:304-311. Price, R. P., Ransom, L., and Penman, S., 1974, Identification of a small subfraction of hnRNA with the characteristics of a precursor to mRNA, Cell 2:253-258. Puckett, L., Cambers, S., and Darnell, J. E., 1975, Short-lived messenger RNA in HeLa cells and its impact on the kinetics of accumulation of cytoplasmic polyadenylate, Proc. Natl. Acad. Sci. USA 72:389-393. Puvion, E., and Bernhard, W., 1975, Ribonucleoprotein components in liver cell nuclei as visualized by cryoultramicrotomy, J. Cell Bioi. 67:200-214. Rabbitts, T. H., 1978, Evidence for splicing of interrupted immunoglobulin variable and constant region sequences in nuclear RNA, Nature (London) 275:291-296. Racevskis, J., and Webb, T. E., 1974, Processing and release of ribosomal RNA from isolated nuclei: Analysis of the ATP dependence and cytosol dependence, Eur. J. Biochem. 49:93100.

246

Anatoly V. Lichtenstein et al.

Raskas, H. J., and Rho, Y.-C., 1973, ATP requirement for release of adenovirus mRNA from isolated nuclei, Nature New BioI. 245:47-49. Remington, J. A., 1979, Attenuation as a general mechanism for the regulation of differential gene transcription in eukaryotes, FEBS Lett. 100:225-229. Roberts, R., 1980, Small RNAs and splicing (news and views), Nature (London) 283:132-133. Rolton, H. A., Birnie, G. D., and Paul, J., 1977, The diversity and specificity of nuclear and polysomal poly(A)+ RNA populations in normal and MSV-transformed cells, Cell Differ. 6:25-39. Royal, A., Garapin, A., Cami, B., Perrin, F., Mandel, J. L., Le Meur, M., Bregegegre, F., Gannon, F., LePennec, J. P., Chambon, P., and Kourilsky, P., 1979, The ovalbumin gene region: Common features in the organization of three genes expressed in chicken oviduct under hormonal control, Nature (London) 279:125-132. Rutter, W. J., Pictet, R. L., and Morris, P. W., 1973, Towards molecular mechanisms of developmental processes, Annu. Rev. Biochem. 42:601-646. Ryan, R., Shupnik, M. A., and Gorski, J., 1979, Effect of estrogen on preprolactin messenger ribonucleic acid sequences, Biochemistry 18:2044-2048. R yffel, G. u., 1976, Comparison of cytoplasmic and nuclear poly(A)-containing RNA sequences in Xenopus liver cells, Eur. J. Biochem. 62:417-423. Ryffel, G. U., and McCarthy, B. J., 1975, Complexity of cytoplasmic RNA in different mouse tissues measured by hybridization of polyadenylated RNA to complementary DNA, Biochemistry 14:1379-1385. Sacher, J. A., and Leo, P. D., 1977, Wound-induced RNase in senescing bean pod tissue: Posttranscriptional regulation of RNase, Plant Cell Physiol. 18: 161-171. Samal, B., and Bekhor, I., 1979, Kinetic estimation of the base sequence complexity of poly(A)containing mRNA in Ehrlichcascites-tumor cells and its abundance in nuclear RNA, Eur. J. Biochem 94:51-57. Samarina, o. P., and Zbarsky, I. 8., 1964, Studies on release of RNA from nuclear structures, Biokhimiya 29:321-330. Samarina, O. P., Lukanidin, E. M., Molnar, J., and Georgiev, G. P., 1968, Structural organization of nuclear complexes containing DNA-like RNA, J. Mol. Bioi. 33:251-263. Sasaki, R., Goto, H., Arima, K., and Sasaki, Y., 1974, Effect of polyribonucleotides on eukaryotic DNA-dependent RNA polymerases, Biochim. Biophys. Acta 366:435-442. Savage, M. J., Sala-Trepat, J. M., and Bonner, J., 1978, Measurement of the complexity and diversity of poly(adenylic acid) containing messenger RNA from rat liver, Biochemistry 17:462-467. Sawada, H., and Saunders, G. F., 1974, Transcription of nonrepetitive DNA in human tissues, Cancer Res. 34:516-520. Scheller, R. H., Costantini, F. D., Kozlowski, M. R., Britten, R. J., and Davidson, E. H., 1978, Specific representation of cloned repetitive DNA sequences in sea urchin RNAs, Cell 15: 189-203. Scherrer, K., and Marcaud, L., 1968, Messenger RNA in avian erythroblasts at the transcriptional and translational levels and the problem of regulation in animal cells, J. Cell. Physiol. 72(SuppI.1 ):181-212. Schumm, D. E., and Webb, T. E., 1972, Transport of informosomes from isolated nuclei of regenerating rat liver, Biochem. Biophys. Res. Commun. 48:1259-1265. Schumm, D. E., and Webb, T. E., 1974a, The in vivo equivalence of a cell-free system for RNA processing and transport, Biochem. Biophys. Res. Commun. 58:354-360. Schumm, D. E., and Webb, T. E., 1974b, Modified messenger ribonucleic acid release from isolated hepatic nuclei after inhibition of polyadenylate formation, Biochem. J. 139:191196.

Control of RNA Transport from Nucleus to Cytoplasm

247

Schumm, D. E., and Webb, T. E., 1975a, Differential effect of plasma fractions from normal and tumour-bearing rats on nuclear RNA restriction, Nature (London) 256:508-509. Schumm, D. E., and Webb, T. E., 1975b, Differential effect of ATP on RNA and DNA release from nuclei of normal and neoplastic liver, Biochem. Biophys. Res. Commun. 67:706713. Schumm, D. E., and Webb, T. E., 1978, Effect of adenosine 3':5'-monophosphate and guanosine 3';5'-monophosphate on RNA release from isolated nuclei, J. Bioi. Chem. 253:8513-8517. Schumm, D. E., McNamara, D. J., and Webb, T. E., 1973a, Cytoplasmic proteins regulating messenger RNA release from nuclei, Nature New Bioi. 245:201-203. Schumm, D. E., Morris, H. P., and Webb, T. E., 1973b, Cytosol-modulated transport of messenger RNA from isolated nuclei, Cancer Res. 33:1821-1828. Schumm, D. E., Hanausek-Walaszek, M., Yannarell, A., and Webb, T. E., 1977, Changes in nuclear RNA transport incident to carcinogenesis, Eur. J. Cancer 13:139-147. Schlitz, G., Nguyen-Huu, M. c., Giesecke, K., Hynes, N. E., Groner, B., Wurtz, T., and Sippel, A. E., 1978, Hormonal control of egg white protein messenger RNA synthesis in the chicken oviduct, Cold Spring Harbor Symp. Quant. Bioi. 42(2):617 -624. Sehgal, P. B., and Tamm, I., 1976, An evaluation of messenger RNA competition in the shutoff of human interferon production, Proc. Natl. Acad. Sci. USA 73:1621-1625. Sehgal, P. B., Dobberstein, B., and Tamm, I., 1977, Interferon messenger RNA content of human fibroblasts during induction, shutoff, and superinduction of interferon production, Proc. Natl. Acad. Sci. USA 74:3409-3413. Seifert, H., Scheurlen, M., Northemann, W., and Heinrich, P. C., 1979, Low molecular weight RNAs as components of nuclear ribonucleoprotein particles containing heterogeneous nuclear RNA, Biochim. Biophys. Acta 564:55-66. Shearer, R. W., I 974a, Irreversibility of the alteration in RNA transport induced by an azo-dye carcinogen in the rat liver, Biochem. Biophys. Res. Commun. 57:604-613. Shearer, R. W., 197 4b, Specificity of chemical modification of ribonucleic acid transport by liver carcinogens in the rat, Biochemistry 13:1764-1769. Shearer, R. W., 1977, Differences and similarities in RNA regulation between tumors induced by chemicals and by DNA and RNA viruses, Vestn. Akad. Med. Nauk SSSR 3:64-72. Shearer, R. W., 1979, Altered RNA transport without derepression in a rat kidney tumor induced by dimethyl nitrosoamine (single injection), Chem. Bioi. Interact. 27:91-98. Shearer, R. W., and McCarthy, B. J., 1967, Evidence for ribonucleic acid molecules restricted to the cell nucleus, Biochemistry 6:283-289. Shearer, R. W., and McCarthy, B. J., 1970, Characterisation of RNA molecules restricted to the nucleus in mouse L-cells, J. Cell. Physiol. 75:97- 107. Shearer, R. W., and Mayer, L. A., 1974, The heterogeneity of host gene transcription and transport of host RNA to the cytoplasm in polyoma virus-induced rat kidney tumors, Biochim. Biophys. Acta 335:437-446. Shearer, R. W., and Smuckler, E. A., 1971, A search for gene derepression in RNA of primary rat hepatoma, Cancer Res. 31:2104-211 I. Shearer, R. W., and Smuckler, E. A., 1972, Altered regulation of the transport of RNA from nucleus to cytoplasm in rat hepatoma cells, Cancer Res. 32:339-346. Shepherd, G. W., and Flickinger, R., 1979, Post-transcriptional control of messenger RNA diversity in frog embryos, Biochim. Biophys. Acta 563:413-421. Shiokawa, K., Misumi, Y., Yasudo, Y., Nishio, Y., Kurata, S., Sameshima, M., and Yamana, K., 1979, Synthesis and transport of various RNA species in developing embryos of Xenopus laevis, Dev. Bioi. 68:503-514. Shuster, R. C., Houdebine, L.-M., and Gaye, P., 1976, Studies on the synthesis of casein messenger RNA during pregnancy in the rabbit, Eur. J. Biochem. 71:193-199.

248

Anatoly V. Lichtenstein et al.

Siegal, G. P., Quinlan, T. J., Moses, H. L., and Getz, M. J., 1979, Frequency differences between nuclear and polysomal sequences of poly(A)-containing RNA from cultured AKR mouse embryo cells, J. Cell. Physiol. 98:283-298. Silagi, S., and Bruce, S. A., 1970, Suppression of malignancy and differentiation in melanotic melanoma cells, Proc. Natl. Acad. Sci. USA 66:72-78. Singer, R. H., and Penman, S., 1973, Messenger RNA in HeLa cells: Kinetics of formation and decay, J. Mol. Bioi. 78:321-334. Sippel, A. E., Groner, B., Hynes, N., and Schiitz, G., 1977a, Size distribution of rat liver nuclear RNA containing mRNA sequences, Eur. J. Biochem. 77:153-164. Sippel, A. E., Hynes, N., Groner, B., and Schiitz, G., 1977b, Frequence distribution of messenger sequences within polysomal mRNA and nuclear RNA from rat liver, Eur. J. Biochem. 77:141-151. Smith, K., and Lingrel, J. B., 1978, Sequence organization of the )3-globin mRNA precursor, Nucleic Acids Res. 5:3295-3301. Smuckler, E. A., and Koplitz, M., 1973, Altered nuclear RNA transport associated with carcinogen intoxication in rats, Biochem. Biophys. Res. Commun. 55:499-506. Soeiro, R., Birnboim, H. C., and Darnell, J. E., 1966, Rapidly labeled HeLa cell nuclear RNA. II. Base composition and cellular localization of a heterogeneous RNA fraction, J. Mol. Bioi. 19:362-372. Spirin, A. S., 1978, Eukaryotic messenger RNA and informosomes: Omnia mea mecum porto, FEBS Lett. 88:15-17. Spirin, A. S., Belitsina, N. V., and Ajtchozhin, M. A., 1964, Informational macromolecules in early embryogenesis, Zh. Obshch. Bioi. 25:321-337. Spohr, G., Imaizumi, T., and Scherrer, K., 1974, Synthesis and processing of nuclear precursor messenger RNA in avian erythroblasts and HeLa cells, Proc. Natl. Acad. Sci. USA 71:5009-5013. Spradling, A., Hui, H., and Penman, S., 1975, Two very different components of messenger RNA in an insect cell line, Cell 4:131-137. Stein, G., Park, W., Thrall, C., Mans, R., and Stein, J., 1975, Regulation of cell cycle stagespecific transcription of histone genes from chromatin by nonhistone chromosomal proteins, Nature (London) 257:764-767. Stein, G., Stein, J., Shephard, E., Park, W., and Phillips, I., 1977, Evidence that the coupling of histone gene expression and DNA synthesis in HeLa S, cells is not mediated at the transcriptionallevel, Biochem. Biophys. Res. Commun. 77:245-252. Stevenin, J., Gallinaro-Matringe, H., Gattoni, R., and Jacob, M., 1977, Complexity of the structure of particles containing heterogeneous nuclear RNA as demonstrated by ribonuclease treatment, Eur. J. Biochem. 74:589-602. Storb, U., Hager, L., Wilson, R., and Putnam, D., 1977, Expression of immunoglobulin and globin genes in Band T lymphocytes and other cells, Biochemistry 16:5432-5438. Strlltling, W. H., and O'Malley, B. W., 1976, Studies on the structure and function of chickoviduct chromatin. 2. Biochemical characterization of two chromatin fractions isolated by ECTHAM-cellulose chromatography, Eur. J. Biochem. 66:435-441. Strlltling, W. H., Van, N. T., and O'Malley, B. W., 1976, Studies on the structure and function of chick-oviduct chromatin. 1. Fractionation of ECTHAM-cellulose chromatography and physico-chemical characterization, Eur. J. Biochem. 66:423-434. Stuart, S. E., Rottman, F. M., and Patterson, R. J., 1975, Nuclear restriction of nucleic acids in the presence of ATP, Biochem. Biophys. Res. Commun. 62:439-447. Sussman, M. 1970, Model for quantitative and qualitative control of mRNA translocation in eukaryotes, Nature (London) 225: 1245-1246.

Control of RNA Transport from Nucleus to Cytoplasm

249

Swaneck, G. E., Nordstrom, J. L., Kreuzaler, F., Tsai, M.-J., and O'Malley, 8. W., 1979, Effect of estrogen on gene expression in chicken oviduct: Evidence for transcriptional control of ovalbumin gene, Proc. Natl. Acad. Sci. USA 76:1049-1053. Tarantul, V. Z., Lipasova, V. A., Baranov, Ju. N., and Gasaryan, K. G., 1974, Study on longlived nuclear RNA of avian erythroid cells, Mol. Bioi. (Moscow) 8:864-870. Tata, J. R., 1978, Induction and regulation of vitellogenin synthesis by estrogen, in: Biochemical Actions of Hormones (G. Litwack, ed.), pp. 397-431, Academic Press, New York. Tata, J. R., and Baker, 8., 1975, Sub-nuclear fractionation. III. Sub-nuclear distribution of poly(A)-rich RNA, Exp. Cell Res. 93:191-201. Tata, J. R., and Baker, B., 1978, Enzymatic fractionation of nuclei: Polynucleosomes and RNA polymerase II as endogenous transcriptional complexes, J. Mol. Bioi. 118:249-272. Tedeschi, M. V., Colbert, D. A., and Fausto, N., 1978, Transcription of the non-repetitive genome in liver hypertrophy and the homology between nuclear RNA of normal and 12-hregenerating liver, Biochim. Biophys. Acta 521:641-649. Therwath, A., and Scherrer, K., 1978, Post-transcriptional suppression of globin gene expression in cells transformed by avian erythroblastosis virus, Proc. Natl. Acad. Sci. USA 75:37763780. Tilghman, S. M., Tiemeier, D. C., Seidman, J. G., Peterlin, 8. M., Sullivan, M., Maizel, J. V., and Leder, P., 1978a, Intervening sequence of DNA identified in the structural portion of a mouse ｾＭｧｬｯ｢ｩｮ＠ gene, Proc. Nat!. Acad. Sci. USA 75:725-729. Tilghman, S. M., Curtis, P. J., Tiemeier, D. C, Leder, P., and Weissman, C, 1978b, The intervening sequence of a mouse ｾＭｧｬｯ｢ｩｮ＠ gene is transcribed within the 15S ｾＭｧｬｯ｢ｩｮ＠ mRNA precursor, Proc. Natl. Acad. Sci. USA 75: 1309-1313. Tobin, A. J., Selvig, S. E., and Lasky, L., 1978, RNA synthesis in avian erythroid cells, Dev. Bioi 67: 11-22. Tomkins, G. M., Gelehter, T. D., Granner, D., Martin, D., Samuels, H. H., and Thompson, E. 8., 1969, Control of specific gene expression in higher organisms, Science 166: 1474-1480. Tomkins, G. M., Levinson, 8. 8., Baxter, Y. D., and Dethlefsen, L., 1972, Further evidence for post-transcriptional control of inducible tyrosine aminotransferase synthesis in cultured hepatoma cells, Nature New Bioi. 239:9-14. Tonegawa, S., Maxam, A. M., Tizard, R., Bernard, 0., and Gilbert, W., 1978, Sequence of a mouse germ-line gene for a variable region of an immunoglobulin light chain, Proc. Nat!. Acad. Sci. USA 75:1485-1489. Towle, H. C., Dillmann, W. H., and Oppenheimer, J. H., 1979, Messenger RNA content and complexity of euthyroid and hypothyroid rat liver, J. Bioi. Chem. 154:2250-2257. Toyoji, S., Ishikawa, K., and Ogata, K., 1977, Factors causing release of ribosomal subunits from isolated nuclei of regenerating rat liver in vitro. Biochim. Biophys. Acta 474:549-561. Truman, D. E. S., 1974, The Biochemistry of Cytodifferentiation. Blackwell, London. Turkington, R. W., and Self, D. 1., 1970, New species of hybridizable nuclear RNA in breast cancer cells, Cancer Res. 30: 1833-1840. Turner, S. H., and Laird, CD., 1973, Diversity of RNA sequences in Drosophila melanogaster. Biochem. Genet. 10:236-274. Van Voorthuizen, W. F., Dinsart, C, Flavell, R. A., De Vijlder, J. 1. M., and Vassart, G., 1978, Abnormal cellular localization of thyroglobulin mRNA associated with hereditary congenital goiter and thyroglobulin deficiency, Proc. Natl. Acad. Sci. USA 75:74-78. Varian, A., and Sacher, J. A., 1978, Wound-induced RNase in bean pod tissue. II. Auxin regulation of RNase synthesis at transcription, Plant Cell Physiol. 19: 1185-1193. Wallace, R. B., Dube, S. K., and Bonner, J., 1977, Localization of the globin gene in the template active fraction of chromatin of Friend leukemia cells, Science 198:1166-1168.

250

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Watts, J. W., and Harris, H., 1959, Turnover of nucleic acids in a non-multiplying animal cell, Biochem. J. 72:147-157. Week, P. K., and Johnson, T. c., 1976, Nuclear-cytosol interactions that modulate RNA synthesis and transcript size of mouse brain nuclei, J. Neurochem. 27:1367-1374. Week, P. K., and Johnson, T. C., 1978, Nuclear-cytosol interactions that facilitate release of RNA from mouse brain nuclei, J. Neurochem. 30:1057-1065. Weinberg, R., and Penman, S., 1968, Small molecular weight monodisperse nuclear RNA, J. Mol. Bioi. 36:289-304. Weintraub, H., and Groudine, M., 1976, Chromosomal subunits in active genes have an altered conformation, Science 193:848- 856. Wilkers, P. R., Birnie, G. D., and Old, R. W., 1978, Histone gene expression during the cell cycle studied by in situ hybridization, Exp. Cell Res. 115:441-444. Wilkers, P. R., Birnie, G. D., and Paul, J., 1979, Changes in nuclear and polysomal polyadenylated RNA sequences during rat-liver regeneration, Nucleic Acids Res. 6:2193-2208. Wold, B. J., Klein, W. H., Hough-Evans, B. R., Britten, R. 1., and Davidson, E. H., 1978, Sea urchin embryo mRNA sequences expressed in the nuclear RNA of adult tissues, Cell 14:941-950. Yannarell, A., Schumm, D., and Webb, T. E., 1976, Nature of the facilitated messenger ribonucleic acid transport from isolated nuclei, Biochem. J. 154:379-385. Young, B. D., Birnie, G. D., and Paul, J., 1976, Complexity and specificity of polysomal poly(A)+ RNA in mouse tissues, Biochemistry 15:2823-2829. Zaboykin, M. M., Sorokin, A. I., Lichtenstein, A. V., and Shapot, V. S., 1978, Post-transcriptional association of nuclear RNP particles with chromatin in Zajdela ascites hepatoma cells, Biokhimiya 43: 1266-1276. Zasloff, M., and Felsenfeld, G., 1977a, Analysis of in vitro transcription of duck reticulocyte chromatin using mercury-substituted ribonucleoside triphosphates, Biochemistry 16:51355145. Zasloff, M., and Felsenfeld, G., 1977b, Use of mercury-substituted ribonucleoside triphosphates can lead to artefacts in the analysis of in vitro chromatin transcripts, Biochem. Biophys. Res. Commun. 75:598-603. Zbarsky, I. B., 1972, Association of nuclear envelope with cytoplasmic structures and mechanisms of nuclear-cytoplasmic interrelationships, Usp. Sovrem. Bioi. 73:3-25. Zbarsky, I. B., 1978, An enzyme profile of the nuclear envelope, Int. Rev. Cytol. 54:295-360.

Chapter 5

Subcellular Mechanisms Involving Vitamin D Hector F. DeLuca Department of Biochemistry College of Agricultural and Life Sciences University of Wisconsin Madison, Wisconsin 53706

1. INTRODUCTION Vitamin D was originally discovered owing to its basic function in providing for the normal mineralization of bone (DeLuca, 1978, 1979; Haussler and McCain, 1977). The deficiency disease in young animals and children is known as rickets and in adults as osteomalacia. Both are characterized by a failure to mineralize the organic matrix of bone (DeLuca, 1967). Although there is considerable sentiment that some form of vitamin D might function directly to promote the transfer of minerals from plasma to the calcifying centers, so far clear evidence on this is not available. The major role of vitamin D in providing for mineralization of bone is the elevation of plasma calcium and phosphorus to supersaturating levels that are in turn required for the normal mineralization process (DeLuca, 1967, 1978, 1979; Haussler and McCain, 1977). To provide calcium and phosphorus in the plasma compartment, it has long been recognized that vitamin D facilitates or promotes intestinal absorption of calcium and phosphorus. Vitamin D is also involved in the mobilization of calcium from bone, and there is some evidence that vitamin D may playa role in the renal conservation of calcium (Sutton and Dirks, 1978; Gran, 1960). These activities are generally believed to be responsible for the elevation of plasma calcium and phosphorus concentrations. In addition to these well-established mechanisms, this review will illustrate possible new sites of vitamin D function heretofore unappreciated. It is also important to realize that the function of vitamin D in regulating mineral metabolism is of a basic nature, and disturbances in this 251

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system can result in a variety of pathological conditions, only two of which are osteomalacia and rickets. Further, the role of vitamin D in elevating plasma calcium concentration prevents muscle and nerve dysfunction known as hypocalcemic tetany. It is also possible that a provision of adequate mineral metabolism results in improved muscle strength and tone. Finally, because of its interaction with other hormones, a disturbance in the vitamin D system may also cause other types of metabolic bone disease or contribute to diseases such as certain types of bone-thinning syndromes known as osteoporosis. To facilitate the intestinal absorption of calcium and phosphorus, the mobilization of calcium and phosphorus from bone, and possibly renal reabsorption of calcium, vitamin D must be metabolically activated (DeLuca, 1967, 1978, 1979; DeLuca and Schnoes, 1976; Haussler and McCain, 1977). As a result of intensive investigation, it is now known that vitamin D must first be 25-hydroxylated primarily in the liver to yield the major circulating form of vitamin D, 25-hydroxyvitamin D3 (25-0H-D3) (DeLuca, 1967, 1978, 1979; Haussler and McCain, 1977). This compound apparently does not act directly in any target tissue, but must be transported to the kidney where it is further activated to form 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. 1,25-(OH)2D3 is believed to be the hormonal form of the vitamin and is transported to the intestine, bone, and perhaps elsewhere in the kidney where it exerts its actions on the regulation of calcium and phosphorus transport reactions (DeLuca, 1967, 1978, 1979; Haussler and McCain, 1977). To complete the picture of the activation of vitamin D, it must also be recognized that vitamin D ordinarily is not required in the diet since it can be manufactured in the skin. In recent years it has been clearly demonstrated that 7-dehydrocholesterol, found in abundant quantities in the epidermis, is photolytically converted to a compound known as previtamin D3 (Esvelt et ai., 1978; Holick et ai., 1979). This process is believed to be strictly a chemical photolytic process not involving proteins or enzymes. The previtamin D3 then slowly equilibrates to form vitamin D3 that is in turn transported to the liver where it begins its metabolic activation sequence. The functional metabolism of vitamin D as far as is conclusively known at the present time by physiologic experiments is illustrated in Figure 1. In addition to these metabolic alterations, vitamin D is subject to other metabolic pathways, the function of which remains unknown. The total known metabolic conversions of vitamin D that have been elucidated to date are illustrated in Figure 2 (DeLuca, 1967, 1978, 1979; DeLuca and Schnoes, 1976; Haussler and McCain, 1977), which includes not only the functional metabolism of vitamin D but also the other metabolic sequences whose physiologic function remains unknown. All metabolism that is currently known apparently stems from the 25-hydroxylated form of vitamin D, although vitamin D esters are known to be formed (Fraser and Kodicek, 1966). 25-0H-D 3 can then be

Vitamin D

253

HO

7-DEHYDROCHOLESTEROL

VITAMIN 0,

Ｌｾｏｈ＠ KIDNEY

ｾＯＭ

6:

..

HO\\' 25-0H-D,

ＢＧｾ＠

II

./

MITOCHONDRiA

CH. OH

I. 25 - ( OH I. 0,

BONE K lONE Y

FIGURE 1. Metabolism of vitamin D required for function.

converted either to 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] (Holick et al., 1972) or to 25,26-dihydroxyvitamin D3 [25,26-(OH)2D3] (Suda et al., 1970). 24,25-(OH)2D3 can be converted to a C-24 carboxylic acid (DeLuca and Schnoes, 1979) or can be I-hydroxylated to form 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3] (Holick et al., 1973). 1,24,25-(OH)3D3 can also be formed from the 24-hydroxylation of 1,25-(OH)2D3 (Tanaka et al., 1977). 1,25-(OH)2D3' the hormonal form of the vitamin, is rapidly converted to a C23 carboxylic acid, calcitroic acid (Esvelt et al.. 1979). This is a major biliary excretory form of the active hormone derived from vitamin D (Onisko et al., 1980). The major route of excretion of the vitamin D compounds is biliary (DeLuca, 1967, 1978, 1979; Haussler and McCain, 1977; LeVan et al., 1981); approximately 4% of the total metabolites are excreted in the urine (Avioli et al., 1967; Norman and DeLuca, 1963). Much remains to be learned concerning the further metabolism of vitamin D, and considerable argument is still raging regarding the physiologic significance of such compounds as 24,25(OH)2D3 (DeLuca, 1979). Due to the magnitude of interest in 24,25-(OH)2D3' its significance will be discussed. This metabolite is found in concentrations second only to 25-0H-

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Hector F. DeLuca

25-0H-0 3-26,23-loctone OH eooH

OH

..

..

..

HO'\

HO'"

25-0H- 03

°3

eOOH

ｾ＠

OH

Ｏｾｏｈ＠

-

.. OH

OH calcitrOIC

FIGURE 2.

OH

acid

1,25-(OH)z03

1,24,25- (OH)3 03

Current understanding of the metabolism of vitamin D.

0 3 in the plasma of man and animals (Gray et al., 1974; Horst et al., 1979; Shepard et al., 1979). In the chicken its biological activity is low (Boris et al., 1977; Holick et al., 1976), whereas in mammals it is almost equal to its precursor 25-0H-0 3 (Tanaka et aI., 1976a). In the bird, however, its biological activity is less than one tenth that of its precursor (Boris et al., 1977; Holick et al., 1976). Its presence has prompted investigators to consider it to have special roles not yet appreciated for vitamin O. Thus, it has been suggested that 24,25-(OH)203 has a special role in the mineralization of bone (Ornoy et aI., 1978; Rasmussen and Bordier, 1978), in embryonic development (Henry and Norman, 1978), in the secretion of parathyroid hormone (Henry et al., 1977), and in the metabolism of cartilage to form chondromucoproteins (Garabedian et al., 1978a). To support these contentions, complicated experiments have been devised that are difficult to interpret. In the case of cartilage metabolism, these changes are found primarily in culture systems, and it is not known whether 24,25-(OH)203 plays a special role in vivo in the development and maturation of cartilage matrix in preparation for mineralization. The recent

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synthesis of 24,24-difluoro-25-0H-D J has provided an important new tool to examine the significance of 24-hydroxylation in the functioning of vitamin D (Kobayashi et af., 1979; Yamada et af., 1979). This difluoro compound is not converted to the 24-hydroxylated form of 25-0H-D J but retains full biological activity in the prevention of rickets in rats, in the healing of rickets and the mineralization of bone in rats and chicks, and in provision for intestinal calcium absorption and the mobilization of calcium from bone (Okamoto et af., 1981; Tanaka et al., 1979a; unpublished results). In short, in the known functions of vitamin D, 24-hydroxylation does not appear to play an obligatory role. However, work is continuing with this and other difluoro compounds to examine the question further. Thus far, convincing evidence of a special role for 24,25-(OH)2DJ has not yet been provided, and its significance in the metabolism, excretion, or function of vitamin D remains unknown. Furthermore, no specific receptor for 24,25-(OH)2DJ has been found despite efforts by the leading laboratories. Of special importance is the fact that the production of the vitamin D hormone, 1,25-(OH)2DJ, is strongly feedback regulated either directly or indirectly by plasma calcium concentration (DeLuca, 1967, 1978, 1979; Haussler and McCain, 1977; Boyle et al., 1971) and by plasma phosphate concentration (Huges et al., 1975; Tanaka and DeLuca, 1973) in true endocrine fashion. It is believed that the plasma calcium concentration, when below normal, stimulates secretion of the parathyroid hormone and that this hormone in turn stimulates production of 1,25-(OH)2DJ in the kidney (Fraser and Kodicek, 1973; Garabedian et aI., 1972). It is not known how the low plasma phosphate concentration increases the circulating level of 1,25-(OH)2DJ, although there is some evidence that very low plasma phosphorus concentration stimulates 25OH-D-l-hydroxylase activity. Thus, this provides a true endocrine system in which low blood calcium or low blood phosphorus stimulates production of 1,25-(OH)2DJ, a very potent calcium and phosphorus mobilizing hormone. In this mobilization, 1,25-(OH)2DJ is transported to the intestine, bone, and kidney, where it stimulates the intestine to actively transport calcium and phosphorus from the lumen of the intestine to the plasma, and to the kidney, where it stimulates the active transport of calcium in the distal tubule, and to the bone membrane, where it stimulates the transfer of calcium and phosphorus to the extracellular fluid compartment. This is then an integrated endocrine system for the regulation of plasma calcium and phosphorus (DeLuca, 1967, 1978, 1979; Haussler and McCain, 1977). In addition to these factors, under conditions in which there are great demands for calcium such as in pregnancy and lactation (Halloran et aI., 1979; Pike et aI., 1979) or in eggshell formation (Castillo et aI., 1979; Kenny, 1979), there is a stimulation of the 25-0H-D-1-hydroxylase in the kidney and in the case of pregnant animals in the placenta (Tanaka et al., 1979b) to produce

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1,25-(OH)2DJ. There is some evidence that hormones such as prolactin (Spanos et af., 1976) and growth hormone (Spanos et af., 1978a) may be involved directly or indirectly in this stimulation. In the case of egg-laying birds, the sex hormones estrogen, progesterone, and testosterone function in a synergistic manner (probably indirectly) to stimulate 25-0H-D-l-hydroxylase to provide the 1,25-(OH)2DJ required to mobilize calcium for eggshell formation (Castillo et af., 1979; Tanaka et af., 1978). The vitamin D endocrine system, therefore, is a central one that is involved in the provision of calcium and phosphorus when required either for special functions, for mineralization of bone, or for the function of the neuromuscular junction. The remainder of this review will be devoted to the molecular aspects of the vitamin D endocrine system.

2.

SUBCELLULAR ASPECTS OF FUNCTIONAL VITAMIN D METABOLISM

2.1. Vitamin D-25-Hydroxylase This system of enzymes is found in liver, intestine, and kidney (Bhattacharyya and DeLuca, 1974a; Tucker et af., 1973), its concentration being low in the latter two organs. As vitamin D J accumulates in the liver following administration or production, hepatic conversion probably is the major system involved in converting vitamin D to its major circulating from, 25-0H-D J (Olson et af., 1976). In the liver there are two 25-hydroxylases known; one is a mitochondrial system and the other a microsomal system. Early in the study of vitamin D metabolism, it was not unequivocally known if vitamin D-25hydroxylase was located in the microsomes or in the mitochondria (Bhattacharyya and DeLuca, 1974b; Horsting and DeLuca, 1969). Subsequent experiments have shown it to be located in both sites. The microsomal system, however, has a low Michaelis constant for vitamin DJ of the order of 10- 8 M (Madhok and DeLuca, 1979), whereas the mitochondrial system will hydroxylate vitamin D J with a Km of about 10- 6 M (Bjorkhem and Holmberg, 1978). The mitochondrial 25-hydroxylase is not specific for vitamin D and appears to hydroxylate cholesterol either in the 26 position or in the 25 position. Nevertheless, this system may operate when vitamin D is present in large amounts. This enzyme system requires succinate and molecular oxygen for activity (Bjorkhem and Holmberg, 1978). It has been solubilized and shown to be a three-component mixed-function monooxygenase involving NADPH, a flavoprotein, an iron-sulfur protein, and a cytochrome P-450 (Pedersen et aI., 1979a,b). The major physiologic 25-hydroxylation, however, is likely carried out by the microsomal system (Madhok and DeLuca, 1979; Bhattacharyya and DeLuca, 1973a). This system requires a cytoplasmic factor, molecular

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oxygen, and magnesium ions. It is inhibited by cytochrome P-450 inhibitors and has recently been solubilized and reconstructed (Yoon and DeLuca, 1980a). This system involves at least two components, a flavoprotein and a cytochrome P-450. The activity of the microsomal system can be suppressed by the administration of vitamin D to vitamin D-deficient animals (Bhattacharyya and DeLuca, 1973b; Madhok and DeLuca, 1979). Whether this suppression has physiologic significance remains uncertain, since the microsomal system can be supplemented by the mitochondrial system when vitamin D is present in larger amounts, thereby eliminating the significance of the feedback control mechanism.

2.2. 25-0H-D-I-Hydroxylase In the nonpregnant animal, 25-0H-D-l-hydroxylase is located exlusively in the kidney, as nephrectomized animals cannot convert radiolabeled 25-0HD J to 1,25-(OHhDJ (Fraser and Kodicek, 1970; Gray et al., 1971). The pregnant female, however, produces 1,25-(OH)2DJ from 25-0H-D J even when nephrectomized (Weisman et aI., 1978). This led to a clear demonstration of the existence of a 25-0H-D-l-hydroxylase in the placenta (Tanaka et aI., 1979b). The 25-0H-D-l-hydroxylase is the key enzyme that is turned on and off by the need for calcium and phosphorus or by the hormones involved in special calcium- and phosphorus-requiring reactions as for example in eggshell formation or in lactation. This enzyme is exclusively mitochondrial (Fraser and Kodicek, 1970; Gray et al., 1972), at least in the chicken. It is located in the proximal convoluted tubules (Brunette et al., 1978) and requires internally generated NADPH, molecular oxygen, and magnesium ions (Ghazarian and DeLuca, 1974). In intact mitochondria, oxidative phosphorylation is required for full activity, probably because of the necessity of energy-linked transhydrogenation of NADP by NADH (Ghazarian and DeLuca, 1974). It is inhibited by cytochrome P-450 inhibitors including carbon monoxide (Ghazarian and DeLuca, 1974). This system has been solubilized, reconstructed, and some of the components purified to homogeneity (Ghazarian et al., 1974; Pedersen et al., 1976; Yoon and DeLuca, 1980b). This system (Figure 3) is a three-component mixed-function monooxygenase in which NADPH reduces a flavoprotein (renal ferredoxin reductase), an iron-sulfur protein [renal ferredoxin; isolated in pure form by Yoon and DeLuca (1980b)], and the cytochrome P-450, which is the specific portion of the enzyme system that interacts with the substrate and molecular oxygen to produce 25-0H-D J. Of the components, only renal ferredoxin has been isolated in a homogeneous state (Yoon and DeLuca, 1980b). It is a 2-iron, 2-sulfur cluster protein having a molecular weight of 11,800 (Yoon and DeLuca, 1980b; Yoon et al., 1980). This component is apparently not regulated by calcium or the parathyroid hormone. It is the

Hector F. DeLuca

258 RENAL ..

NADPH

FP

.. FERREDOXIN

/ Cytochrome P450

*

ｾ＠

la,25-(OH)2D3

C

O· 2

H 2 0* 25-0H-D 3

FIGURE 3. Mechanism of 25-hydroxyvitamin OJ-la-hydroxylation by chick kidney mitochondria. Asterisks indicate the path of 180 into the 25-0H-D 3 molecule to form 1,25(OH)2D 3'

cytochrome P-450 portion of the system that appears regulated but exactly how this occurs is unknown. The regulation of this enzyme has been studied in cultures of kidney cells that have unfortunately contributed little information regarding the molecular or subcellular mechanism (Henry, 1979; Spanos et al., 1978b; Trechsel et al., 1979). The parathyroid-hormone-stimulated 25-0H-DI-hydroxylase may require insulin (Henry, 1980), but this effect is likely mediated by cyclic AMP (Horiuchi et al., 1977). If cyclic AMP is involved, one might expect a phosphorylation-dephosphorylation mechanism. However, this has not yet been studied. 25-0H-D-24-hydroxylase has been found in kidney (Knutson and DeLuca, 1974), intestine (Kumar et al., 1978), and cartilage cells (Garabedian et al., 1978b). Thus far, only the kidney 24-hydroxylase has been studied to any degree (Knutson and DeLuca, 1974). This enzyme in the kidney is found in the mitochondria, although it is uncertain whether it is found in the mitochondria of the same cells that contain 25-0H-D-l-hydroxylase. Under physiologic circumstances where 25-0H-D-l-hydroxylase is suppressed, 25-0H-D24-hydroxylase is increased. In vitamin D deficiency, the 24-hydroxylase is absent, and can be induced by some metabolites of vitamin D, the most active being 1,25-(OH)2D3 (Tanaka et al., 1975). The induction of this enzyme system has been shown both in vivo and in vitro in isolated kidney cells in culture (Juan and DeLuca, 1977). When 1,25-(OH)2D3 stimulates the 24-hydroxylase, it also suppresses the I-hydroxylase (Tanaka et al., 1975). This regulation appears to be mediated by a nuclear mechanism since it is blocked by inhibitors of transcription and protein synthesis (Colston et al., 1977). The mechanism whereby 1,25-(OH)2D3 suppresses the I-hydroxylase and stimulates or induces the 24-hydroxylase remains unknown. The 24-hydroxylase inserts a hydroxyl group in the R position (Tanaka et al., 1976a) and is found exclusively in the mitochondria in the kidney. The

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reaction appears to involve internally generated NADPH and molecular oxygen. 180 experiments support this reaction as being a mixed-function monooxygenase (Madhok et al., 1977); inhibitor studies indicate that carbon monoxide, when present as 90% of the atmosphere, will inhibit the reaction (Ghazarian, personal communication). It is likely to be a cytochrome P-450dependent reaction, although this work has not yet been conclusively demonstrated. It has been suggested that the hormone calcitonin may be a regulator of vitamin D metabolism, especially at the 25-0H-D-l-hydroxylase level. Injection of large amounts of calcitonin in vivo stimulates the conversion of 25-0HD to 1,25-(OH)2D (Galante et al., 1972). However, in parathyroidectomized animals, calcitonin has no such effect (Lorenc et al., 1977). It seems reasonable that calcitonin, when given in large amounts, produces a hypocalcemia that then results in a countersecretion of parathyroid hormone. Parathyroid hormone in turn would stimulate the 25-0H-D-l-hydroxylase. Thus, it is likely that calcitonin is not a regulator of vitamin D metabolism despite these early reports. Injection of prolactin has been reported to stimulate 25-0H-D-lhydroxylase in the bird within 1 hr after its administration (Spanos et al., 1976). Unfortunately, these experiments have not been reproduced in other laboratories, and thus their significance is unclear. However, hypophysectomy does decrease circulating plasma 1,25-(OH)2D levels, and injection of growth hormone to such animals does increase the conversion of 25-0H-D to 1,25(OH)2D (Pahuja and DeLuca, 1981). Furthermore, under conditions of reproduction and lactation wherein prolactin is secreted, 1,25-(OH)2D production is markedly increased (Halloran et al., 1979; Pike et al., 1979). However, patients with hyperprolactinemia do not have increased circulating levels of 1,25-(OH)2D3 (Kumar et al., 1980). It seems likely that some of the hypophyseal hormones may directly or indirectly regulate 1,25-(OH)2D3 production, but as yet the mechanism and the specific hormones are unknown. During the egg-laying cycle and accompanying ovulation, there is a marked stimulation of the 25-0H-D-l-hydroxylase and increased plasma levels of 1,25-(OH)2D3 (Castillo et al., 1979). These subside at the time calcium is mobilized for eggshell formation. Therefore, it is likely that the ovulation cycle in the female bird in some way regulates the 25-0H-D-l-hydroxylase. This stimulation appears likely to be related to the sex hormones. The injection of estradiol to mature male birds markedly stimulates the renal 25-0H-D-lhydroxylase (Baksi and Kenny, 1977; Tanaka et al., 1976b). In castrate male birds, this effect could not be found unless the animals were primed with a dose of testosterone (Castillo et al., 1977). Testosterone could be replaced by progesterone, and the three hormones given together provide maximal stimulation of the 25-0H-D-l-hydroxylase (Tanaka et al., 1978). Thus, in the egg-laying

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Hector F. DeLuca

bird, the sex hormones undoubtedly playa major role in stimulating the 25OH-D-l-hydroxylase. When added in culture to chick kidney cells, the sex hormones do not stimulate the I-hydroxylase, suggesting that the effect of the sex hormones is indirect (Henry, 1980; Horiuchi et al., 1977; Spanos et al., 1978b). In support of this, parathyroidectomized male castrate birds do not respond to testosterone and estradiol (Castillo, Tanaka, and DeLuca, unpublished results). It might be surmised, but is certainly not established, that the sex hormones in the bird may stimulate the I-hydroxylase by a process mediated by the parathyroid glands. It is evident that much work remains to be done regarding the endocrinology of regulation of the vitamin D system, and certainly a great deal of work remains to be done regarding the molecular mechanisms involved in regulation of the 25-0H-D-l-hydroxylase and the 25-0H-D-24-hydroxylase.

3. MOLECULAR MECHANISM OF ACTION OF 1,25-(OH)2D3 Since the only proven functional form of vitamin D is 1,25-(OH)2D3' this section will focus on its molecular mechanism of action. The synthesis of highspecific-activity tritiated 1,25-(OH)2D3 (160 Ci/mmol) (Napoli et al., 1980) has permitted experiments using frozen-section autoradiography, which have clearly demonstrated the localization of 1,25-(OH)2D3 in the nuclei of crypt and villus cells of the mucosa of both small and large intestine (Zile et al., 1978; Stumpf et al., 1979). This localization occurs within half an hour and is specific for 1,25-(OH)2D3' This then confirms subcellular distribution studies carried out prior to the advent to this tool (Chen and DeLuca, 1973; Lawson et al., 1969). In addition to the intestinal villus and crypt cells, specific nuclear localization of 1,25-(OH)2D3 by this technique can be demonstrated in distal kidney tubule cells (Stumpf et al., 1980), in the Malpighian layer of the epidermis of skin (Stumpf et al., 1979), in the parathyroid glands (Stumpf et aI., 1979), in the pancreas (Stumpf et aI., 1981), in the osteoblasts and chondrocytes of bone, in the podocytes of the kidney (Stumpf et al., 1980), in the endocrine cells of the stomach mucosa but not in the parietal cells (Stumpf et al., 1979), and in certain cells of the hypophysis (Stumpf et al., 1979). No such localization could be found in liver, spleen, smooth muscle of small intestine, skeletal muscle, proximal kidney convoluted cells, and bone marrow cells. Specific and strong localization is found especially in the small intestine, in the parathyroid glands, and in the bone cells. The other nuclear localizations are clearly present but perhaps not to as strong a degree. The nuclear localization would argue very strongly that 1,25-(OH)2D3 functions in these tissues by a nucleus-mediated process. It would be expected, therefore, that 1,25-(OH)2D3, being a steroid, functions in a manner similar to other steroid hormones (Jensen et aI., 1968).

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The application of RNA and protein synthesis inhibitors in vivo has been rather disappointing in illustrating the mechanism. There has been considerable controversy as to whether 1,25-(OH)2D/s action in the small intestine can be blocked in vivo by the administration of actinomycin D or cycloheximide (Bikle et al., 1978; Tanaka et al., 1971; Tsai et al., 1973). Unfortunately, in the experiments that failed to block the action of 1,25-(OH)2D3' evidence of total inhibition of mRNA production has not been provided. Therefore, the experiments are inconclusive. The experiments that show a block in vivo with large amounts and frequent dosing with actinomycin D are equally inconclusive since the animals show nonspecific effects of intoxication (Tsai et al., 1973). More recent experiments involving organ cultures are consistent with a nucleus-mediated mechanism of action of 1,25-(OH)2D3 not only in inducing intestinal calcium binding protein but also in stimulating intestinal calcium uptake or transport (Corradino, 1973; Franceschi and DeLuca, 1981a,b). As might be expected, if 1,25-(OH)2D3 functions in the intestine by a nucleus-mediated process, a specific receptor for 1,25-(OH)2D3 must exist. This was first demonstrated by Brumbaugh and Haussler (1973) and conclusively demonstrated by Kream et al. (1976, 1977a) in the small intestine of chicken and rat and also in fetal bones of these animals (Kream et al., 1977b). This protein is of the order of 70,000 daltons and has a sedimentation coefficient between 3.2 and 3.7 S. Recent work has demonstrated this protein to have a Kd with respect to 1,25-(OH)2D3 of 5 X 10- 11 M in rat and chicken intestine (Mellon and DeLuca, 1979) and rat and chicken bone (Mellon and DeLuca, 1980). Both association and dissociation rate constants have been determined, and their ratios give approximately the same Kd as has been determined by Scat chard plot analysis. Since the early experiments of Brumbaugh and Haussler and of Kream et al. and the frozen-section autoradiography experiments, receptors for 1,25-(OH)2D3 have been demonstrated in human intestine (Wecksler et al., 1979), chicken kidney (Simpson et al., 1980), mouse kidney (Colston and Feldman, 1979), rat bone (Kream et al., 1977b; Mellon and DeLuca, 1980; Chen et al., 1979), chicken parathyroid glands (Brumbaugh et al., 1975), and rat skin (Simpson and DeLuca, 1980). The existence of a receptor in the chicken pancreas has been reported (Christakos and Norman, 1979). Although a receptor is probably present in the tissue, the report is not convincing. The intestinal receptor from chicken has been purified to about 80,000fold (Pike and Haussler, 1979), although significant progress in purification of the receptor to homogeneity has not yet been made primarily because of the instability of the receptor (Mellon et al., 1980), its low concentration, and its tendency to aggregate under low-salt conditions (Franceschi and DeLuca, 1979). It has been reported recently that under conditions where the cytosolic receptor for estrogen can be demonstrated, no cytosolic receptor for 1,25(OH)2D3 can be found (Walters et al., 1980). These authors claim that the

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Hector F. DeLuca

1,25-(OH)2D3 receptor is located in the nucleus prior to ligand interaction. This conclusion is unwarranted, as artifactual association of the cytosolic receptor could easily have resulted, especially since the 1,25-(OH)2D3 receptor easily aggregates with other proteins (Franceschi and DeLuca, 1979). It is to be expected that the 1,25-(OH)2D3 receptor plus ligand will interact with nuclear chromatin, although this has not been satisfactorily demonstrated in in vitro systems despite attempts (Procsal et al., 1975; Brumbaugh and Haussler, 1974). Besides identifying the receptors in the various target tissues and characterizing their size and binding constants, little progress has been made in understanding the nature of the receptor and its interaction with the nucleus. It is to be expected that mRNA is formed in response to interaction of the 1,25-(OH)2D3 receptor plus ligand with the nucleus. mRNA for a calcium binding protein has been shown to be formed in intestine but not necessarily by a specific interaction of the 1,25-(OH)2D3 receptor plus ligand with the nucleus (Spencer et al., 1976). The nature of the calcium transport and phosphate transport proteins that are formed in response to 1,25-(OH)2D3 has not yet been determined. Only one protein known to be formed in response to 1,25(OH)2D3 has been characterized; this is a calcium binding protein of 28,000 daltons in the chicken and about 8000-12,000 daltons in mammalian srecies (Wasserman and Feher, 1977). It binds four atoms of calcium per mol of protein, and although it was originally believed to be formed in the goblet cells (Taylor and Wasserman, 1970), more recent work suggests it to be formed in the intestinal villous cells, consistent with the nuclear localization of 1,25(OH)2D3 (Morissey et al., 1978). The calcium binding protein is found in a variety of tissues, only some of which are dependent upon vitamin D (Taylor and Wasserman, 1970). It is vitamin D dependent in kidney (Hermsdorf and Bronner, 1975), but some question exists as to whether it is found in bone and other targets of 1,25-(OH)2D3 action (Christakos and Norman, 1978). Certainly, current kinetic evidence argues against the calcium binding protein as being the transport protein accounting for calcium transport in the small intestine in response to 1,25-(OH)2D3 (Spencer et al., 1978). Thus, the molecular mechanism whereby 1,25-(OH)2D3 initiates intestinal calcium transport remains largely unknown. The most favored model is illustrated in Figure 4 for chick small intestine. Here, 1,25-(OH)2D3 interacts with a cytosolic receptor that is translocated to the nucleus, resulting in transcription of specific mRNAs that code for calcium and phosphate transport proteins. It is believed that these proteins function at the brush border site to facilitate the transfer of these ions into the cell (Martin and DeLuca, 1969a; Schachter et aI., 1966). Exactly how these ions are transferred ｡｣ｲｶｾｳ＠ the cell to the basal-lateral membrane remains unknown, although it is possible that it is mediated by mitochondria or by vesicle formation. Sodium is involved in the expulsion of calcium at the basallateral membrane (Martin and DeLuca, 1969b). On the other hand, sodium is

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--+--------'------1r-- 1,25

FIGURE 4. Current hypothesis regarding the mechanism of action of 1,25-dihydroxyvitamin DJ in stimulating intestinal calcium transport. 1,25 indicates 1,25-dihydroxyvitamin D J • It interacts with the 3.7 S receptor and is transported into the nucleus, where a specific mRNA is transcribed that codes for the calcium transport protein or proteins. These substances are believed to act at the brush border membrane surface to facilitate the transfer of calcium into the intestinal cell. Sodium is required for the expulsion of calcium into the extracellular fluid compartment across the basal-lateral membrane.

involved in the initial uptake of phosphate at the brush border site (Taylor, 1974). Much remains to be learned regarding the molecular mechanism whereby vitamin D or 1,25-(OH)2D3 stimulates intestinal calcium and phosphate transport. Even less is known concerning the molecular mechanism whereby 1,25(OH)2D3 stimulates the transport of calcium across the bone fluid-plasma membrane barrier. The mobilization of calcium from bone in response to 1,25(OH)2D3 is blocked by prior administration of actinomycin D, consistent with a nucleus-mediated process (Tanaka and DeLuca, 1971). Virtually nothing is known concerning the gene products that are transcribed in response to 1,25(OH)2D3 in bone, although there has been a report that one of these proteins is the calcium binding protein (Christakos and Norman, 1978). Besides the receptor for 1,25-(OH)2D3' there have been reports of a so-

Hector F. DeLuca

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called receptor for 25-0H-D J (Haddad and Birge, 1975). In fact, homogenates of all tissues in the body reveal a cytosolic material that binds 25-0H-D J in preference to 1,25-(OH)2DJ. It has, however, been shown quite conclusively that this so-called receptor is nothing more than an artifactual association of the plasma transport protein with a protein believed to be actin of the cytosol to form a 6 S macromolecular substance that binds 25-0H-D J (Kream et al., 1979; Van Baelen et al., 1977). In cells carefully washed free of serum, no such protein can be detected. Furthermore, no one has yet provided evidence that 25-0H-D J functions directly in any target tissue, although this possibility certainly remains. Finally, it should be mentioned that a group of scientists believe that 1,25(OH)2DJ may function in the intestine at least in part by a nonnucleusmediated mechanism to facilitate intestinal calcium transport. To support this view, the failure of actinomycin D to prevent the calcium transport response to 1,25-(OH)2DJ has been put forward. Furthermore, changes in lipid composition of the brush border membrane in response to 1,25-(OH)2DJ have been reported (Goodman et al., 1972), and vesicles have been isolated from the brush border membranes of animals treated with vitamin D metabolites and shown to have increased permeability to calcium (Rasmussen et al., 1979). Whether any of these are direct actions of 1,25-(OH)2DJ or are mediated by the nuclear processes is not entirely clear. Of considerable interest is the fact that the intestinal calcium transport response to 1,25-(OH)2DJ is of a biphasic nature (Halloran and DeLuca, 1981). In the rat there is initially a very rapid (6 hr) response to a single dose of 1,25-(OH)2DJ that diminishes to almost baseline levels by 18 hr only to increase again to high levels at 24 hr. The 6-hr response can be reinduced upon the 24-hr response, suggesting two possible, independent mechanisms whereby 1,25-(OH)2DJ stimulates intestinal calcium transport. One may involve the response of existing villus cells to 1,25-(OH)2DJ, and the other may involve the programming of crypt cells as they migrate up the villus to the tips where they carry out calcium transport. It is evident that the molecular mechanism whereby 1,25-(OH)2DJ affects mineral metabolism is far from elucidated, and one can expect in the future significant advances in this area.

4.

SUMMARY

During the past 15 years a vitamin D endocrine system has been demonstrated in which vitamin D produced normally in the skin is activated first by conversion in the liver and subsequently in the kidney to a hormonal form, 1,25-(OH)2DJ. The production of the hormonal form of vitamin D J is regulated, and much has been learned regarding the molecular mechanism of the

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hydroxylations of vitamin D and regarding the physiologic regulators of the 25-0H-D-I-hydroxylase. Much remains to be learned regarding the mechanism whereby the I-hydroxylase is modulated. 1,25-(OH)2D3 appears to function in the target organs of bone, intestine, kidney, and elsewhere by a nucleusmediated process. Receptors for 1,25-(OHhD3 have been clearly demonstrated and characterized in crude form. How the receptor and ligand interact with the nucleus is not clear, nor are the gene products that result from this interaction known. One product, a calcium binding protein, is known but its role in calcium transport is in debate. Although much has been learned in the last decade and a half, much remains to be learned regarding the molecular mechanisms whereby vitamin D brings about its remarkable changes in mineral metabolism.

ACKNOWLEDGMENTS

Some of the work reported here was supported by National Institutes of Health Program Project Grant AM-14881 and the Harry Steenbock Research Fund of the Wisconsin Alumni Research Foundation.

5. REFERENCES Avioli, L. V., Lee, S. W., McDonald, J. E., Lund, J., and DeLuca, H. F., 1967, Metabolism of vitamin D,-'H in human subjects: Distribution in blood, bile, feces, and urine, J. Clin. Invest. 46:983-992. Baksi, S. N., and Kenny, A. D., 1977, Vitamin D, metabolism in immature Japanese quail: Effects of ovarian hormones, Endocrinology 101: 1216-1220. Bhattacharyya, M. H., and DeLuca, H. F., 1973a, Comparative studies on the 25-hydroxylation of vitamin D, and dihydrotachysterol" J. Bioi. Chern. 248:2974-2977. Bhattacharyya, M. H., and DeLuca, H. F., 1973b, The regulation of rat liver calciferol-25hydroxylase, J. Bioi. Chern. 248:2969-2973. Bhattacharyya, M. H., and DeLuca, H. F., 1974a, The regulation of calciferol-25-hydroxylase in the chick, Biochern. Biophys. Res. Cornrnun. 59:734-741. Bhattacharyya, M. H., and DeLuca, H. F., 1974b, Subcellular location of rat liver calciferol-25hydroxylase, Arch. Biochern. Biophys. 160:58-62. Bikle, D. D., Zolock, D. T., Morrissey, R. L., and Herman, R. H., 1978, Independence of 1,25dihydroxyvitamin D,-mediated calcium transport from de novo RNA and protein synthesis, J. Bioi. Chern. 253:484-488. Bjorkhem, I., and Holmberg, I., 1978, Assay and properties of a mitochondrial 25-hydroxylase active on vitamin D" J. Bioi. Chern. 253:842-849. Boris, A., Hurley, J. F., and Trmal, T., 1977, Relative activities of some metabolites and analogs of cholecalciferol in stimulation of tibia ash weight in chicks otherwise deprived of vitamin D, J. Nutr. 107:194-198.

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Boyle, I. T., Gray, R. W., and DeLuca, H. F., 1971, Regulation by calcium of in vivo synthesis of 1,25-dihydroxycholecalciferol and 21 ,25-dihydroxycholecalciferol, Proc. Natl. Acad. Sci. USA 68:2131-2134. Brumbaugh, P. F., and Haussler, M. R., 1973, la,25-Dihydroxyvitamin DJ receptor: Competitive binding of vitamin D analog, Life Sci. 13: 1737-1746. Brumbaugh, P. F., and Haussler, M. R., 1974, la,25-Dihydroxycholecalciferol receptors in intestine. II. Temperature-dependent transfer of the hormone to chromatin via a specific cytosol receptor, J. Bioi. Chem. 249:1258-1262. Brumbaugh, P. F., Hughes, M. R., and Haussler, M. R., 1975, Cytoplasmic and nuclear binding components for la,25-dihydroxyvitamin DJ in chick parathyroid glands, Proc. Natl. Acad. Sci. USA 72:4871-4875. Brunette, M. G., Chan, M., Ferriere, c., and Roberts, K. c., 1978, Site of 1,25-dihydroxyvitamin DJ synthesis in the kidney, Nature (London) 276:287-289. Castillo, L., Tanaka, Y., DeLuca, H. F., and Sunde, M. L., 1977, The stimulation of 25-hydroxyvitamin DJla-hydroxylase by estrogen, Arch. Biochem. Biophys. 179:211-217. Castillo, L., Tanaka, Y., Wineland, M. J., Jowsey, 1. 0., and DeLuca, H. F., 1979, Production of 1,25-dihydroxyvitamin DJ and formation of medullary bone in the egg-laying hen, Endocrinology 104: 1598-1601. Chen, T. C., and DeLuca, H. F., 1973, Receptors of 1,25-dihydroxycholecalcifero1 in rat intestine, J. Bioi. Chem. 248:4890-4895. Chen, T. L., Hirst, M. A., and Feldman, D., 1979, A receptor-like binding macromolecule for la,25-dihydroxycholecalciferol in cultured mouse bone cells, J. Bioi. Chem. 254:74917494. Christakos, S., and Norman, A. W., 1978, Vitamin DJ-induced calcium binding protein in bone tissue, Science 202:70-71. Christakos, S., and Norman, A. W., 1979, Studies on the mode of action of calciferol. XVIII. Evidence for a specific high affinity binding protein for 1,25-dihydroxyvitamin DJ in chick kidney and pancreas, Biochem. Biophys. Res. Commun. 89:56-63. Colston, K. W., and Feldman, D., 1979, Demonstration of a 1,25-dihydroxycholecalciferol cytoplasmic receptor-like binding in mouse kidney, J. Clin. Endocrinol. Metab. 49:798-800. Colston, K. W., Evans, I. M. A., Spelsberg, T. C., and MacIntyre, I., 1977, Feedback regulation of vitamin D metabolism by 1,25-dihydroxycholecalciferol, Biochem. J. 164:83-90. Corradino, R. A., 1973, 1,25-Djhydroxycholecalciferol: Inhibition of action in organ-cultured intestine by actinomycin D and a-amanitin, Nature (London) 243:41-43. DeLuca, H. F., 1967, Mechanism of action and metabolic fate of vitamin D, Vitam. Horm. (N.Y.) 25:315-367. DeLuca, H. F., 1978, Vitamin D, in: The Fat-Soluble Vitamins. Handbook of Lipid Research (H. F. DeLuca, ed.), pp. 69-132, Plenum Press, New York. DeLuca, H. F., 1979, The vitamin D system in the regulation of calcium and phosphorus metabolism. Nutr. Rev. 37:161-193. DeLuca, H. F., and Schnoes, H. K., 1976, Metabolism and mechanism of action of vitamin D, Annu. Rev. Biochem. 45:631-666. DeLuca, H. F., and Schnoes, H. K., 1979, Recent development in the metabolism of vitamin D, in: Vitamin D: Basic Research and Its Clinical Application (A. W. Norman, K. Schaefer, D. von Herrath, H. G. Grigoleit, J. W. Coburn, H. F. DeLuca, E. B. Mawer, and T. Suda, eds.), pp. 445-458, de Gruyter, Berlin. Esvelt, R. P., Schnoes, H. K., and DeLuca, H. F., 1978, Vitamin DJ from rat skins irradiated in vitro with ultraviolet light, Arch. Biochem. Biophys. 188:282-286. Esvelt, R. P., Schnoes, H. K., and DeLuca, H. F., 1979, Isolation and characterization of lahydroxy-tetranor-vitamin D-23-carboxylic acid: A major metabolite of 1,25-dihydroxyvitamin DJ, Biochemistry 18:3977-3983.

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Franceschi, R. T., and DeLuca, H. F., 1979, Aggregation properties of the 1,25-dihydroxyvitamin D J receptor from chick intestinal cytosol, J. Bioi. Chern. 254:11629-11635. Franceschi, R. T., and DeLuca, H. F., 1981a, Characterization of 1,25-dihydroxyvitamin D Jdependent calcium uptake in cultured embryonic chick duodenum, J. BioI. Chern. 256:3840-3847. Franceschi, R. T., and DeLuca, H. F., 1981 b, The effect of inhibitors of protein and RNA synthesis on la, 25-dihydroxyvitamin DJ-dependent calcium uptake in cultured embryonic chick duodenum, J. Bioi. Chern. 256:3848-3852. Fraser, D. R., and Kodicek, E., 1966, The synthesis of vitamin D esters in the rat, Biochern. J. 100:67P. Fraser, D. R., and Kodicek, E., 1970, Unique biosynthesis by kidney of a biologically active vitamin D metabolite, Nature (London) 228:764-766. Fraser, D. R., and Kodicek, E., 1973, Regulation of 25-hydroxycholecalciferol-I-hydroxylase activity in kidney by parathyroid hormone, Nature New Bioi. 241:163-166. Galante, L., Colston, K. W., MacAuley, S. J., and MacIntyre, I., 1972, Effect of calcitonin on vitamin D metabolism Nature (London) 238:271-273. Garabedian, M., Holick, M. F., DeLuca, H. F., and Boyle, I. T., 1972, Control of 25-hydroxycholecalciferol metabolism by the parathyroid glands, Proc. Natl. Acad. Sci. USA 69: 16731676. Garabedian, M., Lieberherr, M., Nguyen, T. M., Corvol, M. T., Dubois, M. B., and Balsan, S., 1978a, In vitro production and activity of 24,25-dihydroxycholecalciferol in cartilage and calvarium, Clin. Orthop. Relat. Res. 135:241-248. Garabedian, M., Dubois, M. B., Corvol, M. T., Pezant, E., and Balsan, S., 1978b, Vitamin D and cartilage. I. In vitro metabolism of 25-hydroxycholecalciferol by cartilage, Endocrinology 102: 1262-1268. Ghazarian, J. G., and DeLuca, H. F., 1974, 25-Hydroxycholecalciferol-l-hydroxylase: A specific requirement for NADPH and a hemoprotein component in chick kidney mitochondria,

Arch. Biochern. Biophys. 160:63-72. Ghazarian, J. G., Jefcoate, CR., Knutson, J. C, Orme-Johnson, W. H., and DeLuca, H. F., 1974, Mitochondrial cytochrome P 450 : A component of chick kidney 25-hydroxycholecalciferol-Ia-hydroxylase, J. Bioi. Chern. 249:3026-3033. Goodman, D. B. P., Haussler, M. R., and Rasmussen, H., 1972, Vitamin DJ induced alteration of microvillar membrane lipid composition, Biochern. Biophys. Res. Cornrnun. 46:8086. Gran, F. C, 1960, The retention of parenterally injected calcium in rachitic dogs, Acta Physiol.

Scand.50:132-139. Gray, R., Boyle, I., and DeLuca, H. F., 1971, Vitamin D metabolism: The role of kidney tissue, Science 172: 1232-1234. Gray, R. W., Omdahl, J. L., Ghazarian, J. G., and DeLuca, H. F., 1972, 25-Hydroxycholecalciferol-I-hydroxylase: Subcellular location and properties, J. Bioi. Chern. 247:7528-7532. Gray, R. W., Weber, H. P., Dominguez, J. H., and Lemann, J., Jr., 1974, The metabolism of vitamin D J and 25-hydroxyvitamin D J in normal and anephric humans, J. Clin. Endocrinol. Metab. 39: 1045-1 056. Haddad, J. G., and Birge, S. J., 1975, Widespread, specific binding of 25-hydroxycholecalciferol in rat tissues, J. Bioi. Chern. 250:299-303. Halloran, B. P., and DeLuca, H. F., 1981, Intestinal calcium transport: Evidence for two distinct mechanisms of action of 1,25-dihydroxyvitamin D J, Arch. Biochern. Biophys., 208:477-486. Halloran, B. P., Barthell, E. N., and DeLuca, H. F., 1979, Vitamin D metabolism during pregnancy and lactation in the rat, Proc. Nat!. Acad. Sci. USA 76:5549-5553. Haussler, M. R., and McCain, T. A., 1977, Vitamin D metabolism and action. Parts I and II, N. Engl. J. Med. 297:974-983,1041-1050.

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Henry, H. L., 1979, Regulation of the hydroxylation of 25-hydroxyvitamin DJ in vivo and in primary cultures of chick kidney cells, J. Bioi. Chem. 254:2722-2729. Henry, H. L., 1980, Insulin permits parathyroid hormone stimulation of 25·hydroxyvitamin DJ· I-hydroxylase in kidney cell cultures, Endocrine Society Abstract p. 80. Henry, H. L., and Norman, A. W., 1978, Vitamin D: Two dihydroxylated metabolites are required for normal chicken egg hatchability, Science 201:835-837. Henry, H. L., Taylor, A. N., and Norman, A. W., 1977, Response of chick parathyroid glands to the vitamin D metabolites 1,25-dihydroxyvitamin DJ and 24,25-dihydroxyvitamin DJ, J. Nutr. 107:1918-1926. Hermsdorf, C. L., and Bronner, F., 1975, Vitamin D-dependent calcium-binding protein from rat kidney, Biochim. Biophys. Acta 379:553-561. Holick, M. F., Schnoes, H. K., DeLuca, H. F., Gray, R. W., Boyle, I. T., and Suda, T., 1972, Isolation and identification of 24,25-dihydroxycholecalciferol: A metabolite of vitamin DJ made in the kidney, Biochemistry 11:4251-4255. Holick, M. F., Kleiner-Bossaller, A., Schnoes, H. K., Kasten, P. M., Boyle, I. T., and DeLuca, H. F., 1973, 1,24,25-Trihydroxyvitamin DJ: A metabolite of vitamin DJ effective on intestine, J. Bioi. Chem. 248:6691-6696. Holick, M. F., Baxter, L. A., Schraufrogel, P. K., Tavela, T. A., and DeLuca, H. F., 1976, Metabolism and biological activity of 24,25-dihydroxyvitamin DJ in the chick, J. Bioi. Chem. 251:397-402. Holick, M. F., Richtand, N. M., McNeill, S. c., Holick, S. A., Fromer, J. E., Henley, J. W., and Potts, J. T., Jr., 1979, Isolation and identification of previtamin DJ from the skin of rats exposed to ultraviolet irradiation, Biochemistry 18: 1003-1 008. Horiuchi, N., Suda, T., Takahashi, H., Shmiazawa. E., and Ogata, E., 1977, In vivo evidence for the intermediary role of 3',5'-cyclic AMP in parathyroid hormone-induced stimulation of la,25-dihydroxyvitamin D J synthesis in rats, Endocrinology 101:969-974. Horst, R. L., Shepard, R. M., Jorgensen, N. A., and DeLuca, H. F., 1979, The determination of 24,25-dihydroxyvitamin D and 25,26-dihydroxyvitamin D in plasma from normal and nephrectomized man, J. Lab. Clin. Med. 93:277-285. Horsting, M., and DeLuca, H. F., 1969, In vitro production of 25-hydroxycholecalciferol, Biochem. Biophys. Res. Commun. 36:251-256. Hughes, M. R., Brumbaugh, P. F., Haussler, M. R., Wergedal, J. E., and Baylink, D. J., 1975, Regulation of serum la,25-dihydroxyvitamin DJ by calcium and phosphate in the rat, Science 190:578-580. Jensen, E. V., Suzuki, T., Kawashima, T., Stumpf, W. E., Jungblut, P. W., and DeSombre, E. R., 1968, A two-step mechanism for the interaction of estradiol with rat uterus, Proc. Natl. Acad. Sci. USA 59:632-638. Juan, D., and DeLuca, H. F., 1977, The regulation of 24,25-dihydroxyvitamin DJ production in cultures of monkey kidney cells, Endocrinology lOt: 1184-1193. Kenny, A. D., 1976, Vitamin D metabolism: Physiological regulation in egg-laying Japanese quail, Am. J. Physiol. 230:1609-1615. Knutson, J. c., and DeLuca, H. F., 1974, 25-Hydroxyvitamin DJ-24-hydroxylase: Subcellular location and properties, Biochemistry 13: 1543-1548. Kobayashi, Y., Taguchi, T., Terada, T., Oshida, J., Morisaki, M., and Ikekawa, N., 1979, SynDJ, Tetrahedron Lett. 22:2023thesis of 24,24-difluoro- and ＲＴｾＭｦｬｵｯｲＵｨｹ､ｸｶｩｴ｡ｭｮ＠ 2026. Kream, B. E., Reynolds, R. D., Knutson, J. C., Eisman, J. A., and DeLuca, H. F., 1976, Intestinal cytosol binders of 1,25-dihydroxyvitamin D, and 25-hydroxyvitamin DJ, Arch. Biochem. Biophys. 176:779-787. Kream, B. E., Yamada, S., Schnoes, H. K., and DeLuca, H. F., 1977a, Specific cytosol binding protein for 1,25-dihydroxyvitamin D, in rat intestine, J. Bioi. Chem. 252:4501-4505.

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Kream, B. E., Jose, M., Yamada, S., and DeLuca, H. F., 1977b, A specific high-affinity binding macromolecule for 1,25-dihydroxyvitamin D J in fetal bone, Science 197:1086-1088. Kream, B. E., DeLuca, H. F., Moriarity, D. M., Kendrick, N. C., and Ghazarian, 1. G., 1979, Origin of 25-hyrdoxyvitamin D J binding protein from tissue cytosol preparations, Arch. Biochem. Biophys. 192:318-323. Kumar, R., Schnoes, H. K., and DeLuca, H. F., 1978, Rat intestinal 25-hydroxyvitamin D J- and 1a,25-dihydroxyvitamin D r 24-hydroxylase, J. Bioi. Chem. 253:3804-3809. Kumar, R., Abboud, C. F., and Riggs, B. L., 1980, The effect of elevated prolactin levels on plasma 1,25-dihydroxyvitamin D and intestinal absorption of calcium, Mayo Clin. Proc. 55:51-53. Lawson, D. E. M., Wilson, P. W., Barker, D. c., and Kodicek, E., 1969, Isolation of chick intestinal nuclei. Effect of vitamin D J on nuclear metabolism, Biochem. J. 115:263-268. LeVan, L. W., Schnoes, H. K., and DeLuca, H. F., 1981, Isolation and identification of 25hydroxyvitamin D,-25-glucuronide: A biliary metabolite of vitamin D, in the chick, Biochemistry 20:222-226. Lorenc, R., Tanka, Y., DeLuca, H. F., and Jones, G., 1977, Calcitonin and regulation of vitamin D metabolism, Endocrinology 100:468-472. Madhok, T. C., and DeLuca, H. F., 1979, Characteristics of the rat liver microsomal enzyme system converting cholecalciferol into 25-hydroxycholecalciferol. Evidence for the participation of cytochrome P-450, Biochem. J. 184:491-499. Madhok, T. c., Schnoes, H. K., and DeLuca, H. F., 1977, Mechanism of 25-hydroxyvitamin D J 24-hydroxylation: Incorporation of oxygen-18 into the 24 position of 25-hydroxyvitamin D J, Biochemistry 16:2142-2145. Martin, D. L., and DeLuca, H. F., 1969a, Calcium transport and the role of vitamin D, Arch. Biochem. Biophys. 134:139-148. Martin, D. L., and DeLuca, H. F., 1969b, Influence of sodium on calcium transport by the rat small intestine, Am. J. Physio/' 216:1351-1359. Mellon, W. S., and DeLuca, H. F., 1979, An equilibrium and kinetic study of 1,25-dihydroxyvitamin D J binding to chicken intestinal cytosol employing high specific activity 1,25-dihydroxy[JH-26,27] vitamin D J, Arch. Biochem. Biophys. 197:90-95. Mellon, W. S., and DeLuca, H. F., 1980, A specific 1,25-dihydroxyvitamin D J binding macromolecule in chicken bone, J. Bioi. Chern. 255:4081-4086. Mellon, W. S., Franceschi, R. T., and DeLuca, H. F., 1980, An in vitro study of the stability of the chicken intestinal cytosol 1,25-dihydroxyvitamin DJ-specific receptor, Arch. Biochem. Biophys. 202:83-92. Morrissey, R. L., Zolock, D. T., Bikle, D. D., Empson, R. N., Jr., and Bucci, T. J., 1978, Intestinal response to la,25-dihydroxycholecalciferol. II. A timed study of the intracellular localization of calcium binding protein, Biochim. Biophys. Acta 538:34-42. Napoli, J. L., Mellon, W. S., Fivizzani, M. A., Schnoes, H. K., and DeLuca, H. F., 1980, Direct chemical synthesis of 1a,25-dihydroxy[26,27-JH]-vitamin DJ with high specific activity: Its use in receptor studies, Biochemistry 19:2515-2521. Norman, A. W., and DeLuca, H. F., 1963, The preparation of HJ-vitamins D, and DJ and their localization in the rat, Biochemistry 2: 1160-1168. Okamoto, S., Tanaka, Y., DeLuca, H. F., Yamada, S., and Takayama, H., 1981, 24,24-Difluoro25-hydroxyvitamin Drenhanced bone mineralization in rats: Comparison with 25-hydroxyvitamin DJ and vitamin D J, Arch. Biochem. Biophys. 206:8-14. Olson, E. 8., Jr., Knutson, J. C., Bhattacharyya, M. H., and DeLuca, H. F., 1976, The effect of hepatectomy on the synthesis of 25-hydroxyvitamin D J, J. Clin. Invest. 57:12131220. Onisko, B. L., Esvelt, R. P., Schnoes, H. K., and DeLuca, H. F., 1980, Metabolites of la,25dihydroxyvitamin D J in rat bile, Biochemistry 19:4124-4130.

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Ornoy, A., Goodwin, D., Noff, D., and Edelstein, S., 1978, 24,25-Dihydroxyvitamin D is a metabolite of vitamin D essential for bone formation, Nature (London) 276:517-519. Pahuja, D. N., and DeLuca, H. F., 1981, Role of the hypophysis in the regulation of vitamin D metabolism, Mol. Cell. Endocrinol. (in press). Pedersen, 1. I., Ghazarian, 1. G., Orme-lohnson, N. R., and DeLuca, H. F., 1976, Isolation of chick renal mitochondrial ferredoxin active in the 25-hydroxyvitamin Dr 1a-hydroxylase system, J. Bioi. Chern. 251:3933-3941. Pedersen, 1. I., Bjorkhem, I., and Gustafsson, 1., 1979a, 26-Hydroxylation of C,,-steroids by soluble liver mitochondrial cytochrome P-450, J. Bioi. Chern. 254:6464-6469. Pedersen, 1. I., Holmberg, I., and Bjorkhem, I., 1979b, Reconstitution of vitamin DJ 25-hydroxylase activity with a cytochrome P-450 preparation from rat liver mitochondria, FEBS Lett. 98:394-398. Pike, 1. W., and Haussler, M. R., 1979, Purification of chicken intestinal receptor for 1,25-di,hydroxyvitamin D J, Proc. Natl. Acad. Sci. USA 76:5485-5489. Pike, 1. W., Parker, 1. B., Haussler, M. R., Boass, A., and Toverud, S. U., 1979, Dynamic changes in circulating 1,25-dihydroxyvitamin D during reproduction in rats, Science 204:1427-1429. Procsal, D. A., Okamura, W. H., and Norman, A. W., 1975, Structural requirements for the interaction of 1a,25-(OH),-vitamin DJ with its chick intestinal receptor system, J. Bioi. Chern. 250:8382-8388. Rasmussen, H., and Bordier, P., 1978, Vitamin D and bone, Metab. Bone Dis. Relat. Res. 1:7-

13. Rasmussen, H., Fontaine, 0., Max, E., and Goodman, D. B. P., 1979, The effect of 1a-hydroxyvitamin D J administration on calcium transport in chick intestine brush border membrane vesicles, J. Bioi. Chern. 254:2993-2999. Schachter, D., Kowarski, S., Finkelstein, 1. D., and Wang Ma, R., 1966, Tissue concentration differences during active transport of calcium by intestine, Am. J. Physiol. 211: 1131-1136. Shepard, R. M., Horst, R. L., Hamstra, A. J., and DeLuca, H. F., 1979, Determination of vitamin D and its metabolites in plasma from normal and anephric man, Biochern. J. 182:5569. Simpson, R. u., and DeLuca, H. F., 1980, Characterization of a receptor-like protein for 1,25dihydroxyvitamin D J in rat skin, Proc. Natl. Acad. Sci. USA 77:5822-5826. Simpson, R. U., Franceschi, R. T., and DeLuca, H. F., 1980, Characterization of a specific, high affinity binding macromolecule for la,25-dihydroxyvitamin D J in cultured chick kidney cells, J. Bioi. Chern. 255:10160-10166. Spanos, E., Colston, K. W., Evans, I. M. A., Galante, L. S., MacAuley, S. 1., and Macintyre, I., 1976, Effect of prolactin on vitamin D metabolism, Mol. Cell. Endocrinol. 5:163-167. Spanos, E., Barrett, D., Macintyre, I., Pike, 1. W., Safilian, E. F., and Haussler, M. R., 1978a, Effect of growth hormone on vitamin D metabolism, Nature (London) 273:246-247. Spanos, E., Barrett, D. I., Chong, K. T., and Macintyre, I., 1978b, Effect of oestrogen and 1,25dihydroxycholecalciferolon 25-hydroxycholecalciferol metabolism in primary chick kidneycell cultures, Biochern. J. 174:231-236. Spencer, R., Charman, M., Emtage, 1. S., and Lawson, D. E. M., 1976, Production and properties of vitamin D-induced mRNA for chick calcium-binding protein, Eur. J. Biochern. 71:399-409. Spencer, R., Charman, M., Wilson, P. W., and Lawson, D. E. M., 1978, The relationship between vitamin D-stimulated calcium transport and intestinal calcium-binding protein in the chicken, Biochern. J. 170:93-102. Stumpf, W. E., Sar, M., Reid, F. A., Tanaka, Y., and DeLuca, H. F., 1979, Target cells for 1,25-dihydroxyvitamin D J in intestinal tract, stomach, kidney, skin, pituitary and parathyroid, Science 206: 1188-1190.

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Stumpf, W. E., Sar, M., Narbaitz, R., Reid, F. A., DeLuca, H. F., and Tanaka,Y., 1980, Cellular and subcellular localization of 1,25-(OH),-vitamin D J in rat kidney: Comparison with localization of parathyroid hormone and estradiol, Proc. Natl. Acad. Sci. USA 77:11491153. Stumpf, W. E., and Sar, M., and DeLuca, H. F., 1981, Sites of action of 1,25-(OH), Vitamin D J identified by thaw mount autoradiography, in: Hormonal Control of Calcium Metabolism. Proceedings of the VII Parathyroid Conference, (D. V. Cohn, R. V. Talmage, and J. L. Matthews, eds.), pp. 222-229, Excerpta Medica, Amsterdam. Suda, T., DeLuca, H. F., Schnoes, H. K., Tanaka, Y., and Holick, M. F., 1970, 25,26-Dihydroxycholecalciferol, a metabolite of vitamin D J with intestinal calcium transport activity,

Biochemistry 9:4776-4780. Sutton, R. A. L., and Dirks, 1. H., 1978, Renal handling of calcium, Fed. Proc. 37:2112-2119. Tanaka, Y., and DeLuca, H. F., 1971, Bone mineral mobilization activity of 1,25-dihydroxycholecalciferol, a metabolite of vitamin D, Arch. Biochem. Biophys. 146:574-578. Tanaka, Y., and DeLuca, H. F., 1973, The control of 25-hydroxyvitamin D metabolism by inorganic phosphorus, Arch. Biochem. Biophys. 154:566-574. Tanaka, Y., DeLuca, H. F., Omdahl, J., and Holick, M. F., 1971, Mechanism of action of 1,25dihydroxycholecalciferol on intestinal calcium transport, Proc. Natl. Acad. Sci. USA 68:1286-1288. Tanaka, Y., Lorenc, R. S., and DeLuca, H. F., 1975, The role of 1,25-dihydroxyvitamin D J and parathyroid hormone in the regulation of chick renal 25-hydroxyvitamin DJ-24-hydroxylase,

Arch. Biochem. Biophys. 171:521-526. Tanaka, Y., DeLuca, H. F., Akaiwa, A., Morisaki, M., and Ikekawa, N., 1976a, Synthesis of 24S and 24R-hydroxy-[ 24-JHJ vitamin D J and their metabolism in rachitic rats, Arch.

Biochem. Biophys. 177:615-621. Tanaka, Y., Castillo, L., and DeLuca, H. F., 1976b, Control of the renal vitamin D hydroxylases in birds by the sex hormones, Proc. Natl. Acad. Sci. USA 73:2701-2705. Tanaka, Y., Castillo, L., DeLuca, H. F., and Ikekawa, N., 1977, The 24-hydroxylation of 1,25dihydroxyvitamin D J, J. Bioi. Chem. 252:1421-1424. Tanaka, Y., Castillo, L., Wineland, M. J., and DeLuca, H. F., 1978, Synergistic effect of progesterone, testosterone, and estradiol in the stimulation of chick renal 25-hydroxyvitamin D,I a-hydroxylase, Endocrinology 103:2035-2039. Tanaka, Y., DeLuca, H. F., Kobayashi, Y., Taguchi, T., Ikekawa, N., and Morisaki, M., 1979a, Biological activity of 24,24-difluoro-25-hydroxyvitamin DJ. Effect of blocking of 24-hydroxylation on the functions of vitamin D, J. Bioi. Chem. 254:7163-7167. Tanaka, Y., Halloran, B., Schnoes, H. K., and DeLuca, H. F., 1979b, In vitro production of 1,25-dihydroxyvitamin D J by rat placental tissue, Proc. Natl. Acad. Sci. USA 76:50335035. Taylor, A. N., 1974, In vitro phosphate transport in chick ileum: Effect of cholecalciferol, calcium, sodium and metabolic inhibitors, J. Nutr. 104:489-494. Taylor, A. N., and Wasserman, R. H., 1970, Immunofluroescent localization of vitamin Ddependent calcium-binding protein, J. Histochem. Cytochem. 18: I 07-115. Trechsel, U., Bonjour, J.-P., and Fleisch, H., 1979, Regulation of the metabolism of 25-hydroxyvitamin DJ in primary cultures of chick kidney cells, J. Clin. Invest. 64:206-217. Tsai, H. C., Midgett, R. J., and Norman, A. W., 1973, Studies on calciferol metabolism. VII. The effects of actinomycin D and cycloheximide on the metabolism, tissue and subcellular localization, and action of vitamin DJ, Arch. Biochem. Biophys. 157:339-347. Tucker, G., Ill, Gagnon, R. E., and Haussler, M. R., 1973, Vitamin D J-25-hydroxylase: Tissue occurrence and apparent lack of regulation, Arch. Biochem. Biophys. 155:47-57. Van Baelen, H., Bouillon, R., and DeMoor, P., 1977, Binding of 25-hydroxycholecalciferol in tissues, J. Bioi. Chem. 252:2515-2518.

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Walters, M. R., Hunziker, W., and Norman, A. W., 1980, Unoccupied 1,25-dihydroxyvitamin D3 receptors. Nuclear j cytosol ratio depends on ionic strength, J. Bioi. Chem. 255:67996805. Wasserman, R. H., and Feher, J. J., 1977, Vitamin D-dependent calcium-binding proteins, in: Calcium Binding Proteins and Calcium Function (R. H. Wasserman, R. A. Corradino, E. Carafoli, R. H. Kretsinger, D. H. MacLennan, and S. L. Siegel, eds.), pp. 292-302, Elsevier jNorth-Holland, Amsterdam. Wecksler, W. R., Mason, R. S., and Norman, A. W., 1979, Specific cytosol receptors for 1,25dihydroxyvitamin D3 in human intestine, J. C/in. Endocrinol. M etab. 48:715-717. Weisman, Y., Vargas, A., Duckett, G., Reiter, E., and Root, A., 1978, Synthesis of 1,25-dihydroxyvitamin D in the nephrectomized pregnant rat, Endocrinology 103:1992-1998. Yamada, S., Ohmori, M., and Takayama, H., 1979, Synthesis of 24,24-difluoro-25-hydroxyvitamin D 3, Tetrahedron Lett. 21:1859-1862. Yoon, P. S., and DeLuca, H. F., 1980a, Resolution and reconstitution of soluble components of rat liver microsomal vitamin D3 25-hydroxylase, Arch. Biochem. Biophys. 203:529-541. Yoon, P. S., and DeLuca, H. F., 1980b, Purification and properties of chick renal mitochondrial ferredoxin, Biochemistry 19:2165-2171. Yoon, P. S., Rawlings, J., Orme-Johnson, W. H., and DeLuca, H. F., 1980, Renal mitochondrial ferredoxin active in 25-hydroxyvitamin D3 la-hydroxylase. Characterization of the ironsulfur cluster using interprotein cluster transfer and electron paramagnetic resonance spectroscopy, Biochemistry 19:2172-2176. Zile, M., Bunge, E. c., Barsness, L., Yamada, S., Schnoes, H. K., and DeLuca, H. F., 1978, Localization of 1,25-dihydroxyvitamin D3 in intestinal nuclei in vivo, Arch. Biochem. Biophys.186:15-24.

Chapter 6

Macromolecular Organization of the Nicotinic Acetylcholine Receptors S. K. Malhotra Biological Sciences Electron Microscopy Laboratory University of Alberta Edmonton T6G 2E9, Canada

1.

INTRODUCTION

Our knowledge of the organization of cellular membranes has advanced rapidly during the last decade in particular. Current investigations are aimed at the molecular organization of the membranes themselves and their interactions with other cellular and external components that regulate membrane-related functions. One of the widely investigated systems is that concerned with the receptor for the neurotransmitter acetylcholine (Ach). The Ach receptors are being investigated by neurobiologists, biochemists, pharmacologists, immunologists, and cell and developmental biologists. Several comprehensive reviews have been published in recent years that reflect the current emphasis on the Ach receptors (e.g., The Synapse, Cold Spring Harbor Symposia on Quantitative Biology 40, 1976; Advances in Cytopharmacology 3, 1979; Edwards, 1979; Fambrough, 1979; Frank, 1979; Hider, 1979; Robertson, 1979; Weeds, 1979). The neuromuscular junctional sarcolemma of vertebrate skeletal muscle, where the Ach receptors are confined and concentrated, has been the favorite model of neurobiologists for investigating the molecular events in nervemuscle interactions. The electroplaques [homologous to the neuromuscular junction in vertebrate skeletal muscle (Nachmansohn, 1955)] of certain electric fish, Torpedo and Electrophorus in particular, provide a rich source for isolation and biochemical characterization of the Ach receptors (see reviews by Heidmann and Changeux, 1978; Hider, 1979). 273

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The well-known function of the Ach receptor is to transduce the binding of the transmitter Ach so that a change in the cationic permeability of the excitable membranes takes place (see Karlin et al., 1979). Apart from this, the Ach receptors may serve a regulatory role in the growth and maintenance of the presynaptic terminal in the retinal tectum (Freeman, 1977). Also, in the autoimmune disease characterized as myasthenia gravis in humans, the receptors have been reported to be degraded by antibodies against Ach receptors (Vincent, 1976; Kao and Drachman, 1977; Drachman et al., 1979; Fuchs, 1979). An understanding of the regulation of the autoimmune response to Ach receptors should facilitate the development of a means of suppressing this disease. The Ach receptors in the sarcolemma in the region of the neuromuscular junction and the electroplaques of the electric fish are henceforth referred to as the junctional Ach receptors. Besides these, there are the extrajunctional Ach receptors, i.e., the Ach receptors in the extrajunctional sarcolemma of denervated skeletal muscle of the adult (see Albuquerque and McIssac, 1970; Meledi and Potter, 1971; Tipnis and Malhotra, 1980) and in the sarcolemma of embryonic muscle fibers (see Steinbach et al., 1979). The role(s) served by the extrajunctional Ach receptors is uncertain (see Frank, 1979); so is the chemical nature of the junctional and extrajunctional Ach receptors (see Fambrough, 1979). The molecular structure of the Ach receptor is being investigated (Wise et aI., 1979). The present chapter is a short review of some of the aspects of Ach receptors, particularly the extrajunctional Ach receptors. The macromolecular organization of the sarcolemma of skeletal muscle provides a valuable experimental system for investigating the functional organization of cellular membranes, besides contributing to our understanding of the role(s) of nerve-muscle interactions. In addition to the Ach receptors, specific acetylcholinesterase is the other major protein concentrated in the sarcolemma in the region of the neuromuscular junction in adults. It has been reported to undergo a marked decrease in denervated skeletal muscle (Hall, 1973; Malhotra and Tipnis, 1978). Physicochemical studies of the Ach receptors have been greatly facilitated by the use of postsynaptic neurotoxins from snake venom. One such toxin is the well-known a-bungarotoxin (a-BGT), which binds specifically and irreversibly to the nicotinic Ach receptors in muscle and electroplaques (Chang and Lee, 1963; Green et al., 1975; Lee, 1979; Ravdin and Berg, 1979). The binding of the neurotoxin to the receptor has been reported to be the result of multiple electrostatic and hydrophobic interactions (Lee, 1979). a-BGT can be conjugated to ferritin (Hourani et aI., 1974; Tipnis and Malhotra, 1979b), tritium, 125 1, or horseradish peroxidase (reviewed by Daniels and Vogel, 1978) for

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characterization of the receptors. Immunochemical techniques are also being applied for structural studies (Klymkowsky and Stroud, 1979) as well as for studies on cross-reactivity between possible subunits of Ach receptors (Lindstrom, 1979). A number of antagonists and affinity agents for Ach receptors are available and widely in use (Karlin et al., 1976; Heidmann and Changeux, 1978; Advances in Cytopharmacology, Vol. 3, 1979). In this article the term Ach receptor is applied to the entire macromolecular membrane complex, which includes the Ach receptor protein and the Ach ionophore, described as the Ach regulator (Heidmann and Changeux, 1978; Sobel et al., 1979). The binding of Ach to its recognition site is followed by a conformational change of the Ach receptor that leads to the opening of an ionic channel through the membrane (see Nachmansohn, 1955; Heidmann and Changeux, 1978; Karlin et al., 1979). Nicotinic Ach receptors can be distinguished from the muscarinic type as they react with different groups of the Ach molecule; the muscarinic type binds to the methyl side of Ach and the nicotinic type to the carbonyl side (Chothia, 1970). There are various agonists and antagonists that differentiate between nicotinic and muscarinic Ach receptors. The commonly used ones are curare and atropine, which block the nicotinic and the muscarinic types, respectively (see Koelle, 1970). The Ach receptors are widely distributed among invertebrates (see Dudai, 1979) and vertebrates (see Vogel and Nirenberg, 1976; Fambrough, 1979). But our knowledge of the functioning of Ach receptors is very largely derived from studies on the neuromuscular junction of vertebrate skeletal muscle and the homologous electro plaques in the electric organ of Torpedo and Electrophorus; the latter provide a rich source of Ach receptors for biochemical and biophysical characterization (Heidmann and Changeux, 1978; Advances in Cytopharmacology, Vol. 3, 1979). However, in recent years, extrajunctional Ach receptors have been explored in denervated mammalian skeletal muscles in situ and in cultured myotubes. Though the function(s) of these Ach receptors is not known, they are of interest with respect to our understanding of the significance of nerve-muscle interactions, besides their usefulness as a model system in studies related to the correlation of the structure and function of membranes. It is also of interest to understand differences, if any, between the molecular structure of the junctional and extrajunctional Ach receptors. Though subtle differences have been reported between the receptors at the two locations (Brockes and Hall, 1975a,b; Tipnis and Malhotra, 1980), it is not certain whether such differences arise from the structure of the receptor molecules themselves or from differences in the membrane structure or membraneassociated cytoskeleton [microfilaments and microtubules (Karlin et al., 1979; Heuser and Salpeter, 1979)].

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S. K. Malhotra

DISTRIBUTION OF ACETYLCHOLINE RECEPTORS Innervated Skeletal Muscle and Electroplaques

The Ach receptors are generally recognized to be concentrated in the sarcolemma in the region of the neuromuscular junction in adult animals (Figure lB). However, a wide variation in the number of Ach receptors has been reported for different muscles (see Porter et al., 1973; Edwards, 1979). For example, estimates for mammalian muscles range from approximately 8700/ p,m 2 (Albuquerque et aI., 1974) to 46,000 ± 27,000/p,m2 (Fertuck and Salpeter, 1976). This variation may reflect differences in the type of muscle and species or differences in the method of application of a-BGT used to estimate the receptor number (Albuquerque et al., 1974; Fertuck and Salpeter, 1976). These estimates are based on the assumption that there is one toxin binding site per receptor complex, though there are reported to be two such binding sites (Albuquerque et al., 1974; Karlin et al., 1978). The number of receptors declines with the distance from the neuromuscular junction. At the bottom of the junctional folds, the density of the receptors is only about 3% of that at the top of the folds and may reach the level of extrajunctional receptors at an equal distance from the nerve ending (Fertuck and Salpeter, 1976). In innervated muscle the extrajunctional receptors appear to vary in number depending on the type of muscle studied. In slow muscle the extrajunctional sarcolemma has high and low patches of Ach sensitivity (Miledi and Zelena, 1966; Albuquerque and Thesleff, 1968; Albuquerque and McIssac, 1970), and the number of receptors is estimated to be 22-25 binding sites/ p,m 2• In diaphragm, where the sensitivity to Ach is restricted to the neuromuscular junction (1695 receptors/ p,m 2), and in the regions surrounding it, there are 5 extrajunctional a-BGT binding sites/ p,m 2 (Hartzell and Fambrough, 1972). In the electroplaques of Electrophorus electricus, there are 50,000 ± 16,000 Ach receptors/ p,m 2 (Heidmann and Changeux, 1978). 2.2.

Denervated Skeletal Muscle (Extrajunctional Ach Receptors)

It should be recalled that the denervation of the electric organ of E. electricus does not affect the density of extrajunctional receptors (Bourgeois et al., 1973). It has been speculated that the intracellular calcium concentration may control the receptor number by influencing muscle activity, as an inverse relation between muscle activity and receptor density seems to exist (Edwards, 1979). There is an increase in intracellular levels of calcium with muscle activity, and this might inhibit the synthesis of the receptors. In the electric organ, denervation may not lead to a large increase in intracellular calcium, as is found in skeletal muscle (Edwards, 1979).

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In contrast to innervated muscle, denervated skeletal muscle responds to the application of Ach in the extrajunctional region (Axelsson and Thesleff, 1959). An increase in chemosensitivity begins 4 days after de nervation and appears to spread over the extrajunctional membrane by 7 days (Axelsson and Thesleff, 1959). After the denervation of fast muscle, the increase in the chemosensitive zone does not occur centrifugally, but first appears near the endplate and then near the myotendinous region, the mid portion of the extrajunctiona I region being the last to acquire chemosensitivity. Soleus, which in the innervated state has a high Ach sensitivity in the neuromuscular junction and myotendinous junction, exhibits regions of relatively high and low Ach sensitivity in the extrajunctional sarcolemma. This muscle, when denervated, exhibits a uniform increase in Ach sensitivity along the length of the fiber. Also, the appearance of chemosensitivity occurs earlier in soleus than in extensor digitorum longus (EDL) (Albuquerque and McIssac, 1970). The increase in extrajunctional Ach sensitivity, as shown in denervated rat diaphragm, occurs linearly with the increase in the number of Ach receptors in the extrajunctional sarcolemma (Hartzell and Fambrough, 1972). Such an appearance of extrajunctional Ach receptors in denervated EDL muscle is shown in Figure lA, which is an autoradiograph of 12sI-a-BGT-treated material. The Ach supersensitivity following de nervation is accompanied by a 5- to 50-fold increase in the number of Ach receptors per muscle (Dolly and Barnard, 1977; see Fambrough, 1979), and most of these receptors are in the extrajunctional sarcolemma. 2.3.

Biosynthesis of Extrajunctional Ach Receptors

It is now well known that denervation of skeletal muscle fibers leads to the progressive degeneration of muscle and that the muscle fibers develop an increased sensitivity to Ach (Axelsson and Thesleff, 1959; Miledi, 1960; reviewed by Edwards, 1979; Fambrough, 1979; Tipnis and Malhotra, 1979b). This phenomenon has been referred to as denervation supersensitivity (see Axelsson and Thesleff, 1959). It is also established that the increased sensitivity to Ach in the denervated muscle results from the spread of Ach sensitivity to the extrajunctional sarcolemma, and the entire sarcolemma becomes sensitive to Ach in the chronically denervated muscle (Alexsson and Thesleff, 1959; Miledi, 1960). However, the pattern of emergence of chemosensitivity differs in slow and fast muscles (Albuquerque and McIssac, 1970), and this subject has been reviewed by Tipnis and Malhotra (1980). The mechanism and the factors that influence the appearance or disappearance are being studied. The lack of extrajunctional Ach sensitivity in normal innervated muscle is generally attributed to muscle activity, which represses the biosynthesis of extrajunctional Ach receptors (see Betz and Changeux, 1979; Edwards, 1979).

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Electrical stimulation of denervated muscle prevents the appearance of extrajunctional sensitivity, and the blockage of nerve impulses causes the entire sarcolemma to become sensitive to the application of Ach (Lcj>mo and Rosenthal, 1972; Reiness and Hall, 1977; reviewed by Betz and Changeux, 1979). "Trophic" factors have also been implicated in the appearance of denervation changes in muscle (see Miledi, 1960; Hofmann and Thesleff, 1972; Betz and Changeux, 1979; Braithwaite and Harris, 1979; Schalow and Schmidt, 1979). Notwithstanding the specific nerve-muscle events leading to the appearance of extrajunctional sensitivity of Ach, it is commonly accepted that this sensitivity results from the synthesis of the receptors and their incorporation into the extrajunctional sarcolemma (Hartzell and Fambrough, 1973; Brockes and Hall, 1975c; Sakmann, 1975; Devreotes and Fambrough, 1976; Fambrough, 1979). These conclusions have been confirmed by studies involving inhibitors of RNA and protein synthesis, which block the appearance of extrajunctional sensitivity (Gramp et al., 1972; Chang and Tung, 1974; Hartzell and Fambrough, 1973). Also, the extrajunctional sensitivity reappears in denervated muscle or in developing myotubes if a-BGT has been used to block the preexisting receptors. The reappearance of sensitivity was inhibited by cycloheximide (Sakmann, 1975). The increase in extrajunctional Ach sensitivity, as shown in denervated rat diaphragm, occurs linearly with the increase in the number of Ach receptors in the extrajunctional sarcolemma (Hartzell and Fambrough, 1972). In this case the receptor density increases from approximately 5 receptors/ p,m 2 in the normal diaphragm to approximately 1695 receptors/ p,m 2 at 14 days after denervation. (Subsequently, the density drops to approximately 529/ p,m 2 at 45 days.) That the extrajunctional Ach sensitivity results from the incorporation of newly synthesized receptors is supported by observations on the freeze-fractured sarcolemma of denervated rat skeletal muscles. The protoplasmic half (P face) of the extrajunctional sarcolemma shows intramembranous particles (approximately 15 nm in diameter) in denervated muscle (Figure 2B). These particles are similar to those on the corresponding fractured face in the junc-

.

90 parameters were found to be a = 9.08 ± 0.41 nm, C = 9.10 ± 0.43 nm, and {3 = 118 ± 4. Although the unit cell angles are close to 120 both hexagonal and trigonal lattices have been ruled out because the amplitudes and the phases in the transform are not characteristically symmetric. [In a trigonal lattice, a = b = c and a = {3 = 'Y < 120 if rhombohedral axes are chosen, and a = b =F c, a = {3 = 90 'Y = 120 when referred to a hexagonal lattice (Henry and Lonsdale, 1965).] The Ach receptor oligomer has a molecular mass of approximately 370,000 daltons. The Ach-receptor-rich membranes give rise to a sharp reflection at d = 0.52 nm, which is indicative of a helices in coiled coil conformation (Henderson, 1975). These a helices appear to be approximately 8 nm long and oriented perpendicular to the membrane surface. Another reflection noted at 0.63 nm poses some interesting possibilities. It can be accounted for by a number of possible structures (Ross et al., 1977): 0

.)

0

0

,

0

0

0

,

1. 2.

A coiled polypeptide of rather large diameter cross-linked as a paralei {3 sheet structure. A twisted antiparallel {3 sheet forming a barrel-like structure.

Acetylcholine Receptors

3. 4.

291

A twisted anti parallel pleated sheet. Since such a 0.63 nm reflection has also been noted in the X-ray diffraction patterns of other membranes prepared from Torpedo and Electrophorus, including those enriched in acetylcholinesterase or ATPase, it raises the likely possibility that 0.63 nm reflection arises due to some regular lipid structure in the membranes.

Of these possibilities, (I) and (2) can easily define an energetically stable channel of approximate dimensions 0.5-1 nm (Ross et al., 1977). Since uranyl acetate, in negative staining, easily penetrates the center of each Ach receptor molecule, it appears to be a plausible location for the ionophoretic channel. Klymkowsky and Stroud (1979) have recently labeled the Ach receptor molecules in Ach-receptor-rich membranes by employing antibodies raised against Ach receptors and coupled to colloidal gold particles. Electron micrographs of such preparations have confirmed some of the conclusions of their previous work. Ach receptor molecules indeed extend above the. extracellular surface by approximately 5.5 nm and little on the cytoplasmic side. Recently, Brisson (1978) has reported tubular structures produced from Ach-receptor-rich membranes that exhibit a hexagonal type of packing. So far, published images of the Ach-receptor-rich membranes do not have sufficient resolution to elucidate the symmetry of the Ach receptor molecule itself. However, optical filtering experiments suggest that the stain-excluding regions are arranged in a hexagonal pattern, though the constituting subunits in these regions were not resolved. Klymkowsky and Stroud (1979) have suggested that perhaps Ach receptor particles have no internal symmetry, based on the assumption that they are made up of four different subunits (Raftery et al., 1979; see Hider, 1979). On the other hand, Changeux has presented evidence that the Ach receptor is a hexamer of six identical subunits of 40,000 daltons (see Hider, 1979; Sobel et al., 1979). If this is indeed the case, it would be more likely for these particles to have a six fold internal symmetry (since there is only one Ach receptor molecule per unit cell, the lattice symmetry cannot be greater than the symmetry within the Ach receptor molecule and must arise from it). However, the polypeptide structure of the receptor molecule is discussed on p. 289. Recently, Wise et al. (1979) have reported a radius of gyration of 4.6 nm for the Ach receptor alone, based on their analysis of the neutron-scattering results for the Ach receptor-Triton X-IOO complex. An analysis of the extended scattering curve was not possible due to low-coherence scattering and high-incoherence background. Nothing much could be said about the shape and structure of the Ach receptor because only a single parameter is known. The value of the radius of gyration and the molecular volume (approximately 3 X 105 A3), however, suggest two alternative simple shapes, namely oblate

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and prolate ellipsoids and cylinders. Alternatives about the shapes and sizes of the Ach receptor can be further narrowed down by incorporating the existing information on the structure of Ach receptors obtained from a variety of techniques such as electron microscopy, X-ray diffraction, and single-channel ionconductance measurements. The most consistent model that Wise et af. (1979) have proposed consists of three stacked concentric cylinders and a cylindrical pore running through them. The pore has a diameter of 3 nm within the top cylinder and then narrows down to 1 nm through the middle and bottom cylinders (see Figure 7). Zingsheim et al. (1980) have obtained a projection of Ach-receptor-rich membranes of Torpedo at a resolution of 1.8 nm. The negatively stained membranes were examined with a scanning transmission electron microscope by using a low electron dose « 10 e/ N, which is one tenth of the critical dose for most negatively stained biological specimens). The data were computed by digital imaging processing in which an average over many Ach receptor particles was obtained. The projection indicates that the Ach receptor protein

FIGURE 7. A diagrammatic representation of Ach receptor complex showing five (2:1:1:1) polypeptides arranged around an ionophoretic channel that traverses the membrane. This hypothetical model is derived from data published by Raftery et af. (1980) and Wise et af. (1979), and reviewed by Stevens (1980). Not to scale.

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extends into the aqueous medium and is an asymmetric structure. At the resolution of this projection (i.e., 1.8 nm), individual subunits could not be resolved, though a threefold symmetry is suggested. Zingsheim and co-workers, therefore, suggest that the Ach receptor protein molecule is made up of at least three (probably different) structural regions.

5.

MORPHOLOGICAL CORRELATES OF ACETYLCHOLINE RECEPTORS

A correlation of the number of Ach receptors with the macromolecular assemblies visualized as membrane surface protrusions and as intramembranous particles in the freeze-etch replicas is under investigation in several laboratories. One such study has been conducted on the electroplaques of Torpedo (Heuser and Salpeter, 1979). Collectively, there are approximately 5500 particles (8-10 nm) on the P (protoplasmic) and E (exoplasmic) fractured faces of the postsynaptic membrane. This is about half of the approximately 10,000 surface protrusions (approximately 8.5 nm) present on the ES face of the postsynaptic membrane (i.e., extracellular surface). Counts of the surface protrusions closely match the estimates for the density of Ach receptors on the postsynaptic membrane deduced from C\'-BGT binding (i.e., two C\'-BGT binding sites per Ach receptor). The reason for incongruence between the number of surface protrusions and the number of intramembranous particles is not clear at the moment. Such detailed studies are still lacking for the mammalian postsynaptic sarcolemma, though a spatial correlation between the intramembranous particles and the Ach receptor has been attempted (Yee et al., 1978; Bridgman and Greenberg, 1979; Cohen and Pumplin, 1979; Tipnis and Malhotra, 1980). Vee et al. (1978) showed that regions of sarcolemma containing identifiable Ach hot spots in cultured chicken muscle (uninnervated) also show clusters of 20-50 intramembranous particles (10-19 nm in diameter). The P face had approximately 2000 particles/ ｾｭＲ＠ vs. approximately 700/ ｾｭＲ＠ on the E face. (Some of these intramembranous particles appeared to be composed of five or six subunits arranged cylindrically around a central dark spot.) Cohen and Pumplin (1979) also reported a spatial correlation between clusters of 10nm particles and identifiable regions of hot spots in cultured chick myotubes. Bridgman and Greenberg (1979) have reported a temporal correlation between the appearance of 11- to 19-nm particles and the appearance of Ach sensitivity in embryonic muscle of Xenopus. Embryos that did not exhibit Ach sensitivity showed smaller (8.2 nm in diameter) intramembranous particles. In rat denervated skeletal muscle examined at a stage when the extrajunctional Ach sensitivity is known to be at an optimum, i.e., 2 weeks after

294

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nerve sectioning (Hartzell and Fambrough, 1972; Fambrough, 1974), the extrajunctional sarcolemma shows particles of approximately 15 nm (range 15-18 nm) on the P face. Such particles are lacking from the corresponding fractured face of the innervated sarcolemma, which has an abundance of 8-nm particles [2000 particles/ /oLm2 (Tipnis and Malhotra, 1976, 1977, 1979a,b; Malhotra and Tipnis, 1978; Malhotra, 1978, 1980)]. The large particles manifest two types of spatial distribution, i.e., as dispersed single particles and as clusters (up to as many as 50 particles per cluster). By using a-BGT conjugated to ferritin (for visualization in electron micrographs) or 1251 (for autoradiographic visualization), a spatial correlation between the 15-nm particles and the a-BGT binding sites (Ach receptors) has been observed (Tipnis and Malhotra, 1979a,b). Electron micrographs of thin sections and freeze-etch preparations of sarcolemma incubated with ferritin a-BGT indicate that the a-BGT binding sites extend into the extracellular material (Figures 4, 5). These findings are supportive of the idea that the approximately 15-nm particles in denervated sarcolemma are related to the extrajunctional Ach receptor molecules. However, a precise relation between the density of these intramembranous particles and the Ach receptor protein remains to be ascertained, as the reported number of intramembranous particles (approximately 500/ /oLm2) is two- to threefold lower than the number of a-BGT binding sites, i.e., approximately 1600//oLm2 (Tipnis and Malhotra, unpublished observations on EDL muscle). Hartzell and Fambrough (1972) also reported 1695 1251_a_BGT binding sites/ J.Lm 2 for extrajunctional sarcolemma of the rat diaphragm denervated for 2 weeks. Fambrough (1974) subsequently revised this density to 635 ± 29 125J-a-BGT binding sites/ /oLm 2. Also, in the neuromuscular junctional region, an exact relation between the density of intramembranous particles and the a-BGT binding sites is not yet certain (reviewed by Fambrough, 1979). As many as 5- to 10-fold more aBGT binding sites than intramembranous particles have been reported. Furthermore, the size of the intramembranous particles (the putative Ach receptors) has been reported to differ in replicas from various sources, i.e., 8-10 nm in frog (Heuser et al., 1974; Peper et al., 1974), 10 nm in chick (Cohen and Pumplin, 1979), and 10-19 nm in chicken and mammalian muscles (Tipnis and Malhotra, 1976,1980; Malhotra and Tipnis, 1978; Vee et al., 1978; Bridgman and Greenberg, 1979; Fambrough, 1979). This contrasts with the rather uniform 8- to 9-nm intramembranous particles generally reported from the receptor-rich electroplaques and considered to be components of the receptor molecules (Cartaud et al., 1978; Heidmann and Changeux, 1978; Heuser and Salpeter, 1979). Some of the variations in size might arise from the varying thickness of the metal(s) used for making replicas for which allowances can be made by using an appropriate correction factor (see Garber and Steponkus, 1974; Sikerwar and Malhotra, 1979; Tewari and Malhotra, 1979).

Acetylcholine Receptors

6.

295

DIFFERENCES BETWEEN JUNCTIONAL AND EXTRAJUNCTIONAL ACETYLCHOLINE RECEPTORS

It appears from the existing literature that the junctional and extrajunctional Ach receptors are very similar molecules (Brockes and Hall, 197 Sa; Dolly and Barnard, 1977). They may, however, differ in their functioning, which could arise from differences in the structure of the surrounding membrane (Merlie et aI., 1979). It should be of interest to know what such differences are. Also, although extrajunctional receptors from embryonic and denervated muscle have many common characteristics, they have been reported to differ in their response to curare. In intercostal muscles of rat embryo and neonatal rats, curare produced localized contractions and action potentials. These are resistant to tetrodotoxin but can be blocked by a-BGT, thereby suggesting that the action of curare is receptor specific. In parallel experiments with denervated muscle, curare did not produce any depolarization. These results suggest either the existence of a third category of Ach receptor in embryonic muscle or the difference in molecular environments in embryonic and denervated muscle (Ziskind and Dennis, 1978). With a view to compare the junctional and extrajunctional Ach receptors, Brockes and Hall (1975a,b) isolated the receptors from normal rat diaphragm muscle, extrajunctional regions of the denervated diaphragm, and neonatal diaphragm muscle. These receptors reacted with Con A, had an identical reaction with an antiserum to the eel Ach receptors, and had a similar sedimentation coefficient (9 S). The junctional receptors had a different isoelectric focusing peak (pH 5.1) than the extrajunctional receptors (pH 5.3; Brockes and Hall, 1975b), which suggests a difference of 10-30 charges per receptor molecule (Fambrough, 1979). Differences have been reported between junctional and extrajunctional receptors in their affinity for d-tubocurarine (e.g., Lapa et al., 1974; Brockes and Hall, 1975b), but these have not yet been confirmed (Almon and Appel, 1976b; Fambrough, 1979). Almon and Appel (1976b) have reported two classes of a-BGT binding sites with affinity constants of 109 M-' in rat skeletal muscle. In denervated muscle there was a 28-fold increase in the high-affinity site and as-fold increase in the low-affinity site. Long-term exposure of normal muscle to the nonionic detergent Triton X-100 for 8-10 days converted all binding to a highaffinity set of sites (10 9 M-') with no change in the total number of sites (Almon el aI., 1974). In a parallel experiment with denervated muscle, there was no change in the binding affinity of Ach receptors. Such differences in receptors from innervated and denervated muscles have been suggested to reflect different molecular interaction of the receptor with the environment, rather than a difference in the primary structure of the receptor protein (Almon et al., 1974).

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Also, Merlie et al. (1979) found that the degradation of the two types of receptors (in organ-cultured adult rat diaphragms) was similar in many ways, the point of difference being the rate constant (10-fold difference) for the degradation requires energy; both are inhibited by colchicine and by specific lysosomal protease inhibitors (leupeptin + pepstatin); and both are stimulated by anti-Ach receptor antibodies. It was speculated that the difference in the rate of internalization of both receptors was due to some structural difference in the membrane or the membrane-associated proteins. That the junctional and extrajunctional Ach receptors differ with respect to their turnover in the membrane is generally agreed (see Fambrough, 1979). The stability of the toxinreceptor complex of rat diaphragm muscles studies in vivo (Berg and Hall, 1975) or in vitro (Berg and Hall, 1975; Chang and Huang, 1975; Devreotes and Fambrough, 1975) indicated a turnover of the receptor with a half-life of 14-18 hr. The junctional receptors turn over more slowly, i.e., a half-life of about 6 days (see reviews by Edwards, 1979; Fambrough, 1979; Steinbach et al., 1979). In chick, the junctional and extrajunctional receptors turn over at similar rates (half-lives of about 30 hr) even 1 week after hatching, but the two classes of receptors show distinct turnover rates 3 weeks after hatching, with the junctional receptors turning over slower (half-life of about 5 days) than the extrajunctional receptors (half-life of about 30 hr; Burden, 1977). Consistent with the similarity in turnover rates in the two classes of receptors in the earlier stages of chick development are the findings of Schuetze et al. (1978) that the junctional and extrajunctional receptors (in cultured chick myotubes) have similar mean channel open time (about 3.0-3.5 msec at 24°C). It therefore appears that synapse formation may be a long drawn-out process in chick as compared to that in rat or frog (Schuetze et al., 1978). Even in a single species different skeletal muscles may mature at different rates, presumably depending on the activity. In this context, the findings of Sakmann and Brenner (1978) are relevant: the shift in the mean channel open time (see below) from the extrajunctional to the junctional receptor type occurs earlier in the omohyoideous muscle than in the soleus muscle in embryonic rat. Beyond this, observations made by Steinbach et al. (1979) on developing rat diaphragm muscle suggest that different properties of Ach receptors, i.e., location, degradation rate, and mean channel open time, may change at different times during development of the muscle. They found that some junctional receptors were metabolically stable even when their mean channel open time was characteristic of adult extrajunctional Ach receptors. It is of obvious interest to know how these alterations take place and whether the receptor molecules undergo alterations or whether they reflect the membranous environment of the receptors. The fluctuation analysis used for determining the size and duration of the elementary current indicates that the lifetime of the Ach-induced channel is

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much shorter in innervated muscle. The various values reported are 3.2 msec in frog (Colquhoun et aI., 1975), 1 msec in rat (Sakmann, 1978), and 1.54 msec in human muscles (Cull-Candy et al., 1978). Four- to fivefold higher values have been reported for extrajunctional channels in human myotubes (Bevan and Kullberg, 1978) and in denervated muscles of frog (Neher and Sakmann, 1976) and rat (Sakmann, 1978). Boulter and Patrick (1979) have examined the Con A-induced inhibition of [ 125 I]_a_BGT binding in a nonfusing muscle cell line (BC 3Hl) with a view to determine if there is more than one class of Ach receptor. Con A inhibits only about 35% of the toxin binding sites in the whole cell, whereas more than 90% of the toxin binding sites in purified Ach receptors are sensitive to Con A. Experimental data then led Boulter and Patrick to conclude that there is only one class of Ach receptor and that Con A binds to all of the Ach receptor molecules, sometimes producing complete blockage of a-BGT binding, sometimes partial inhibition, and sometimes no blockage. It appears, therefore, that Con A inhibition of the Ach receptor is a random event. Nevertheless, Weinberg and Hall (1979) have reported that the extrajunctional Ach receptors contain determinants that can be detected by sera from myasthenic patients. However, all of the determinants of the junctional receptors detected by myasthenic sera were present on the extrajunctional receptors so that myasthenic sera contained two classes of antibodies, one directed against determinants present in both junctional as well as extrajunctional receptors and the other directed against determinants present only on the extrajunctional receptors. It is noteworthy that antisera from several animals immunized with rat extrajunctional receptors or eel or Torpedo receptors could not detect differences in the two classes of receptors. In this respect, myasthenic sera may provide a valuable system to distinguish the molecular species of the Ach receptors.

7. SIGNIFICANCE OF EXTRAJUNCTIONAL ACETYLCHOLINE RECEPTOR AGGREGATES Apart from the concentration of the receptors at the top of the junctional folds in the neuromuscular junction, clusters of Ach receptors occur in the extrajunctional sarcolemma in denervated muscle and in embryonic muscle (Vogel et al., 1972; Sytkowski et al., 1973; Fischbach and Cohen, 1973; Ko et aI., 1977). Such extrajunctional patches of the sarcolemma manifest an Ach response several degrees higher than elsewhere on the muscle and are therefore often described as Ach hot spots (Fischbach and Cohen, 1973). The size of these regions may vary from less than 1 Mm to 30 Mm in different muscles and with the period of de nervation (Ko et al., 1977), and there may be one or more clusters on each fiber (see Frank, 1979). The morphological correlates of these

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Ach receptors, namely the clusters of intramembranous particles seen in freeze-fracture replicas, have been described by a number of workers and are discussed on page 293 (see Figure 6). The photobleaching technique used in the study of lateral motion of Ach receptors marked with fluorescent a-BGT indicates that the uniformly dispersed receptors are mobile (diffusion constant of approximately 5 x 1O-1l cm 2/sec at 22 C), whereas those occurring in clusters are immobile (Axelrod et al., 1976). Selective photobleaching of the patch shows that the receptors in the immobile and mobile phases do not exchange even after 10 hr in culture. The entire patch of receptors moves slowly at a rate of 4 pm/hr. The mechanism of the stability or the formation of the Ach clusters is not understood except that the receptors in the cluster gradually disappear under electrical stimulation. Axelrod et al. (1978) have proposed that receptors in the patch may be stabilized by an immobile intra- or submembranous filamentous structure composed of molecules other than the receptors themselves. Microtubule and microfilament disrupting agents had no effect on the mobility of Ach receptors, whether in a mobile state or in clusters. Braithwaite and Harris (1979) have reported that electrical activity of the nerve and/or muscle is required to suppress the appearance of extrajunctional receptor clusters and for the normal progress of muscle growth. These findings are based on experiments on embryonic rat muscle in which nerve and muscle activity were paralyzed by the application of tetrodotoxin. In untreated embryonic rat muscle, clusters of Ach receptors Uunctiona1) appear synchronously after day IS! of gestation. Tetrodotoxin application at day 16 of gestation did not alter the events of normal junctional Ach receptor appearance, but extrajunctional receptors appeared as they do in the denervated muscle. Several laboratories have reported that extracts from nervous tissue, when added to muscle cultures, increased the number of Ach receptors (reviewed by Frank, 1979; Podleski et al., 1978; Christian et al., 1978; Jessell et al., 1979). These extracts influence the number of dispersed receptors as well as clustered receptors. Podleski et al. (1978) have tentatively identified their active factor as a protein(s) of 100,000 daltons, and Jessell et al. (1979) describe their active factor as a small molecule, possibly a peptide of less than 2000 daltons. With respect to the mechanism of formation of receptor clusters, it is interesting that a factor produced by a cholinergic neuroblastoma-glioma hybrid cell line has been reported to increase the Ach receptor clusters on myotube cultures from mouse, rat, or chick (Christian et al., 1978). This factor has been hypothesized to act either by aggregating mobile dispersed receptors or by stabilizing receptor clusters once formed. In this respect, the aggregation of the receptors in the sarcolemma may be comparable to the lectin-immunoglobulin-induced caps or the patch formation of dispersed surface receptors (see Christian et al., 1978). Also, Anderson and Cohen (1977) have proposed that the Ach receptor clus0

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ters in the extrajunctional sarcolemma as well as in the junctional sarcolemma are formed by aggregation of individual mobile receptors. Though the mechanism involved in the formation of clusters of Ach receptors is not known, they suggested that certain cytoplasmic structures (microtubules, microfilaments) may mediate the formation of these clusters. The rationale for their suggestion is the knowledge that drugs that disrupt microtubules and microfilaments in lymphocytes affect the clustering of receptors for Con A and immunoglobulins (Edelman, 1976). However, microtubule and microfilament disrupting drugs appear to have no effect on the mobility of Ach receptors (Axelrod et al .. 1978; see earlier in this section), and other filaments insensitive to such drugs may be involved in the organization of the sarcolemma. The significance of the receptor aggregates is of current interest. The suggestion that the clusters may be the sites of synapse formation has not been substantiated (see Frank, 1979). Bevan and Steinbach (1977) have reported that the high density of receptors at the neuromuscular junction develops after the formation of the junction. Braithwaite and Harris (1979) also found that developing muscles contract in response to nerve impulses at least 6 hr before the receptor clusters could be resolved by autoradiography. Jacob and Lentz (1979) have described electrondense regions of the plasma membrane in the developing myogenic cells of the chick embryo. These regions are thought to serve as recognition sites for nerve, and after the initial contact of nerve with the muscle a high concentration of Ach receptors appears in the area of the junction. The Ach receptors were localized by using a horseradish peroxidase-a-BGT conjugate. Anderson et al. (1979) have reported the results of their studies on nervemuscle cultures from Xenopus larvae; their observations, made 1-3 days after adding dissociated neural tube cells, indicate that the development of synaptic transmission precedes the onset of Ach receptor localization. The latter was examined by using a tetramethyl rhodamine-a-BGT conjugate, and receptor clusters could be detected elsewhere on muscle cells but not necessarily in the region of nerve-muscle contact. Earlier, Anderson et al. (1977) reported that innervation causes Ach receptors to accumulate at sites of nerve-muscle contact, and this process can operate independently of muscle activity. Cohen (1980) has reported that the stimulus-evoked release of Ach over the postsynaptic membrane has a low Ach sensitivity in embryonic chick muscle cultured with explants from spinal cord. The release was detectable 30 min after a nerve process first came in contact with muscle fiber. The response (synaptic potential) varied from 25 to 200 J.L V and could be detected only after waveform averaging in order to improve the signal-to-noise ratio. This synaptic potential could be blocked reversibly by the local application of d-tubocurarine. No high-density areas of Ach receptors, i.e., hot spots, were found in the region of initial nerve-muscle contact. Hot spots were present in the extrajunctional regions of the sarcolemma. With respect to the function of hot spots, it is of

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interest that nerve-muscle contacts were not found in these areas of relatively high Ach-sensitive sarcolemma. Also, in the embryonic skeletal muscle of the rat, clusters of Ach receptors (as shown by [ 125 I]-a-BGT autoradiography) appear after day 15! of gestation, and this first appearance of the clusters is not affected by the application of tetrodotoxin (at day 16 of gestation), which causes paralysis of nerve and muscle (Braithwaite and Harris, 1979). Also, neither the progressive increase in the size of the clusters nor the appearance of extrajunctional receptors was affected. Prives et al. (1976) reported that the clusters of Ach receptors on the surface of developing chick myotubes in vitro are transient structures that disappear upon maturation (in the absence of innervation). Based on a study of autoradiographs, the appearance of these clusters on myotubes of developing chick embryo seems to coincide with the appearance of cross-striations. As the cross-striations increase and are localized sharply within the myotubes, the clusters of Ach receptors disappear from the surface. Such a transient occurrence of the clusters without neuronal influence has been suggested to reflect a sequential process during muscle differentiation. Prives et al. (1976) also speculated that the clustering of Ach receptors might be an event in the disappearance of the extrajunctional receptors by a process of internalization. That the Ach receptors are degraded and internalized has been demonstrated by Devreotes and Fambrough (1976) and Carbonetto and Fambrough (1979).

8. CONCLUSION The sarcolemma of a skeletal muscle is a valuable model system for investigating the correlation between membrane structure and function. There is an obvious mosaicism of function along the sarcolemma, as manifested by the neuromuscular junction, which results from interactions as yet not understood between nerve and muscle. In the junctional region it has two major proteins, Ach receptor and acetylcholinesterase (see Malhotra and Tipnis, 1978), and thus has a relatively simple composition as compared to the membranous organelles of eukaryotic cells. Denervation of skeletal muscle results in the biosynthesis of new Ach receptors and their incorporation into the sarcolemma (see Fambrough, 1979), and the latter thus becomes sensitive to Ach. It is of interest to know the functional parameters of extrajunctional Ach receptors, the differences between the dispersed and the clustered receptors, and the mechanism of formation of clusters. Whether the extrajunctional Ach receptors in denervated sarcolemma differ from the receptors in embryonic muscle is not certain. How does the organization of junctional receptors differ from that of the extrajunctional receptors? Do the microfilaments and microtubules

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or other intracellular or extracellular structures playa role in the organization of the receptors? Denervation also produces a decrease in the activity of specific acetylcholinesterase localized at the junctional sarcolemma, i.e., the 16 S form in rat (Hall, 1973; Malhotra and Tipnis, 1978). The sarcolemma is amenable to investigations by a variety of biochemical and biophysical techniques currently in vogue that can be applied to investigations in vivo and in cultures. Cyclic nucleotides are implicated in the regulatory control of the synthesis of extrajunctional receptors (Betz and Changeux, 1979). In cultured chick myoblasts, dibutyryl cyclic GMP repressed the synthesis of Ach receptors and abolished the development of Ach sensitivity, whereas dibutyryl cyclic AMP increased the number of a-BGT binding sites. The repression of Ach receptor synthesis by cyclic GMP may be a result of an increase in CaH , which enters the cell during the opening of Na+ channels by electrical activity (Betz and Changeux, 1979). Cyclic GMP appears to mimic the effects of muscle activity, which has been reported to be involved in the accumulation of acetylcholinesterase in the neuromuscular junction. a-BGT and curare, which are known to block synaptic activity (not the formation of synapses), also block the appearance of acetylcholinesterase at the junctional region in nerve-muscle cultures of chick embryos (Rubin et al., 1980). The synthesis of specific acetylcholinesterase at the neuromuscular junction is induced by interaction of the muscle cell with the nerve (Koenig and Vigny, 1978). It has been suggested that in vivo this activity is controlled by substances that are moved in axonal transport and released from the nerve upon its stimulation (Younkin et al., 1978). In frog sartorius muscle, the extrajunctional receptors are subject to seasonal variation. They are absent in summer, appear in autumn, and increase in winter. No such variation is reported to occur with respect to the junctional receptors. The observed variation in the extrajunctional receptors has therefore been suggested to be due to variation in motor activity (Feltz and Mallart, 1971 ). Ach receptors appear to undergo organizational alterations during development (Burden, 1977; Steinbach et al., 1979). It is likely that nonmembranous components, such as microfilaments and microtubules (Heuser and Salpeter, 1979), playa role in the architectural organization of the sarcolemma. They may exercise regulatory control on the mobility of Ach receptor complexes within the sarcolemma. Antibodies influence the rate of degradation of Ach receptors as evidenced by studies on myasthenic neuromuscular junctions (see Heinemann et al., 1978; Drachman et al., 1979; Fuchs, 1979; Lindstrom, 1979). An electric field applied to an embryonic muscle cell perturbs the Ach receptors, which then accumulate at one pole (Orida and Poo, 1978). It is apparent that the interplay of such diverse nerve and muscle cell activities regulates the biology of Ach receptors and other components associated with the sarcolemma.

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ACKNOWLEDGMENTS

I am grateful to my research colleagues, Dr. U. R. Tipnis, Dr. J. P. Tewari, Dr. S. S. Sehgal, and Mr. S. S. Sikerwar, for their valuable help and discussions. Mr. S. D. Ross provided invaluable technical help. The author's research work included in this article has been supported by Grant A 5021 from the Natural Sciences and Engineering Research Council of Canada. Mrs. D. Arbuthnott has taken great pains in handling the typing of this manuscript and the preparation of the references.

9. REFERENCES Albuquerque, E. X., and Mclssac, R. J., 1970, Fast and slow mammalian muscle after denervation, Exp. Neurol. 26:183-202. Albuquerque, E. X., and Thesleff, S., 1968, A comparative study of membrane properties of innervated and chronically denervated fast and slow skeletal muscles of the rat, Acta Phys-

iol. Scand. 73:471-480. Albuquerque, E. X., Barnard, E. A., Porter, C. W., and Warnick, J. E., 1974, The density of acetylcholine receptors and their sensitivity in the postsynaptic membrane of muscle endplates, Proc. Natl. Acad. Sci. USA 71:2818-2822. Almon, R. R., and Appel, S. H., 1976a, Cholinergic sites in skeletal muscle. I. Denervation effects, Biochemistry 15:3662-3666. Almon, R. R., and Appel, S. H., 1976b, Cholinergic sites in skeletal muscle. II. Interaction of an agonist and two antagonists with the acetylcholine site, Biochemistry 15:3667-3671. Almon, R. R., Andrew, C. G., and Appel, S. H., 1974, Acetylcholine receptor in normal and denervated slow and fast muscle, Biochemistry 13:5522-5528. Anderson, M. J., and Cohen, M. W., 1977, Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells, J. Physiol. 268:757-773. Anderson, M. J., Cohen, M. W., and Zorychta, E., 1977, Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells, J. Physiol. 268:731-756. Anderson, M. J., Kidokoro, Y., and Gruener, R., 1979, Correlation between acetylcholine receptor localization and spontaneous synaptic potentials in cultures of nerve and muscle, Brain

Res. 166:185-190. Axelrod, D., Ravdin, P., Koppel, D. E., Schlessinger, J., Webb, W. W., Elson, E. L., and Podleski, T. R., 1976, Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers, Proc. Natl. Acad. Sci. USA 73:4594-4598. Axelrod, D., Ravdin, P. M., and Podleski, T. R., 1978, Control of acetylcholine receptor mobility and distribution in cultured muscle membranes, Biochim. Biophys. Acta 511:23-38. Axelsson, J., and Thesleff, S., 1959, A study of supersensitivity in denervated mammalian skeletal muscle, J. Physiol. 147:178-193. Berg, D. K., and Hall, Z. W., 1975, Loss of a-bungarotoxin from junctional acetylcholine receptors in rat diaphragm muscle in vivo and in organ culture, J. Physiol. 252:771-789. Betz, H., and Changeux, J.P., 1979, Regulation of muscle acetylcholine receptor synthesis in vitro by cyclic nucleotide derivatives, Nature (London) 278:749-752. Bevan, S., and Kullberg, R. W., 1978, Acetylcholine-induced conductance fluctuations in cultured human myotubes, Nature (London) 273:469-471.

Acetylcholine Receptors

303

Bevan, S., and Steinbach, J. H., 1977, The distribution of a-bungarotoxin binding sites on mammalian skeletal muscle developing in vivo, J. Physiol. 267:195-213. Blunt, R. J., Jones, R., and Vrbova, G., 1975, Inhibition of cell division and development of denervation hypersensitivity in skeletal muscle, Pfiuegers Arch. 355: 189-204. Boulter, J., and Patrick, J., 1979, Concanavalin A inhibition of a-bungarotoxin binding to a nonfusing muscle cell line, J. Bioi. Chem. 254:5652-5657. Bourgeois, J. P., Popot, J.-L., Ryter, A., and Changeux, J.-P., 1973, Consequences of denervation on the distribution of the cholinergic (nicotinic) receptor sites from Electrophorus electricus revealed by high resolution autoradiography, Brain Res. 62:557-563. Braithwaite, A. W., and Harris, A. J., 1979, Neural influence on acetylcholine receptor clusters in embryonic development of skeletal muscle, Nature (London) 279:549-551. Bridgman, P. c., and Greenberg, A. S., 1979, A correlation between the appearance of 11-19 nm intermembrane particles and acetylcholine sensitivity in Xenopus embryonic muscle, Abstracts, Society for Neuroscience, 9th Annual Meeting, Atlanta (November 2-6, 1979), Society for Neuroscience, Bethesda, p. 476. Brisson, A., 1978, Acetylcholine receptor protein structure, Ninth International Congress on Electron Microscopy, Vol. II, pp. 180-181. Brockes, J. P., and Hall, Z. W., 1975a, Acetylcholine receptors in normal and denervated rat diaphragm muscle. I. Purification and interaction with [ 125 I]-a-bungarotoxin, Biochemistry 14:2092-2099. Brockes, J. P., and Hall, Z. W., 1975b, Acetylcholine receptors in normal and denervated rat diaphragm muscle. II. Comparison of junctional and extrajunctional receptors, Biochemistry 14:2100-2106. Brockes, J. P., and Hall, Z. W., 1975c, Synthesis of acetylcholine receptor by denervated rat diaphragm muscle, Proc. Natl. Acad. Sci. USA 72:1368-1372. Burden, S., 1977, Development of the neuromuscular junction in the chick embryo: The number, distribution and stability of acetylcholine receptors, Dev. Bioi. 57:317-329. Carbonetto, S., and Fambrough, D. M., 1979, Synthesis, insertion into the plasma membrane and turnover of a-bungarotoxin receptors in chick sympathetic neurons, J. Cell Bioi. 81:555-569. Cartaud, J., Benedetti, E. L., Sobel, A., and Changeux, J.-P., 1978, A morphological study of the cholinergic receptor protein from Torpedo marmorata in its membrane environment and in its detergent-extracted purified form, J. Cell Sci. 29:313-337. Chang, C. C., and Huang, M. c., 1975, Turnover of junctional and extrajunctional acetylcholine receptors of rat diaphragm, Nature (London) 253:643-644. Chang, C. c., and Lee, C. Y., 1963, Isolation of neurotoxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action, Arch. Int. Pharmacodyn. Ther. 144:241-257. Chang, C. C., and Tung, L. H., 1974, Inhibition by actinomycin D of the generation of acetylcholine receptors induced by denervation in skeletal muscle, Eur. J. Pharmacal. 26:386388. Changeux, J.-P., Heidmann, T., Popot, J.-L., and Sobel, A., 1979, Reconstitution of a functional acetylcholine regulator under defined conditions, FEBS Lett. 105: 181-187. Chothia, c., 1970, Interaction of acetylcholine with different cholinergic nerve receptors, Nature(London) 225:36-38. Christian, C. N., Daniels, M. P., Sugiyama, H., Vogel, Z., Jacques, L., and Nelson, P. G., 1978, A factor from neurons increases the number of acetylcholine receptor aggregates on cultured muscle cells, Proc. Natl. Acad. Sci. USA 75:4011-4015. Cohen, S. A., 1980, Early nerve-muscle synapses in vitro release transmitter over postsynaptic membrane having low acetylcholine sensitivity, Proc. Natl. Acad. Sci. USA 77:644-648.

304

S. K. Malhotra

Cohen, S. A., and Pumplin, D. W., 1979, Clusters of intramembrane particles associated with binding sites for a-bungarotoxin in cultured chick myotubes, J. Cell Bioi. 82:494-516. Colquhoun, D., Dionne, V. E., Steinbach, J. H., and Stevens, C. F., 1975, Conductance of channels opened by acetylcholine-like drugs in muscle end-plate, Nature (London) 253:204-206. Cull-Candy, S. G., Miledi, R., and Trautmann, A., 1978, Acetylcholine-induced channels and transmitter release at human end plates, Nature (London) 271:74-75. Daniels, M. P., and Vogel, Z., 1978, Localization of acetylcholine receptors, in: Principles and Techniques of Electron Microscopy (M. A. Hayat, ed.), pp. 107-125, Van Nostrand-Reinhold, Princeton, N.J. Devreotes, P. N., and Fambrough, D. M., 1975, Acetylcholine receptor turnover in membranes of developing muscle fibers, J. Cell Bioi. 65:335-358. Devreotes, P. N., and Fambrough, D. M., 1976, Synthesis of acetylcholine receptors by cultured chick myotubes and denervated mouse extensor digitorum longus muscles, Proc. Natl. Acad. Sci. USA 73:161-164. Dolly, J. 0., and Barnard, E. A., 1977, Purification and characterization of an acetylcholine receptor from mammalian skeletal muscle, Biochemistry 16:5053-5060. Drachman, D. B., Pestronk, A., and Stanley, E. F., 1979, Pathogensis of myasthenia gravis as a disorder of acetylcholine receptors, in: Advances in Cytopharmacology (B. Ceccarelli and F. Clementi, eds.), Vol. 3, pp. 237-244, Raven Press, New York. Dudai, Y., 1979, Cholinergic receptors in insects, Trends Biochem. Sci. 4:40-44. Edelman, G. M., 1976, Surface modulation in cell recognition and cell growth. Some new hypotheses on phenotypic alteration and transmembranous control of cell surface receptors, Science 192:218-226. Edwards, c., 1979, The effects of innervation on the properties of acetylcholine receptors in muscle, Neuroscience 4:565-584. Fambrough, D. M., 1974, Acetylcholine receptors. Revised estimates of extrajunctional receptor density in denervated rat diaphragm, J. Gen. Physiol. 64:468-472. Fambrough, D. M., 1979, Control of acetylcholine receptors in skeletal muscle, Physiol. Rev. 59: 165-227. Feltz, A., and Mallart, A., 1971, An analysis of acetylcholine responses of junctional and extrajunctional receptors of frog muscle fibers, J. Physiol. 218:85-100. Fertuck, H. C., and Sal peter, M. M., 1976, Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-a-bungarotoxin binding at mouse neuromuscular junctions, J. Cell Bioi. 69:144-158. Fischbach, G. D., and Cohen, S. A., 1973, The distribution of acetylcholine sensitivity over un innervated and innervated muscle fibers grown in cell culture, Dev. Bioi. 31:147-162. Frank, E., 1979, Acetylcholine receptor clusters, Nature (London) 278:599-600. Freeman, J. A., 1977, Possible regulatory function of acetylcholine receptor in maintenance of retinotectal synapses, Nature (London) 269:218-222. Froehner, S. C., Karlin, A., and Hall, Z. W., 1977, Affinity alkylation labels two subunits of the reduced acetylcholine receptor from mammalian muscle, Proc. Natl. Acad. Sci. USA 74:4685-4688. Fuchs, S., 1979, Immunochemical analysis of acetylcholine receptor and its relevance to specific treatment of experimental autoimmune myasthenia gravis, in: Advances in Cytopharmacology (B. Ceccarelli and F. Clementi, eds.), Vol. 3, pp. 279-285, Raven Press, New York. Garber, M. P., and Steponkus, P. L., 1974, Identification of chloroplast coupling factor by freezeetching and negative-staining techniques, J. Cell Bioi. 63:24-34. Gramp, W., Harris, J. B., and Thesleff, S., 1972, Inhibition of de nervation changes in skeletal muscle by blockers of protein synthesis, J. Physiol. 221:743-754.

Acetylcholine Receptors

305

Green, D. P. L., Miledi, R., Perez de la Mora, M., and Vincent, A., 1975, Acetylcholine receptors, Phil. Trans. R. Soc. London Ser. B 270:551-559. Hall, Z. W., 1973, Multiple forms of acetylcholinesterase and their distribution in endplate and non-endplate regions of rat diaphragm muscle, J. Neurobiol. 4:343-361. Harhorne, A. J., and Smith, M. E., 1979, Agonist-induced potentiation of acetylcholine sensitivity in denervated skeletal muscle, Nature (London) 282:85-87. Hartzell, H. c., and Fambrough, D. M., 1972, Acetylcholine receptors: Distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity, J. Gen Physiol.60:248-262. Hartzell, H. C., and Fambrough, D. M., 1973, Acetylcholine receptor production and incorporation into membranes of developing muscle fibers, Dev. BioI. 30:153-165. Heidmann, T., and Changeux, J .-P., 1978, Structural and functional properties of the acetylcholine receptor protein in its purified and membrane-bound states, Annu. Rev. Biochem. 47:317-357. Heinemann, S., Merlie, J., and Lindstrom, J., 1978, Modulation of acetylcholine receptor in rat diaphragm by anti-receptor sera, Nature (London) 274:65-68. Henderson, R., 1975, The structure of the purple membrane from Halobacterium halobium. Analysis of the X-ray diffraction pattern, J. Mol. BioI. 93:123-138. Henry, N. F. M., and Lonsdale, K., 1965, International Tables for X-ray Crystal-Iography, Vol. I, The International Union of Crystallography, The Kynoch Press, Birmingham, England. Heuser, J. E., and Salpeter, S. R., 1979, Organization of acetylcholine receptors in quick-frozen, deep-etched and rotary-replicated Torpedo postsynaptic membrane, J. Cell BioI. 82:150173. Heuser, J. E., Reese, T. S., and Landis, D. M. D., 1974, Functional changes in frog neuromuscular junctions studied with freeze-fracture, J. Neurocytol. 3:109-131. Hider, R. C., 1979, Neurotoxins in Nice, Nature (London) 281:340-341. Hofmann, W. W., and Thesleff, S., 1972, Studies on the trophic influence of nerve on skeletal muscle, Eur. J. Pharmacol. 20:256-260. Hourani, B. T., Torain, B. F., Henkart, M. D., Carter, R. L., and Marchesi, V. T., 1974, Acetylcholine receptors of cultured muscle cells demonstrated with ferritin a-bungarotoxin conjugates, J.Cel/ Sci. 16:473-479. Jacob, M., and Lentz, T. L., 1979, Localization of acetylcholine receptors by means of horseradish peroxidase-a-bungarotoxin during formation and development of the neuromuscular junction in the chick embryo, J. Cell BioI. 82:195-211. Jessell, T. M., Siegel, R. E., and Fischbach, G. D., 1979, Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord, Proc. Natl. Acad. Sci. USA 76:5397-5401.

Kao, I., and Drachman, D. B., 1977, Myasthenic immunoglobulin accelerates acetylcholine receptor degradation, Science 196:527-529. Karlin, A., Weill, C. L., McNamee, M. G., and Valderrama, R., 1976, Facets of the structures of acetylcholine receptors from Electrophorus and Torpedo, Cold Spring Harbor Symp. Quant. BioI. 40:203-209.

Karlin, A., Damle, V., Valderrama, R., Hamilton, S., Wise, D., and McLaughlin, M., 1978, Interactions among binding sites on acetylcholine receptors in membrane and in detergent solution, Fed. Proc. 37:121-122. Karlin, A., Damle, V., Hamilton, S., McLaughlin, M., Valderrama, R., and Wise, D., 1979, Acetylcholine receptors in and out of membranes, in: Advances in Cytopharmacology (B. Ceccarelli and F. Clementi, eds.), Vol. 3, pp. 183-189, Raven Press, New York. Klymkowsky, M. W., and Stroud, R. M., 1979, Immunospecific identification and three-dimen-

306

S. K. Malhotra

sional structure of a membrane-bound acetylcholine receptor from Torpedo californica. J. Mol. BioI. 128:319-334. Ko, P. K., Anderson, M. H., and Cohen, M. W., 1977, Denervated skeletal muscle fibers develop discrete patches of high acetylcholine receptor density, Science 196:540-542. Koelle, G. B., 1970, Neuromuscular blocking agents, in: The Pharmacological Basis of Therapeutics (L. S. Goodman and A. Gilman, eds.), pp. 601-619, Macmillan, London. Koenig, J., and Vigny, M., 1978, Neural induction of the 16S acetylcholinesterase in muscle cell cultures, Nature (London) 271:75-77. Lapa, A. J., Albuquerque, E. X., and Daly, J., 1974, An electrophysiological study of the effects of d-tubocurarine, atropine and a-bungarotoxin on the cholinergic receptor in innervated and chronically denervated mammalian skeletal muscles, Exp. Neurol. 43:375-398. Lee, C. Y., 1979, Recent advances in chemistry and pharmacology of snake toxins, in: Advances in Cytopharmacology (8. Ceccarelli and F. Clementi, eds.), Vol. 3, pp. 1-16, Raven Press, New York. Lindstrom, J., 1979, Antibodies to the acetylcholine receptor molecule and its component polypeptide chains, in: Advances in Cytopharmacology (8. Ceccarelli and F. Clementi, eds.), Vol. 3, pp. 245-253, Raven Press, New York. Lindstrom, J., Einarson, 8., and Merlie, J.-P., 1978, Immunization of rats with polypeptide chains from Torpedo acetylcholine receptor causes an autoimmune response to receptors in rat muscle, Proc. Natl. Acad. Sci. USA 75:769-773. Lindstrom, J., Cooper, J., and Tzartos, S., 1980, Acetylcholine receptors from Torpedo and Electrophorus have similar subunit structures, Biochemistry 19: 1454-1458. Lq,mo, T., and Rosenthal, J., 1972, Control of ACh sensitivity by muscle activity in the rat, J. Physiol. 221:493-513. Malhotra, S. K., 1978, Molecular structure of biological membranes: Functional characterization, in: Subcellular Biochemistry (D. 8. Roodyn, ed.), Vol. 5, pp. 221-259, Plenum, New York. Malhotra, S. K., 1980, Organization, composition, and biogenesis of animal cell membranes, in: Membrane Structure and Function (E. E. Bittar, ed.), Vol. 1, pp. 1-72, Wiley, New York. Malhotra, S. K., and Tipnis, u., 1978, Alterations in the structure of sarcolemma in a denervated skeletal muscle, Proc. R. Soc. London Ser. B 203:59-68. Merlie, J.-P., Heinemann, S., and Lindstrom, J. M., 1979, Acetylcholine receptor degradation in adult rat diaphragms in organ culture and the effect of anti-acetylcholine receptor antibodies, J. BioI. Chern. 254:6320-6327. Miledi, R., 1960, Junctional and extrajunctional acetylcholine receptors in skeletal muscle fibres, J. Physiol. 151:24-30. Miledi, R., and Potter, L. T., 1971, Acetylcholine receptors in muscle fibres, Nature (London) 233:599-603. Miledi, R., and Zelena, J., 1966, Sensitivity to acetylcholine in rat slow muscle, Nature (London) 210:855-856. Nachmansohn, D., 1955, Metabolism and function of the nerve cell, in: The Harvey Lectures. pp. 57-99, Academic Press, New York. Nathanson, N. M., and Hall, Z. W., 1980, In situ labelling of Torpedo and rat muscle acetylcholine receptor by photoaffinity derivative of a a-bungarotoxin, J. BioI. Chern. 255: 16981703. Neher, E., and Sakmann, B., 1976, Noise analysis of drug induced voltage clamp currents in denervated frog muscle fibres, J. Physiol. 258:705-729. Nelson, N., Anholt, R., Lindstrom, J., and Montal, M., 1980, Reconstitution of purified acetylcholine receptors with functional ion channels in planar lipid bilayers, Proc. Natl. Acad. Sci. USA 77:3057-306\.

Acetylcholine Receptors

307

Neubig, R. R., Krodel, E. K., Boyd, N. D., and Cohen, J. B., 1979, Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides, Proc. Natl. Acad. Sci. USA 76:690-694. Orida, N., and Poo, M., 1978, Electrophoretic movement and localisation of acetylcholine receptors in the embryonic muscle cell membrane, Nature (London) 275:31-35. Peper, K. F., Dreyer, F., Sandri, c., Akert, K., and Moor, H., 1974, Structure and ultras.tructure of the frog motor endplates, Cell Tissue Res. 149:437-455. Podleski, T. R., Axelrod, D., Ravdin, P., Greenberg, 1., Johnson, M. M., and Sal peter, M. M., 1978, Nerve extract induces increase and redistribution of acetylcholine receptors on cloned muscle cells, Proc. Natl. Acad. Sci. USA 75:2035-2039. Popot, J.-L., Demel, R. A., Sobel, A., Van Deenen, L. L. M., and Changeux, J.-P., 1978, Interaction of the acetylcholine (nicotinic) receptor protein from Torpedo marmorata electric organ with monolayers of pure lipids, Eur. J. Biochem. 85:27-42. Porter, C. W., Barnard, E. A., and Chiu, T. H., 1973, The ultrastructure localization and quantitation of cholinergic receptors at the mouse motor endplate, J. Membr. BioI. 14:383-402. Prives, J., Silman, 1., and Amsterdam, A., 1976, Appearance and disappearance of acetylcholine receptor during differentiation of chick skeletal muscle in vitro. Cell 7:543-550. Raftery, M., Blanchard, S., Elliott, J., Hartig, P., Moore, H.-P., Quast, U., Schimerlik, M., Witzemann, V., and Wu, W., 1979, Properties of Torpedo cali/arnica acetylcholine receptor, in: Advances in Cytopharmacology (B. Ceccarelli and F. Clementi, eds.), Vol. 3, pp. 159-182, Raven Press, New York. Raftery, M. A., Hunkapiller, M. W., Strader, C. D., and Hood, L. E., 1980, Acetylcholine receptor: Complex of homologous subunits, Science 28: 1454-1457. Ravdin, P. M., and Berg, D. K., 1979, Inhibition of neuronal acetylcholine sensitivity by a-toxins from Bungarus multicinctus venom, Proc. Natl. Acad. Sci. USA 76:2072-2076. Reiness, C. G., and Hall, Z. W., 1977, Electrical stimulation of denervated muscles reduces incorporation of methionine into ACh receptor, Nature (London) 268:655-657. Robertson, M., 1979, Nerves, molecules and embryos, Nature (London) 278:778-780. Ross, M. J., Klymkowsky, M. W., Agard, D. A., and Stroud, R. M., 1977, Structural studies of a membrane-bound acetylcholine receptor from Torpedo cali/arnica. J. Mol. BioI. 116:635-659. Rubin, L. L., Schuetze, S. M., Weill, C. L., and Fischbach, G. D., 1980, Regulation of acetylcholinesterase appearance at neuromuscular junctions in vitro. Nature (London) 283:264267. Sakmann, 8., 1975, Reappearance of extrajunctional acetylcholine sensitivity in denervated rat muscle after blockage with ll'-bungarotoxin, Nature (London) 255:415-416. Sakmann, B., 1978, Acetylcholine-induced ionic channels in rat skeletal muscle, Fed. Proc. 37:2654-2659. Sakmann, B., and Brenner, H. R., 1978, Change in synaptic channel gating during neuromuscular development, Nature (London) 276:401-402. Salvaterra, P. M., Gurd, J. M. and Mahler, H. R., 1977, Interactions of the nicotinic acetylcholine receptor from rat brain with lectins, J. Neurochem. 29:345-348. Sator, V., Raftery, M. A., Thomas, J. K., and Martinez-Carrion, M., 1979, Effect of cholinergic ligands and local anesthetics on acetylcholine receptor enriched membrane preparations from Torpedo califarnica electroplax, Arch. Biochem. Biophys. 192:250-259. Schalow, G., and Schmidt, H., 1979, Local development of action potentials in slow muscle fibres after complete or partial denervation, Proc. R. Soc. London Ser. B 203:445-457. Schuetze, S. M., Frank, E. F., and Fischbach, G. D., 1978, Channel open time and metabolic stability of synaptic and extrasynaptic acetylcholine receptors on cultured chick myotubes, Proc. Natl. Acad. Sci. USA 75:520-523.

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Shorr, R. G., Dolly, J. 0., and Barnard, E. A., 1978, Composition of acetylcholine receptor protein from skeletal muscle, Nature (London) 274:283-284. Sikerwar, S. S., and Malhotra, S. K., 1979, Visualization of mitochondrial coupling factor F, (ATPase) by freeze-drying, Cell Biophys. 1:55-63. Sobel, A., Hofler, J., Heidmann, T., and Changeux, J.-P., 1979, Structural and functional properties of the acetylcholine regulator, in: Advances in Cytopharmacology (8. Ceccarelli and F. Clementi, eds.), Vol. 3, pp. 191-196, Raven Press, New York. Steinbach, J. H., Merlie, J.-P., Heinemann, S., and Bloch, R., 1979, Degradation of junctional and extrajunctional acetylcholine receptors by developing rat skeletal muscle, Proc. Natl. Acad. Sci. USA 76:3547-355l. Stevens, C. F., 1980, The acetylcholine receptor, Nature (London) 287:13-14. Sumikawa, K., and O'Brien, R. D., 1979, A comparison of the subunits of the acetylcholine receptor from electric eel and Torpedo californica. FEBS Lett. 101:395-398. Sytkowski, A. J., Vogel, Z., and Nirenberg, M. W., 1973, Development of acetylcholine receptor clusters on cultured muscle cells, Proc. Natl. Acad. Sci. USA 70:270-274. Tarrab-Haxdai, R., Geiger, 8., Fuchs, S., and Amsterdam, A., 1978, Localization of acetylcholine receptor in excitable membrane from the electric organ of Torpedo: Evidence for exposure of receptor antigenic sites on both sides of the membrane, Proc. Natl. Acad. Sci. USA 75:2497 -250 l. Tarrab-Hazdai, R., Bercovici, T., Goldfarb, V., and Gitler, C., 1980, Identification of the acetylcholine receptor subunit in the lipid bilayer of Torpedo electric organ excitable membranes, J. Bioi. Chem. 255:1204-1209. Tewari, J. P., and Malhotra, S. K., 1979, Visualization of intrinsic proteins in cross-fractured membranes in rotary-shadowed freeze-fracture replicas, Microbios Lett. 9:35-46. Tipnis, u., and Malhotra, S. K., 1976, Denervation of skeletal muscle: Changes in the structure of nonsynaptic sarcolemma, FEBS Lett. 69:141-143. Tipnis, V., and Malhotra, S. K., 1977, Nerve-muscle interaction: Changes in the cellular organization of a skeletal muscle upon denervation, Cytobios 19:181-227. Tipnis, V., and Malhotra, S. K., 1979a, Acetylcholinesterase (AchE) and acetylcholine receptor (AchR) localization in adult mammalian skeletal muscle, Abstract, 8th Annual ICNVCLA Symposia, Molecular and Cellular Biology, Keystone, Colorado, p. 114. Tipnis, V. R., and Malhotra, S. K., 1979b, a-Bungarotoxin binding sites (acetylcholine receptors) in denervated mammalian sarcolemma, J. Supramol. Struct. 12:321-334. Tipnis, V. R., and Malhotra, S. K., 1980, Junctional and extrajunctional acetylcholine receptors, Can. J. Physiol. Pharmacol. 58:445-458. Vandlen, R. L., Wu, W. c.-S., Eisenach, C., and Raftery, M. A., 1979, Studies of the composition of purified Torpedo californica acetylcholine receptor and of its subunits, Biochemistry 18: 1845-1854. Vincent, A., 1976, Experimental myasthenia gravis-A new autoimmune model, Trends Biochem. Sci. 1:289-29l. Vogel, Z., and Daniels, M. P., 1976, Ultrastructure of acetylcholine receptor clusters on cultured muscle fibers, J. Cell Bioi. 69:501-507. Vogel, Z., and Nirenberg, M., 1976, Localization of acetylcholine receptors during synaptogenesis in retina, Proc. Natl. Acad. Sci. USA 73:1806-1810. Vogel, Z., Sytkowski, A. J., and Nirenberg, M. W., 1972, Acetylcholine receptors of muscle grown in vitro. Proc. Natl. Acad. Sci. USA 69:3180-3184. Weeds, A., 1979, Protean muscle, Nature (London) 282:232-233. Weinberg, C. G., and Hall, Z. W., 1979, Antibodies from patients with myasthenia gravis recognize determinants unique to extrajunctional acetylcholine receptors, Proc. Natl. Acad. Sci. USA 76:504-508.

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Wise, D. S., Karlin, A., and Schoenborn, B. P., 1979, An analysis by low-angle neutron scattering of the structure of the acetylcholine receptor from Torpedo californica in detergent solution, Biophys. J. 28:473-496. Yee, A. G., Fischbach, G. D., and Karnovsky, M. J., 1978, Clusters of intramembranous particles on cultured myotubes at sites that are highly sensitive to acetylcholine, Proc. Natl. Acad. Sci. USA 75:3004-3008. Younkin, S. G., Brett, R. S., Davey, B., and Younkin, L. H., 1978, Substances moved by axonal transport and released by nerve stimulation have an innervation-like effect on muscle, Science 200:1292-1295. Zingsheim, H. P., Neugebauer, D.-C. H., Barrantes, F. J. and Frank, A., 1980, Structural details of membrane-bound acetylcholine receptor from Torpedo marmorata, Proc, Natl. Acad. Sci. USA 77:952-956. Ziskind, L., and Dennis, M. J., 1978, Depolarising effect of curare on embryonic rat muscles, Nature (London) 276:622-623.

Chapter 7

Immunological Studies of Tissue Proteinases A. R. Poole Joint Diseases Laboratory Shriners Hospital for Crippled Children McGill University Montreal, Quebec, Canada

1.

INTRODUCTION

In this survey, only tissue proteinases that catalyze the degradation of proteins in mammalian tissues are considered. Proteinases that are involved in digestion within the intestine will not be included. The development of immunological studies of tissue proteinases has permitted, as in other fields, a better understanding of the biochemistry and physiology of these enzymes. Many proteinases share similar if not almost identical substrate specificities, rendering identification by biochemical methods often difficult and inconclusive. Immunological methods have permitted the resolution and identification of discrete proteinases in mixtures and tissues. Inactive proteinases can also be detected and assayed. The use of antibodies to localize proteinases has likewise circumvented problems of identification encountered in histochemistry, again arising from similar substrate specificities. The immunological approach permits a definitive identification to be made if antisera are well characterized and monospecific: both precipitating and nonprecipitating reactions must be studied in this characterization. Polyvalent antisera (which react with more than one molecular species) can be very useful provided they are used with care and with regard to their lack of monospecificity. The ability of antibodies to inhibit proteinase activity against high-molecular-weight substrates permits analyses of the importance and significance of proteinases. Since low-molecular-weight substrates can usually be hydrolyzed in immune 311

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complexes, precipitated proteinases can be identified in immune complexes, such as precipitin lines, by their activities. In this chapter I will review work on tissue proteinases that has usually employed an immunological approach alongside more conventional biochemical methods. The advantages are in many cases obvious and yet it is surprising how little has been done to exploit the full potential of an immunological approach. Frequently, only limited efforts have been and still are made to assess the specificities of antisera, a precise knowledge of which provides the backbone of all work with antibodies. The survey concludes with a brief review of immunological methods used to study proteinases, with recommendations for their use. It is hoped that this review will help to extend our knowledge of proteinases by encouraging better and more common use of immunological methods, and so ultimately help to improve our understanding of the intracellular biochemistry of these important enzymes.

2. 2.1.

CATHEPSIN D Antiserum Production

The lysosomal proteinase cathepsin D occupied increasing attention in the 1960s. A key role in the catabolism of proteoglycans of cartilage was proposed by the Strangeways Research Laboratory group of Fell, Dingle, Barrett, and their collaborators (for reviews see Barrett, 1968; Dingle, 1969). This resulted from a variety of studies based mainly on correlations between excessive cartilage proteoglycan degradation induced in culture and enhanced cathepsin D secretion. As a result of a direct requirement for a means of inhibiting this enzyme, and thereby seeing if cartilage degradation was· inhibited, it was decided to raise an antibody to cathepsin D. So started an important avenue of research into tissue proteinases employing new immunological methods. Antisera were first raised to chicken cathepsin D (Weston, 1969), which had been purified by Barrett (1970). This species was chosen since the majority of the experimental work on cathepsin D and cartilage degradation at that time involved the use of chick embryonic cartilages. Antisera were later also raised against cathepsin D purified from rabbit and human livers (Dingle et at., 1971). Some antisera were raised in rabbits and others in sheep. In general, potent precipitating antisera were obtained in sheep after only two intramuscular injections of a total of 3-5 mg of soluble enzyme with complete Freund's adjuvant. Injection of precipitin lines, made up of an earlier nonspecific antiserum and purified antigen, was also used to produce monospecific antisera using only 0.25 mg of antigen. At this time an antiserum was designated spe-

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cific only if it gave a single precipitin line, containing cathepsin D, after 24 hr diffusion at room temperature. Tissue homogenates and possible contaminant proteins of the immunizing antigen were used together with purified cathepsin D. Enzyme preparations shown to be homogeneous by isoelectric focusing (Barrett, 1970) often produced monospecific antisera (Dingle et al., 1971).

2.2.

Inhibition by Antisera

The resultant antisera were subsequently shown in vitro to inhibit activity of the purified enzyme on hemoglobin. Inhibition was maximal at pH 5.0 and barely detectable at pH 3.0; only 50 percent inhibition was detected at pH 4.0. Analyses of immunoprecipitates revealed that when only three or four antibody molecules were bound to one molecule of cathepsin D, little inhibition was observed. All activity was lost when six or seven antibody molecules were bound, although up to 11 molecules of antibody could bind to one molecule of enzyme (Dingle et al., 1971). Monovalent Fab antibody fragments were also inhibitory. Hence, inhibition and immunoprecipitation are not interrelated. Instead, enzyme inhibition was considered to result from a steric blockade of substrate accessibility created by immunoglobulin bound to the enzyme. This conclusion was supported by the recent observations of Whitaker (1980), who showed that pepstatin, an active-site-directed reagent, could totally inhibit cathepsin D activity and yet not affect immunoreactivity. Antisera to cathepsin D were shown to be species specific but not tissue or organ specific (Dingle et al., 1971; Whitaker, 1980). Moreover, the various isozymes proved to be immunologically similar. As a basis for work to investigate the importance of cathepsin D in proteoglycan degradation in living cartilage and hemoglobin digestion within cells, the antiserum was shown to be capable of completely inhibiting the degradation of purified cartilage proteoglycan and of hemoglobin (Figure 1) (Dingle et al., 1971). The importance of cathepsin D in intracellular digestion was studied in a novel series of experiments by Dingle et al. (1973). They demonstrated that living rabbit alveolar macrophages endocytosed both [3H] hemoglobin and monospecific antibody to rabbit cathepsin D. Hemoglobin was concentrated in secondary lysosomes containing cathepsin D. The antibody, however, was also present in some of these secondary lysosomes and inhibited the subsequent intracellular digestion of hemoglobin (Figure 2). This arrest of digestion was associated with ultrastructural changes indicative of cells suffering a storage disease, resulting from an enzyme deficiency. Many large vacuoles appeared containing membranous whorls. Removal of exogenous antibody rapidly led to a return to normal morphology and normal hemoglobin digestion. This demonstration of the functional importance of intracellular cathepsin D in a living cell is one of the most striking and successful applications of the use of anti-

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FIGURE 9. Extracellular cathepsin B in human rheumatoid synovium. Tissue was cultured with a sheep antibody to human cathepsin B, and extracellular immunoprecipitates near the natural edge of the synovium were demonstrated as described in Figure 4. From Mort and Poole (unpublished, 1981).

used earlier by Poole et al. (1976) to demonstrate extracellular cathepsin D in rheumatoid synovium. Thus, both proteinases have an extracellular presence and may be implicated in the degradation of connective tissue macromolecules in rheumatoid joints. Immunohistochemical methods have also been used to localize cathepsin B in intracellular sites (Figure 10) (Mort et al., 1981). The fine particulate distribution of enzyme observed in human fibroblasts closely resembles that determined for another lysosomal proteinase, cathepsin D (Figure 3). Studies of rabbit cathepsin Bin myocytes of the left ventricle have revealed some striking differences in localization to cathepsin D (Poole, Mort, and Decker, unpublished observations, 1980). Whereas cathepsin D is normally located in lysosomes with paranuclear concentrations, cathepsin B appears to be present in only a few lysosomal sites away from the nucleus. Electron microscopy is now being used to resolve these differences. It is clear that there is a heterogeneity of lysosomal proteinase distribution in some cells that was not previously imagined.

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FIGURE 10. Intracellular localization of cathepsin B in human fibroblasts using a monospecific antiserum to human cathepsin B. Cells were fixed in formaldehyde and extracted with acetone. They were treated with sheep IgG antibody to cathepsin B followed by fluorescein-labeled pig IgG antibody to sheep IgG. A typical lysosomal distribution resembling that seen for cathepsin D (see Figure 3) was observed. See also Mort et al. (1981).

4.

COLLAGENASE

Collagenase is the only mammalian enzyme capable of cleaving the helical portion of collagen. It is a metalloproteinase and produces characteristic "f' and "!" helical fragments from tropocollagen, which can then "unwind" and be degraded by other proteinases. Since collagen is the most ubiquitous extracellular macromolecule, providing tissues such as cartilage, ligament, tendon, skin, and bone with much of their physical strength, it is no wonder that an enzyme capable of degrading this molecule has attracted considerable interest. As in the case of other proteinases where existing methods were limited in their application and sensitivity, the development of immunological means with which to study the enzyme has provided the motivation behind the work of several laboratories. Human skin collagenase was purified by Bauer et al. (1970) by chromatography on Sephadex G-150 followed by Sephadex G-7S. Two "peaks" of col-

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lagenase activity were recovered, one of which migrated faster on polyacrylamide gel electrophoresis and appeared as a single band free of general proteolytic activity. This preparation was used to immunize rabbits. The resultant antisera were pooled and fractionated with 33 percent saturated ammonium sulfate to remove inhibitory serum proteins. The antiserum reacted in immunodiffusion both with the fast-moving skin collagenase and with the slower-moving, less pure species. No differences in identity were observed. Partially purified preparations of tadpole collagenase, and of collagenase secreted from human gingiva and rheumatoid synovia, and collagenases purified from postpartum rat uterus and crustacean hepatopancreas were also examined. Antisera to the human skin collagenase precipitated human collagenase in gels and solution but did not react with nonhuman species; antibodies to the rat and tadpole enzymes showed no reaction with human collagenase. None of the antibodies reacted with crustacean or bacterial collagenases, indicating the species-specific character of these antibodies. These studies were later extended by the development of a radioimmunoassay for collagenase employing radioiodinated purified human skin collagenase (Bauer et al., 1972). It was confirmed that human synovial and gingival collagenases were immunologically closely related and, in addition, showed that human granulocyte collagenase is also related. Baboon gingival collagenase was shown to be less related and rat skin unrelated. Collagenase was detected with this assay in human skin tissue extracts where no collagenase was measurable by enzymatic assay. These early observations suggested the presence of either a precursor or an enzyme-inhibitor complex, both of which are immunologically detectable. The studies of Harper et al. (1971) and Vaes (1972) have also suggested that zymogens of collagnase may be present in the tadpole and mouse bone, respectively. Harper et al. (1971) also made an immunological examination of tadpole collagenase using IgG purified from rabbit antisera. These antibodies also precipitated the enzyme and inhibited its ability to cleave native guinea pig collagen in solution, measured both viscometrically and in a 14C-labeled collagen fibril assay. The antibodies also inhibited collagen gel lysis by live ex plants of tadpole fin tissue. Extracts of tadpole tail fin tissue contained no detectable collagenase activity and yet antibody was removed when it was incubated as IgG with these extracts; this was not due to proteolytic degradation of the antibody. These observations thus suggested the presence of an inactive form of collagenase. Polyacrylamide gel analysis of the crude extract and of collagenase purified from this extract revealed that the extract contained a band that co-migrated with the active purified collagenase and was immunologically identical to it, as shown by double immunodiffusion. This latent enzyme was subsequently shown by Harper and co-workers to be activatable with culture medium in which living tadpole tail fin tissue had been maintained for several

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days. The activated enzyme was also inhibited by IgG antibody. Hence, these authors described the latent enzyme as a zymogen of collagenase. Ohlsson and Olsson (1973) purified two collagenolytic enzymes of different electrophoretic mobility from leukocytes obtained from a patient with chronic myeloid leukemia. Immunodiffusion and crossed immunoelectrophoresis revealed that these species were essentially immunologically identical. Unfortunately, no attempts were made to study the cross-reactivity of this antiserum with other human collagenases. Moreover, these collagenolytic enzymes also degraded fibrinogen and cartilage proteoglycan, and hydrolyzed collagen to give characteristic i and ｾ＠ pieces. They had molecular weights of 76,000 and probably each contained two subunits of 42,000 and 33,000, revealed by treatment with SDS followed by electrophoresis. Werb and Burleigh (1974) reported the purification of collagenase from rabbit fibroblasts grown in monolayer culture. Antisera were prepared in sheep (Werb and Reynolds, 1975a). In one case the purified enzyme was reacted with lX2 macroglobulin before injection to enhance purification. The resultant antisera were used to prepare immunoprecipitates containing collagenase, and these were used to raise another antiserum that was again reacted with collagenase to produce a further immunoprecipitate that was injected into a third sheep. This precipitin line approach had been used earlier by Dingle et al. (1971) to prepare specific antisera to cathepsin D when other approaches had failed. The antisera gave only one precipitin line (in dye-stained gels) in double immunodiffusion or Laurell immunoelectrophoresis against purified and crude preparations of collagenase. The purified IgG from the final antiserum to rabbit collagenase was shown to be capable of inhibiting the activity of this enzyme. In addition, the Fab' fragment prepared with pepsin was also shown to be inhibitory (mol/mol) (Werb and Reynolds, 1975a). These workers subsequently showed (197 5b) that an antiserum to rabbit synovial fibroblast collagenase would precipitate and inhibit collagenase secreted from rabbit skin, synovial fibroblasts, ear fibrocartilage, from subchondral bone, and from inflamed synovia of rabbits with experimental arthritis. A collagenase extracted from a rabbit carcinoma also reacted with the antiserum. Collagenase in human synovium and macrophages proved to be immunologically dissimilar. Moreover, unlike the earlier findings of Bauer et al. (1972), Werb and Reynolds found that collagenase in rabbit polymorphonuclear leukocytes was nonreactive, indicating that the collagenase present within these cells is an enzyme distinct from that found in other cells and tissues. A rabbit antiserum to human skin collagenase was used by Reddick et al. (1974) to study this enzyme in human skin. They found that immunoreactive collagenase was mainly concentrated in the upper or papillary portion of the human dermis, apparently in extracellular sites bound to collagen. In cultured skin fibroblasts, enzmye was also localized intracellularly in an unusual gran-

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ular and reticulated staining pattern consistent with its association with the endoplasmic reticulum. An immunochemical study of collagenase in 21 cases of basal cell carcinoma revealed that there was an approximately twofold increase in the enzyme detected by radioimmunoassay in tissue extracts (Bauer et al., 1977). Localization studies revealed that enzyme was not detectable within or around the tumor cells but was seen in connective tissue at the edge and within the tomors. Again, the collagenase was extracellular and appeared to be bound to collagen. The importance of collagenase in the erosion of articular cartilage in rheumatoid arthritis has been the subject of much investigation since Evanson et al. (1968) demonstrated that rheumatoid synovium in culture secretes collagenase. Woolley et al. (1976) subsequently purified collagenase from such cultures and prepared an antiserum in sheep. Using immunodiffusion, immunoelectrophoresis, and immunoinhibition, they demonstrated that this antiserum was not tissue specific, since it reacted strongly with collagenase purified from human gastric mucosa, cornea, and skin. The enzyme in granulocytes did not detect ably react with this antibody, indicating differences in structure. This observation agreed with that of Werb and Reynolds (197 5b), who studied rabbit collagenases, and disagreed with the observation of Bauer et al. (1972). Moreover, immunoinhibition studies also revealed that the antibody to human synovial collagenase also reacted with rabbit corneal and dog gingival collagenases. The binding of enzymatically active collagenase to insoluble collagen (Woolley et al., 1976) and to soluble collagen (Harper et al., 1971) in suspension and in tissue sections (Woolley et al., 1977) was also demonstrated. These were important observations since the immunohistochemical studies of Reddick et al. (1974) had earlier indicated that this probably occurs in tissues. Many studies have recently been concerned with the latency of collagenase, secreted from cells and tissues, which was discussed earlier. Collagenase is frequently present in culture media in an enzymatically inactive form and can be activated chemically by the addition of 4-aminophenyl mercuriacetate or trypsin (Sellers et al., 1977). In order to be able to use a monospecific antibody to study an enzyme such as collagenase, it is very important to determine whether such latent enzyme species are detected by antibody. Woolley et al (1976, 1977) found that when synovial collagenase was totally inhibited by whole serum or by the serum inhibitors of collagenase, namely (\'2 macroglobulin and {3t anticollagenase, the proteinase was no longer precipitated by antibody in immunodiffusion. More recently, these workers found that treatment of sections with the above-mentioned activators of collagenase failed to reveal any more immunohistochemically demonstrable collagenase (Woolley et al., 1977). They also observed that when collagenase was secreted in a latent form from synovial cell cultures, collagenase was not detectable with precipitating antibody until active enzyme was secreted (Woolley et al., 1978). In later reports,

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it was stated that four out of six enzymatically latent collagenase preparations (presumably prepared from secreted enzymes) were reactive with precipitating anticollagenase antibodies (Woolley et al., 1979). These reactions were enhanced after activation with trypsin, suggesting that activation unmasked antigenic sites. Previously, immunoreactive latent collagenase was demonstrated in tissue extracts by Bauer et al (1972) and by Harper et al. (1971). The question of immunoreactivity versus latency may be related to the nature of the latent enzyme and the reactivity of the antibody. If the latent collagenase is in the form of a zymogen, it would appear to be immunoreactive, whereas if latency is the result of binding of an inhibitor to the active enzyme, then immunoreactivity may also be masked by steric hindrance caused by the inhibitor. In studies of the binding of latent and active collagenases to tissue sections, only binding of active enzyme was detected after reaction with tissue sections (Woolley et al., 1979). This observation is essentially in agreement with that of Harper et al. (1971), who showed immunologically that only enzymatically active tadpole tail collagenase would bind to collagen fibrils in solution. Thus, these studies together suggest that only enzymatically active collagenase may bind to extracellular collagen and yet latent enzyme is immunologically detectable. The earlier work of Bauer et al. (1972) used a sensitive radioimmunoassay to detect inactive collagenase. This method detects both non precipitating and precipitating antibodies to collagenase. The studies of Woolley and colleagues on latent collagenase and its direct reactivity with antibody were restricted to studies of precipitating antibodies. Further work is needed before a true evaluation of the immunological reactivity of latent collagenase is possible. Moreover, one must always remember that different animals invariably produce different antisera, in that their site-reactive specificities for antigen can differ. Hence, it is not possible to directly compare antisera and to expect to see similar reactivities. Allowance for this must always be made when comparing similar work from different laboratories. Ultrastructural studies of cartilage erosion at the edges of rheumatoid synovia have revealed that collagen is grossly destroyed adjacent to infiltrating synovial cells (Harris et al., 1970, 1977). Often an amorphous-looking degraded matrix results in which there are no longer recognizable collagen fibrils. Using immunolocalization, human synovial collagenase was frequently demonstrated at the junctions of cartilage and synovium freshly excised from rheumatoid joints (Figure 11) (Woolley et al., 1977). More remote from this site, collagenase was never detected in cartilage ahead of this synovium and was only localized in synovium in scattered discrete pericellular sites. These observations together indicate a primary involvement of collagenase in cartilage destruction in rheumatoid arthritis. They also indicate, together with those of Poole et al. (1976), that the proteinases detected immunohistochemically at

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FIGURE 11. Immunolocalization of collagenase at the junction of articular cartilage and invading rheumatoid synovium (pannus) in a rheumatoid joint. The diffuse bright-green immunofluorescent staining for collagenase is concentrated in the cartilage at and close to this junction (asterisks). There are also some small discrete patches of staining for collagenase in the invading synoyium close to this junction (arrows). Modified from Woolley et af. (I977) with permission.

the edges of rheumatoid synovium invading articular cartilage are almost certainly derived from synovial cells. There is no good reason to dismiss an earlier contribution of proteinases from chondrocytes ahead of the invasion edge, as is suggested by the more recent work from Dingle and colleagues (1979). They have observed that synovia release a group of molecules that have the capacity to activate chondrocytes, leading to the destruction of the cartilage matrix surrounding them. However, the gross degradation observed at the interface between synovium and cartilage would seem to result from the direct activities of synovial cell proteinases. These immunohistochemical studies were later extended to determine the cellular origin of collagenase in rheumatoid synovia. By enzymatic dispersion of rheumatoid synovium, cells of a fibroblastic, macrophagelike, and dendritic (stellate) morphology were observed after attachment to collagen coated coverslips. The collagen permitted the capture, by binding, of any collagenase secreted by these cells and the demonstration, by antibody localization, that it was the dendritic cells around which immunoreactive collagenase was often

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detected (Woolley et al., 1978). Thus, these cells are mainly responsible for the secretion of detectable enzyme, which mayor may not be active. In an effort to understand more about the importance of collagenase in the process of bone resorption mediated by osteoclasts, Sakamoto et al. (1978) have purified mouse bone collagenase from tissue culture medium and raised an antiserum against it in a rabbit. The antiserum was characterized using immunodiffusion and immunoelectrophoresis. It reacted with the inhibited collagenase in other mouse tissues but only partly reacted with rat and chick collagenase; there was no cross-reaction with human gingival collagenase. They also observed that both latent mouse collagenase and trypsin-activated collagenase reacted with the antibody. Earlier studies on collagenase localization by Montford and PerezTamayo (1975a,b) involved the use of antisera that were only characterized to a limited degree. Rat uterine collagenase was purified and injected into a rabbit. No photographic evidence was provided in support of antisera specificities, which were only assessed by immunodiffusion, a method so often used but of very limited specificity and sensitivity even when gels are stained for precipitin lines to enhance sensitivity. No mention was made of precipitin line staining by these workers; neither did Sakomoto et al. (1978) report any attempt to stain precipitin lines after immunodiffusion. Montford and Perez-Tamayo reported that only one precipitin line was observed (by dark ground observation) with various enzyme preparations and that the reaction failed to appear after absorption with the crude enzyme preparation. This observation adds nothing to one's understanding of antiserum specificity. The authors proceeded to use their antiserum to produce staining of a variety of rat tissues including brain, myocardium, lung, liver, spleen, kidney, uterus, cartilage, tendon, skin, and skeletal muscle. Staining was so commonplace as to suggest that collagenase is distributed in a wide variety of tissues bound to collagen, presumably in a latent form. Otherwise, one might imagine that the collagen in these tissues would readily disintegrate if the enzyme was active. However, in view of the lack of data to support the specificity of this antiserum, a conclusive understanding of the significance of these data must await a more detailed description of anteserum specificity.

5.

ELASTASE AND CATHEPSIN G

These are two major neutral proteinases of polymorphonuclear leukocytes each with the capacity to degrade both collagen and proteoglycan (Starkey et al., 1977). These workers showed that both enzymes can solubilize insoluble cartilage type II collagen. Elastase could also solubilize less effectively type I collagen from tendon, but cathepsin G had little or no effect on type I collagen

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of bone or tendon. Cartilage proteoglycan is also cleaved by both proteinases, which attack the protein core (Starkey et al., 1977; Keiser et al., 1976). A variety of other proteins are also hydrolyzed and, of course, elastase is capable of degrading elastin, the insoluble protein of connective tissue that is normally extremely resistant to proteolytic attack (Starkey, 1977). Three elastases (of different electrophoretic mobility) were purified from human leukemic myeloid cells by Ohlsson and Olsson (1974). Rabbit antisera raised against each of these enzymes gave a single precipitin reaction of complete identity in double immunodiffusion and crossed immunoelectrophoresis when the three elastases were compared with each other using the three different antisera. A single line was also obtained when anyone of the antisera was tested against a partially purified elastase preparation isolated from normal human granulocytes, which also contained three elastase species. Elastase and cathepsin G were later purified from human spleen (Starkey and Barrett, 1976a-c). Antisera to elastase were prepared both to elastase bound to ovoinhibitor and to elastase in an immune complex with antibody as precipitin lines (Starkey and Barrett, 1976b). Partially purified antibody to elastase was shown to be capable of inhibiting the lysis of elastin by elastase in agarose diffusion gels. Cathepsin G was also purified by Feinstein and Janoff (197 5a) from human polymorphonuclear leukocytes and shown to be nonreactive with a monospecific antiserum prepared to human polymorphonuclear leukocyte elastase (Feinstein and Janoff, 1975b). These latter authors reported only immunodiffusion analyses to assess antisera specificity, and only described results for unstained immunodiffusion gels using purified enzyme and crude granular extracts. Their antiserum to elastase was later used to demonstrate the penetration of purified elastase into rabbit articular cartilage. The enzyme penetrated both intact articular surfaces and cut cartilage surfaces, and this was associated with the release of 35S04-labeled proteoglycan from the cartilage as a result of proteoglycan degradation. This was an important observation since it demonstrated that elastase released from polymorphonuclear leukocytes, which are present in large numbers in the synovial fluids of inflamed joints, could degrade and penetrate cartilage to a significant depth and hence play an important role in the pathophysiology of cartilage destruction. The strong basicity of elastase no doubt plays an important role in this ability of elastase to penetrate and degrade the acidic cartilage matrix. As with all proteinases, the expression of proteinase activity is dependent on the effects of various proteinase inhibitors which may be capable of inhibiting proteinases both within cartilage and outside it. Thus, in a parallel study it was demonstrated by Janoff and co-workers (1976) that the major serum inhibitor of elastase, namely a 1antitrypsin inhibitor (Ohlsson and Olsson, 1974), failed to penetrate articular cartilage under similar conditions. This would suggest retention of the activity of elastase that has already penetrated the cartilage matrix, whereas elastase

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reactive with this inhibitor in synovial fluid or in other extraarticular sites may be rapidly inactivated by this and other inhibitors such as lX2 macroglobulin, which are too large to penetrate the matrix. The extracellular activity of elastase in the lung normally appears to be under physiological control as a result of the presence of an excess of lXI-antitrypsin inhibitor. Under certain conditions, elastase activity may exceed the inhibitory activity in the lung. This would favor proteolytic destruction of alveolar septal connective tissue and resultant hyperinflation of respiratory airways. There are genetic forms of the disease emphysema associated with reduced levels of lXI-antitrypsin inhibitor (Janoff et al., 1977), which result in such pathological changes. It was thought that this resulted in uncontrolled activity of elastase in extracellular sites. This hypothesis was supported by the observation that purified human neutrophil elastase instilled into dog lungs produced anatomic evidence of emphysema after 90 min (Janoff et al., 1977). By using the specific antiserum to elastase described previously (Feinstein and Janoff, 197 5b), Janoff et al. (1977) showed that elastase was bound to elastic fibers in lung septa in sites showing clear anatomic lesions. They also observed an uptake of elastase into alveolar macrophages, suggesting that these cells might act to remove excess elastase as a protective measure.

6.

SERINE PROTEINASES OF SKIN AND MUSCLE

The major neutral proteinase of rat skin was purified by Seppa and Jarvinen (1978) using 2 M KCl salt solubilization, and precipitation followed by chromatography on Sephadex G-200 and hydroxyapatite. The enzyme is a serine proteinase and resembles bovine chymotrypsin in its substrate specificity and inhibition. Treatment of rats with compound 48/80, which stimulates mast cell degranulation, decreased the amount of this proteinase to 2 percent that of control levels. A monospecific antiserum to this enzyme was then prepared and used to investigate further the localization of this proteinase in rat skin. By using immunofluorescence microscopy (Seppa, 1978), the enzyme was specifically localized in skin in mast cell granules, confirming the biochemical observations. These scattered cells were localized beneath the epidermis along subdermal vessels and also in the dermis. The cells also stained with the cytochemical substrate napthol AS-D chloroacetate, which is hydrolyzed by the purified neutral protease of rat skin (Seppa and Jarvinen, 1978). The proteinase was also demonstrated in the granules of isolated rat mast cells. The poor solubility of the enzyme in dilute salt solutions permitted localization of the proteins in unfixed tissue sections. The specificity of the antiserum was assessed using double immunodiffusion in agarose containing 1 M NaCI to keep the enzyme in solution. The anti-

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serum produced a single line of precipitation with the purified protease and with skin extracts that contained biochemically measurable enzyme activity. In skin extracts where no enzyme activity was detectable, no antiserum reaction was observed. The antiserum was also tested against the neighboring fractions from the last stage of purification, namely hydroxyapatite chromatography, as they would contain substances that would most probably be contaminants of the purified enzyme: no precipitates were observed. Absorption of the antiserum with the purified enzyme also blocked the histochemical reaction. Chymotrypsinlike serine proteinases in muscle homogenates have been studied by many wokers (see review by Bird et al., 1980). Noguchi and Kandatsu (1971) observed similarities between a muscle serine proteinase and the chymotrypsinlike enzyme of mast cells. The possibility that this muscle proteinase was actually present in mast cells was indicated by the work of Park et al. (1973), who showed that in rats treated with compound 48/80, which can degranulate mast cells, the serine proteinase was not detected in homogenates of skeletal muscle. Recently, Woodbury et al. (1978) demonstrated identical physical, chemical, and immunological properties for the muscle serine proteinase and the chymotrypsinlike protein isolated from peritoneal mast cells.

7. ACROSIN Mammalian spermatozoa possess an acrosomal proteinase that is involved in fertilization (Morton, 1977). The enzyme closely resembles pancreatic trypsin and like this enzyme hydrolyzes benzoylarginine ethyl ester and tosylarginine methyl ester. Both proteinases have optima at pH 8.0 and are stable at pH 7.0. In order to establish the relative identities of these two proteinases, Zaneveld et al. (1973) prepared antisera in rabbits to lyophilized human acrosomal extract and to purified human pancreatic trypsin. IgG antibodies from antisera were partially purified by ammonium sulfate precipitation to remove proteinase inhibitors that would otherwise interfere in the assessment of inhibitory activity against these proteinases. Using a gelatinase plate assay to assess inhibitory activities of antisera, they showed that the antiserum to human purified trypsin inhibited only pancreatic trypsin and that the antiserum to the human acrosomal extract inhibited only the proteinase activity within this extract. This lack of cross-reaction was confirmed by double-immunodiffusion experiments, which showed no sign of cross-reactivity between the pancreatic enzyme and the acrosomal extract. At a later date, Morton (1975) purified acrosin from ram spermatozoa and raised a monospecific antiserum to this proteinase. Initially, he obtained an impure enzyme that resulted in the production of a multivalent rabbit anti-

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serum. Monospecific antisera were subsequently raised in rabbits by cutting out rabbit antibody-sheep acrosin precipitin lines from immunodiffusion gels and using them as immunogens. These antisera were vigorously tested against possible contaminant antigens as well as against purified enzyme by using immunodiffusion, and Laurell immunoelectrophoresis gels* were stained for protein and for proteinase activity. Antibody to ovine acrosin cross-reacted with bovine acrosin. The antiserum to acrosin was used to localize this proteinase in freshly ejaculated, or epididymal, ram spermatozoa. Formaldehyde-fixed cells were treated with Fab monomer fragments of IgG, used both for the first step (immune rabbit serum to acrosin) and for the second step where peroxidase or fluorescein-labeled pig anti-rabbit Fab was employed. The acrosomes (anterior regions of sperm heads) were stained, particularly in the equatorial region. Combined with biochemical data, the results indicated that acrosin is mainly bound to the inner acrosomal membrane.

8.

PLASMINOGEN ACTIVATORS

Plasminogen activators are a group of enzymes capable of converting the plasma precursor plasminogen into plasmin, a serine proteinase. One such enzyme is urokinase, which has been isolated from urine and from fetal kidney cells. Urokinase is secreted by a variety of cells and has been implicated in malignancy since it is often excessively secreted from oncogenic virus-transformed cells in culture (see review by Christman et al., 1977). Virtually all of the earlier work done on this enzyme was concerned with its characterization, purification, and, in particular, mechanism of secretion. Earlier immunological studies on plasminogen activators have been reviewed by Christman et al., (1977). The main conclusion from a variety of studies employing antibodies to urokinase from different species in intraspecies studies was that plasminogen activators from different tissues of the same species are immunologically different. However, the work of Bernik and colleagues (1969, 1973, 1974) concluded, contrary to other findings, that rabbit antibodies to human urokinase inhibit this enzyme and plasminogen activators extracted from heart and blood vessels and plasminogen activators produced by cultured human cells. Christman et al. (1977) prepared a precipitating antiserum to plasminogen activator purified from the culture media of SV40-transformed hamster embryo cells. The antibody inhibited fibrinolysis in agar gels containing purified plasminogen, plasminogen activator, and fibrin. Other plasminogen acti*In this procedure, antigen is electrophoresed into an antibody-containing gel to form a "rocket" of precipitation.

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vators were also inhibited: these were contained in another line of hamster lung cells as well as that produced in primary explants of newborn hamster lung. The plasminogen activators produced by primary explants of newborn hamster kidney or by an established line of hamster cells were not inhibited. In addition, two lines of Rous sarcoma virus-transformed hamster cells and a line of benzpyrene-transformed hamster cells also produced plasminogen activator that was immunologically different from that produced by hamster lung cells and SV40-transformed hamster embryo cells. These studies indicate that there is immunological and hence structural diversity in the plasminogen activators produced by cells and that transformed cells may produce an enzyme different from that seen in some normal cells. Shakespeare and Wolf (1979) prepared an antiserum to human urokinase that inhibited the fibrinolytic activity of this proteinase. Using immunodiffusion, they usually detected single major immunoreactive species in human sera that showed a reaction of identity with urokinase. Addition of urokinase to plasma or serum samples enhanced the reaction. There was no detectable urokinase activity in plasma fractionated on Ultragel AcA44, even though antigen was clearly detectable. Moreover, there was no correlation with urinary urokinase activity. These observations, in the light of other data, led to the conclusion that there is a species immunologically related to urokinase present in serum but that the urokinase in urine is of a purely renal origin. The serum enzyme would appear to be either bound to an inhibitor or represents a proenzyme. Recent work on kidney cell cultures has shown that a pro-urokinase is probably produced that can be converted by proteolytic enzymes to active urokinase; the inactive and active forms appear to be immunologically similar (Bernik, 1973; Nolan et al., 1977). Plasminogen activator has recently been purified from the tissue culture medium of colchicine-treated porcine kidney cells (Paul et al., 1979). A rabbit antiserum to this enzyme was prepared, and immunoglobulin was partially purified free of plasminogen activator inhibitors by two precipitations with 35% saturated ammonium sulfate. This antibody did not react with urokinase in Ouchterlony immunodiffusion. No other tests for immunological reactivity or specificity were described, leaving the specificity of this antiserum open to question. Using electron microscopy to examine the kidney cells from which the enzyme was isolated, these workers found that staining was primarily on the outer side of the plasma membrane. Frequently, membrane bleb bing and vesicle formation were observed in zones of membrane staining. Other work on other cells involving the use of the fibrin overlay method at the ultrastructural level (Warren and Khan, 1974) and biochemical fractionation studies (Dvorak et al., 1978) also came to similar conclusions regarding a plasma membrane localization for this proteinase. While the reactivity of the antiserum used by Paul et al. (1979) needs further description, these results clearly merit further

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study since they describe a mechanism for neutral proteinase secretion quite unlike that considered to be operative for the secretion of lysosomal proteinases. The pathways of secretion of other neutral proteinases, such as collagenase, from cells other than polymorphonuclear leukocytes are not yet understood.

9.

9.1.

IMMUNOLOGICAL METHODS FOR THE STUDY OF PROTEINASES

Purification of Proteinases

Proteinases should be purified to the highest possible specific activity before being used for antibody production. The use of preparative isoelectric focusing in flat beds of Sephadex (Radola, 1974) is a final step worthy of careful consideration. It was recently successfully used by Mort et al. (1981) in the purification of cathepsin B from human liver and resulted in the production of a monospecific sheep antiserum to cathepsin B. Earlier, isoelectric focusing was used by Barrett (1970) to purify cathepsin D.

9.2. Preparation of Antisera For most immunological work, antisera can be raised in rabbits or sheep with the use of from 0.3 to 3.0 mg of antigen. These are very approximate figures and are intended only as a rough guideline. In general, proteinases purified from rabbits, man, and mice are potent antigens and produce good results. Antisera may be raised in rabbits, sheep, and goats if the following guidelines are followed. Antigens are usually injected in the form of an aqueous emulsion with Freund's adjuvant (from Difco, for example). This emulsion (which should contain between 50 and 75% adjuvant) can be conveniently prepared with a small motor-driven homogenizer such as a Sorvall Omnimixer with a Micro-attachment (Ivan Sorvall Inc.) or with an Ultra-Turrax homogenizer (Janke and Kunkel, Staufen, West Germany; United States distributor, Tekmar Co., Cincinnati). Primary injections are made with Mycobacterium present (complete Freund's adjuvant), and subsequent injections can usually be made with or without added Mycobacterium (incomplete Freund's adjuvant). The requirement for a good immune stimulus rests mainly with the primary injection. Subsequent injections at at least 14-day intervals should be in multiple intramuscular and subcutaneous sites. Often only two injections, 14 days apart, are sufficient to elicit a good response (Dingle et al., 1971). Test bleeds can be prepared from marginal ear veins in rabbits (by dripping blood from cut veins) or from ear veins in pigs (using 18-gauge indwelling catheters

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with a 20-gauge needle) or from the jugular vein in sheep and goats (using syringes, or bleeding sets consisting of 18-gauge 1Hnch needles attached to flexible plastic tubing). These should be taken prior to immunization in order to provide adequate control sera, and from 7 to 14 days after an injection. Larger volumes of blood can be collected by cardiac puncture of rabbits under anesthesia with sodium pentobarbital. Larger volumes may be removed from pig ear veins using indwelling catheters combined with the use of a "nosenoose" to restrain the animal. This can be prepared from a loop of rope that passes through a 4-foot-long hollow metal tube. The loop extends at each end. One end of the loop is placed around the snout and through the mouth of the pig. The noose is gently tightened by pulling and holding that part of the loop extending from the other end of the hollow tube. 9.3.

Preparation of Antibodies from Antisera

Antisera should be allowed to clot in air at 4 C. The clot should be freed from the wall of the vessel and cut to permit adequate contraction. After 2-24 hr, serum can be removed and centrifuged to remove small clots and cells. The clear supernatant should be stored frozen at - 20 C or below. Immunoglobulin should be partially purified before use by precipitation with ammonium sulfate at room temperature for 15 min between 33 and 50% saturation. This procedure should be repeated twice before dialysis to remove salt. This is an important step when working with antibodies to proteinases since it is used to remove proteinase inhibitors, the presence of which would otherwise complicate measurements of antibody inhibiting and precipitating capacities. Conditions of salt precipitation should be examined for different species since conditions for preparing inhibitor-free precipitates vary within the range indicated. Often it is necessary to purify IgG from antisera: antibodies in hyperimmune sera are invariably IgG. This is achieved by ion-exchange chromatography using O-(diethylaminoethyl)-cellulose chromatography (Aalund et al., 1965). Purified immunoglobulin is identified by immunodiffusion or immunoelectrophoresis (method of Grabar and Williams) using anti-whole sera and anti-IgG sera. Antibody to the proteinase should be identified by its immunoreactivity with the proteinase in question. Wherever possible, antibodies reacting specifically with a proteinase should be purified by affinity chromatography. Purified proteinase is not essential if antisera are monospecific, but it is necessary to purify the enzyme as much as possible to ensure efficient chromatography. AH Sepharose is now often employed for preparing affinity columns; antigen may be coupled to 6-aminohexyl-Sepharose using glutaraldehyde (Cambiaso et al., 1975). Antibodies are generally bound from ammonium sulfate concentrates of sera and eluted at either pH 2.5 or pH 3.0, followed by rapid neutralization after recovery. 0

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Precipitated antisera can be stored frozen (and thawed as little as possible) or stored at 4·C with 0.05% sodium azide as a preservative (this should be replenished every 3 months). In all cases, precipitate should be removed by centrifugation prior to use. Often, proteinases purified to biochemically assessed purity provide polyvalent antisera. In these cases, monospecific antisera can be obtained as follows. The precipitin line containing the proteinase is initially identified [it is usually the major line and may be stainable for enzyme with a low-molecularweight substrate (see Section 9.5)]. The precipitin line can be separated from other precipitin lines by immunodiffusion between parallel troughs containing antiserum and antibody. Alternatively, precipitin lines can be separated by Laurell immunoelectrophoresis (lmawari and Goodman, 1977). This approach can utilize as little as 200 Jlg of proteinase to give sufficient precipitin lines to produce a good second antiserum in an animal such as a sheep. The second antiserum is raised in another animal of the same species as was used for the first antiserum. This step may have to be repeated twice to achieve specificity.

9.4.

Preparation of Antibodies Using Hybridomas

Kohler and Milstein (1975) were the first to introduce the use of hybridomas to prepare monoclonal antibodies. The method is essentially as follows. Mice are immunized in a conventional manner (Section 9.2) with the proteinase, a total of about 20 Jlg antigen per mouse being used over a course of at least three injections. Up to a dozen mice are used at anyone time. The animals are killed and their spleens removed. Spleen cells are recovered in Hanks' balanced salt solution, and are fused with a suitable mouse myeloma cell population that is grown in culture. The myeloma line must lack hypoxanthine phosphoribosyl transferase (EC 2.4.2.8) and be killed in culture medium containing aminopterin. Thus, malignant plasma cells, which replicate rapidly, are fused with immune spleen cells to produce heterokaryons, which replicate and synthesize and secrete antibodies directed against the immunizing antigen. Cells are fused with polyethylene glycol. A useful method is as follows. 10 7 washed spleen cells and 106 myeloma cells in 20 ml of enriched minimal essential medium (MEM) are pelleted by centrifugation at 600g for 7 min. The supernatant is removed and 0.8 ml of 30-35% (w Iv) polyethylene glycol 1000 is added while stirring. After 1 min at room temperature, a further 1 ml of medium is added. After 1 min, a total of 20 ml of medium is added in four equal volumes over a period of 1 min. After centrifuging as before, the cell pellet is washed twice in medium and finally resuspended in 1 ml of medium with heat-inactivated fetal calf serum. After 1 hr at 37" C, the fused cells are resuspended in 50 ml of medium containing hypoxanthine, aminopterin, and

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thymidine (medium A) and dispersed into Linbro wells ( I ml/well). Heterokaryons that survive and replicate are maintained by removing 0.5 ml of medium every 3-4 days and replacing it with fresh medium A. After 14 days, medium A is replaced with enriched MEM with 10% fetal calf serum. Colonies can usually be observed 2 weeks after seeding. Cells producing antibody can be identified by enzyme-linked immunosorbent assay (see Section 9.7). In the case of proteinases, culture medium can be replaced every 48 hr with serumfree media to which 0.5% lactalbumin hydrolyzate has been added to permit antibody collection in the absence of serum inhibitors. Antibodies may then be assayed by proteinase inhibition using high-molecular-weight substrates. Cells producing antibody can be cloned by dilution in culture medium, or in soft agar, to produce populations that are subsequently tested for antibody production. When antibody production is detected in all the individual subpopulations derived from a single culture, a clone of antibody-producing cells has probably been established. Some of this population should then be frozen in liquid nitrogen (for future use) and the remaining population can be subcultured in plastic flasks. By injecting such cells into the peritoneal cavities of nude mice, antibodies can be produced in high concentrations in sera and ascitic fluids (Linsen mayer et al., 1979). Periodically, myeloma cells will revert in culture (as may be shown by an increased adherence). Revertants can be destroyed by culturing for 1 week in 8-azaguanine or 6-thioguanine at 10 JLM. The monoclonal method, like other methods, relies for its success on the initial use of highly purified proteinases to identify clones producing antibodies to the proteinase in question. Here, assessment of antibody specificity is dependent, as always, on the antigen and methodology used. However, once the production of an antibody-producing clone has been proven, impure proteinases may be used for immunoassay. For further practical information, the work of Linsenmayer et al. (1979), Sharon et al. (1979), and Luben et al. (1979) should be consulted.

9.5.

Immunoprecipitation in Gels and Solution

The precipitating capacities of antisera should be investigated by immunoprecipitation in gels using both immunodiffusion and immunoelectrophoresis. Ouchterlony double immunodiffusion in agarose gels (0.7 -1.0%) should always be analyzed by staining precipitin lines, usually with Coomassie brilliant blue. Proteinases should be detected, wherever possible, using low-molecular-weight substrates and appropriate histochemical methods. These methods result in the staining only of that precipitin line containing the proteinase under study. Precipitin lines should be carefully washed and dried at 37"C before staining. Immunodiffusion is used not only to study reactions of antisera with

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purified immunizing proteinases but also reactions with protein preparations isolated during the purification procedure and which might contain potential contaminants of the purified proteinase, not previously detectable by biochemical methods. Preparations suspected of containing the proteinase in question (or a related proteinase) can also be examined by this method since it offers the possibility to clearly evaluate immunological reactions of identity (fused precipitin lines), partial identity (one precipitin line forms a spur over another), or nonidentity (two precipitin lines cross each other). For basic studies of identity, immunodiffusion has much to offer. In general, however, it is much less sensitive than most electrophoretic methods. The method of Grabar and Williams permits comparison of electrophoretic mobility and immunological reactivity. After electrophoresis of the antigen, arcs of precipitation are formed by immunodiffusion at right angles against antibody. This method is less sensitive than the methods of crossed immunoelectrophoresis and Laurell immunoelectrophoresis. In the crossed method, antigen is electrophoresed first without antibody and then at right angles into an antibody-containing gel. Precipitin "rockets" are formed that cross if they are unrelated or fuse if related. With direct rocket (Laurell) electrophoresis, rockets of precipitation formed against multiple antigens are arranged about a common central axis; resolution is highly dependent on the conditions of electrophoresis, such as voltage, buffers, gel porosity, gel antibody content, and antigen amount used. All of these methods should be used at some stage in characterizing antibody precipitation. In addition, the technique of counterimmunoelectrophoresis is also valuable. Antigen and antibody are electrophoresed against each other from sample wells and a precipitin line is formed where the two components meet and react. Methods used for preparing gels for electrophoresis (Axelsen et al., 1973) and immunodiffusion, and methods for staining precipitin lines for protein and proteinase activity (Poole, 1977) have been described elsewhere and these references should be consulted for further details. The precipitation of proteinases by antibody in solution is an important step in the quantitative characterization of antisera. Control immunoglobulins must be carefully employed to ensure accurate assessment of specific precipitation. Usually an increasing volume of antibody is added to a fixed amount of proteinase. It is essential to use antibody preparations free of proteinase inhibitors since immunoprecipitation is assayed by determining the residual enzyme activity left in supernatants after removal of precipitates by centrifugation. Non-precipitating antibodies can be removed together with antigen by using a suitable amount of a second purified antibody, directed against the first antibody species, to precipitate this immunoglobulin. Precipitations are normally initiated at neutral pH at 37"C for 1 hr followed by 16-24 hr at 4"C to permit complete precipitation. For example, the work of Dingle et al. (1971) should be consulted.

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Immunoinhibition

Studies of proteinase inhibition again necessitate the use of immunoglobulin preparations free of proteinase inhibitors and the use of similarly prepared control serum immunoglobulins. The addition of antibody to proteinase prior to the addition of substrate can lead to proteinase inhibition as shown by subsequent proteinase assay. Antibodies do not usually react with active sites of proteinases: they inhibit by steric hindrance of proteinase-substrate interaction. Hence, the degradation of high-molecular-weight substrates is usually inhibited while that of low-molecular weight substrates (such as chromogenic substrates) is only partly inhibited or uninhibited. Thus, suitable high-molecular-weight substrates such as casein or hemoglobin should be used for assay. Monovalent Fab antibody fragments, produced by papain or by reduction and alkylation of pepsin-produced F(ab')2' react with proteinases without precipitation, yet usually inhibit them as effectively as whole antibody. The use of affinity-purified antibodies in studies of cathepsin D revealed that approximately 7 molecules of antibody produce complete inhibition of the activity of one molecule of cathepsin D and up to 12 molecules can bind to one enzyme molecule. 9.7.

Nonprecipitating Antibodies and Immunoassay

Antisera may contain nonprecipitating antibodies. By definition, nonprecipitating antibodies react with a molecule at only one binding site. Hence, only two molecules of antigen can be joined by bivalent antibody. The formation of a lattice made up of antigen bridged by antibody is impossible, and thus precipitation does not occur. With monoclonal antibodies in particular, the specificity of the selected immune response would more commonly favor the production of nonprecipitating antibodies. The detection of these antibodies in precipitating sera is critical. If they react with antigens other than the one under investigation, their presence would go undetected if only precipitating analyses are used. Consequently, the specific reactivity of any antibody preparation must be held in question when used in immunoassays and immunolocalization methods that rely only on antibody binding. In order to detect all reactive antibodies, hemagglutination assays became popular. However, these can only be semiquantitative at best and often involve the use of relatively large amounts of antigen. More recently, enzyme-linked immunosorbent assays (ELISA) have been introduced; these use much less antigen and, under the appropriate conditions, are very sensitive. These assays can be used both to investigate the amount of antibody present and to assay for antigen. They were originally introduced by Engvall and Perlmann (1972). The principle is based on that of radioimmunoassay. They differ in that they

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employ an enzyme label rather than a radiochemical label. Enzyme activity is recorded and hence antibody binding is determined. To assay for proteinases, the latter would usually be bound in solution to a plastic tube or a plastic microwell (Gosslau and Barrach, 1979). Unreacted antigen is removed by washing. Antibody to the proteinase at an appropriate dilution for antigen assay or at a series of doubling dilutions for antibody assay is permitted to bind. For antigen assays, the antibody at a fixed dilution (determined by prior analysis) is incubated with varying amounts of the free antigen before addition to immobilized antigen (standard curve); samples containing unknown amounts of antigen are included at appropriate diultions. In both assays, bound antibody is detected with an enzyme-labeled second antibody (for example, pig IgG anti-rabbit IgG) directed against the antiproteinase antibody (for example, rabbit IgG). This final conjugate is used at an appropriate dilution to permit sensitive detection of specific antibody binding and its inhibition by antigen and to avoid detection of nonspecific conjugate binding. Enzyme activity is determined by reaction in a suitable colorimetric assay. Peroxidase is commonly used (Gosslau and Barrach, 1979), and conjugates of peroxidase and second-step antibody are generally prepared by conjugation with glutaraldehyde (Poole, 1977). Alternatively, iJ-galactosidase (Kato et al., 1976) or alkaline phosphatase (Engvall and Perl mann, 1972) has been used with much success. The use of fluorimetry with fluorescent reaction products considerably increases sensitivity. 5-Methylumbelliferone iJ-galactoside has been used for 13galactosidase assays (Kato et al., 1976). When determining antibody content, preabsorption of antibody with soluble antigen is used as a control. By using affinity-purified antibody as a standard, the antibody contents of biological fluids can be precisely determined. In general, this approach to the assay of antibody and antigen is gaining considerably in popularity. Investigations of reactivity against defined antigens that may be contaminants of the immunizing antigen are made possible by this assay. Moreover, by absorbing antisera with purified antigens in solution and using impure antigens for antigen coating, one can determine whether the complete antibody reaction can be blocked with purified antigen. If this is not possible, one must suspect that antibodies to some other molecular species present in the impure immobilized antigen are present, and this should be further investigated. When this happens, affinity purification of specific antibody from the antiserum may be a necessary and successful way of resolving this problem without having to resort to raising new antisera. 9.S.

Immunolocalization

Immunolocalization has perhaps presented more technical problems than most other immunological methods with the exception of obtaining monospe-

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cific antisera. The reactions of both precipitating and nonprecipitating antibodies are detected and hence the importance of clearly determining the antibody specificity. All immunostaining (as it is commonly called) should be blockable by pretreatment of the antibody (or its derivative) with an excess of purified antigen: otherwise antiserum monospecificity is questionable. Even then the purity of the antigen is obviously of critical importance. To localize proteinase, it must first be held in its true tissue site. This is usually achieved by fixation. Naturally, the tissue architecture must be maintained as well as possible. Cellular membrane and tissue permeability are enhanced by fixation. This must be sufficient to permit access of the antibody or its Fab subunit to the proteinase. One of the best fixatives available for light microscopy for almost all antigens studied by the author is formaldehyde, prepared from paraformaldehyde (Poole et al., 1972). It should be kept for no more than 1 week before use. Examples of its application have been cited earlier by the author. The most useful and sensitive label is probably fluorescein isothiocyanate. Far more fine detail can be observed than with peroxidase at the level of the light microscope. Conjugates of fluorescein and peroxidase have been discussed in detail elsewhere (Poole, 1977). In general, the use of monovalent Fab' (pepsin prepared) or Fab (papain prepared) IgG antibody fragments is always desirable: they reduce to a minimum nonspecific binding of immunoglobulin and because of their smaller size permit better penetration of fixed tissues and membranes, which are often impermeable to IgG. The indirect method is preferable and much more sensitive than the direct method. The direct method can be employed for the localization of two proteinases simultaneously: one antibody can be labeled with fluorescein and one with rhodamine isothiocyanate. In direct localization, affinity-purified antibody ensures greatest sensitivity (Mort et al .. 1981). Extracellular enzyme in a soluble form can be captured by antibody with the formation of immunoprecipitates both in vivo and in vitro in culture (see Sections 2 and 3). These can be subsequently demonstrated in fixed tissues by a second-step antibody used to detect the immunoglobulin in the immune complexes. Alternatively, soluble enzyme can be captured with a peroxidaselabeled antibody to the enzyme and immune complexes can be detected simply by reacting the fixed sectioned tissues for peroxidase (Poole, 1975). The method of Sternberger et al. (1970) is a very sensitive technique for immunolocalization. It relies on the binding of antibody to a tissue antigen (for example, rabbit anticathepsin D) to which is subsequently attached an excess of a second-step antibody (for example, sheep anti-rabbit). To this antibody (which has spare binding sites available) is bound a soluble immune complex of peroxidase and rabbit antiperoxidase. Thus, the second antibody acts as a bridge between the first antibody (bound to the antigen in question) and the immune complex with peroxidase. Since this method usually employs whole

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IgG and soluble immune complex, tissue penetration may be very limited. Hence, tissue fixation is very important and measures must be taken to enhance membrane permeability. To this end, investigators have used saponin (Fambrough and Devreotes, 1978) and freezing and thawing of fixed tissues to "punch holes" in cellular membranes, to permit antibody access. Electron microscopy can present major problems in obtaining adequate preservation of tissue and antigen. The use of the fixative formaldehyde in conjunction with periodate and lysine (McLean and Nakane, 1976) has proved to be successful in the electron microscopic study of the lysosomal proteinases such as cathepsin D (Decker et al., 1980a,b): additional membranolytic agents were not necessary in this case. Here, the indirect method was used employing peroxidase-labeled Fab' antibody in the second step. The technique of Sternberger and colleagues furnished similar results. Enhanced visualization of the peroxidase reaction product was achieved by the use of uranyl and lead. This did not complicate the identification of the reaction product and in fact greatly enhanced its demonstration and that of cellular fine detail. Similar findings have been obtained by the author from studies of proteoglycans and related molecules in cartilage (Poole et al., 1981).

10. CONCLUSIONS The purpose of this review has been to furnish the reader with an appreciation of the state of the immunological art concerning tissue proteases. Clearly, the marriage of biochemistry and immunology-two techniques that, in combination, can achieve far more than when they are applied individually-can lead to the formation of many new ideas and concepts as new data are compiled. Yet progress in this area of research has generally been slow and often of limited quality, perhaps because of reluctance on the part of some workers to foster and maintain this special set of scientific skills. It is to be hoped that the promising results that their joint application can offer will override the hesitation of investigators who have not already used and profited from immunological methods.

ACKNOWLEDGMENTS

The author's work is currently funded by the Shriners of North America, The Canadian Medical Research Council, The National Cancer Institute of Canada, and The Arthritis Society of Canada.

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11. REFERENCES Aalund, 0., Osebold, J. W., and Murphy, F. A., 1965, Isolation and characterization of ovine gamma globulins, Arch. Biochem. Biophys. 109:142-149. Axelsen, N. H., Krell, J., and Weeke, B. (eds.), 1973, A manual of quantitative immunoelectrophoresis. Methods and applications, Scand. J. Immunol. 2:Supplement 1. Barrett. J. A., 1968, Cartilage, in: Comprehensive Biochemistry (H. Florkin and E. H. Stotz, eds.), Vol. 26B, pp. 425-475, Elsevier, Amsterdam. Barrett, A. J., 1970, Cathepsin D. Purification of isoenzymes from human and chicken liver, Biochem. J. 177:601-607. Barrett, A. J., 1973, Human cathepsin B. Purification and some properties of the enzyme, Biochem. J. 131:809-822. Barrett, A. J., 1977, Cathepsin D and other carboxyl proteinases, in: Proteinases in Mammalian Cells and Tissues (A. 1. Barrett, ed), pp. 209-248, Elsevier/North-Holland, Amsterdam. Bauer, E. A., Eisen, A. Z., and Jeffrey, 1. 1., 1970, Immunologic relationship of a human purified skin collagenase to other human and animal collagenases, Biochim. Biophys. Acta 206: 152160. Bauer, E. A., Eisen, A. Z., and Jeffrey, J. J., 1972, Radioimmunoassay of human collagenase. Specificity of the assay and quantitative determination of in vivo and in vitro human skin collagenase, J. Bioi. Chem. 247:6679-6685. Bauer, E. A., Gordon, J. M., Reddick, M. E" and Eisen, A. Z., 1977, Quantitation and immunocytochemicallocalization of human skin collagenase in basal cell carcinoma, J. Invest. Dermatol.69:373-367. Bernik, M. B., 1973, Increased plasminogen activator (urokinase) in tissue culture after fibrin deposition, J. Clin. Invest. 52:823-834. Bernik, M. B., and Kwaan, H. c., 1969, Plasminogen activator activity in cultures from human tissues. An immunological and histochemical study, J. Clin. Invest. 48:1740-1753. Bernik, M. B., White, W. F., Oller, E. P., and Kwaan, H. c., 1974, Immunologic identity of plasminogen activator in human urine, heart, blood vessels, and tissue culture, J. Lab. Clin. Med.81:546-558. Bird, J. W. c., Carter, J. H., Friemer, R. E., Brooks, R. M., and Spanier, A. M., 1980, Proteinases in cardiac and skeletal muscle, Fed. Proc. 39:20-25. Burleigh, M. c., Barrett, A. J., and Lazarus, G. S., 1974, Cathepsin Bl. A lysosomal enzyme that degrades native collagen, Biochem. J. 137:387-398. Cambiaso, C. L., Goffinet, J. A., Vaerman, J.-P., and Heremans, J. F., 1975, Glutaraldehydeactivated aminohexyl-derivative of Sepharose 4B as a new versatile immunosorbent, Immunochemistry 12:273-278. Christman, J. K., Silverstein, S. C., and Acs, G., 1977, Plasminogen activators, in: Proteinases in Mammalian Cells and Tissues (A. J. Barrett, ed.), pp. 91-149, Elsevier/North-Holland, Amsterdam. Decker, R. S., Poole, A. R., Griffin, E. E., Dingle, J. T., and Wildenthal, K., 1977, Altered distribution of lysosomal cathepsin D in ischemic myocardium, J. Clin. Invest. 59:911-921. Decker, R. S., Poole, A. R., Dingle, J. T., and Wildenthal, K., 1978, Influence of methylprednisolone on the sequential redistribution of cathepsin D and other lysosomal enzymes during myocardial ischemia in rabbits, J. Clin. Invest. 62:797-804. Decker, R. S., Poole, A. R., Dingle, J. T., and Wildenthal, K., 1979, Lysosomal alterations in autolyzing rabbit heart, J. Mol. Cell. Cardiol. 11:189-196. Decker, R. S., Poole, A. R., and Wildenthal, K., 1980a, Distribution of lysosomal cathepsin D in normal, ischemic and starved rabbit cardiac myocytes, Cir. Res. 46:485-494.

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Decker, R. S., Decker, M., and Poole, A. R., 1980b, The distribution of lysosomal cathepsin D in cardiac myocytes, J. Histochem. Cytochem. 28:231-237. Dingle, J. T., 1969, The extracellular secretion of lysosomal enzymes, in: Lysosomes in Biology and Pathology (J. T. Dingle and H. B. Fell, eds.), Vol. 2, pp. 421-436, North-Holland, Amsterdam. Dingle, J. R., Barrett, A. J., and Weston, P.D., 1971, Cathepsin D. Characteristics of immunoinhibition and the confirmation of a role in cartilage breakdown, Biochem.l. 123: 1-13. Dingle, J. T., Poole, A. R., Lazarus, G. S., and Barrett, A. J., 1973, Immunoinhibition of intracellular protein digestion in macrophages, J. Exp. Med. 137:1124-1141. Dingle, J. T., Saklatvala, J., Hembry, R. M., Tyler, J., Fell, H. 8., and Jubb, R., 1979, A cartilage catabolic factor from synovium, Biochem. J. 184: 177 -180. Dvorak, H. F., Orenstein, N. S., Rypysc, J., Colvin, R. B., and Dvorak, A. M., 1978, Plasminogen activator of guinea pig basophilic leukocytes: Probable localization to the plasma membrane, J. Immunol. 120:766-773. Engvall, E., and Perlmann, P., 1972, Enzyme-linked immunosorbent assay ELISA. III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes, J. Immunol. 109:129-135. Erickson, A. H., and Blobel, G., 1979, Early events in the biosynthesis of the lysosomal enzyme cathepsin D, J. Bioi. Chern. 254: 11771-11774. Evanson, J. M., Jeffrey, J. J., and Krane, S. M., 1968, Studies on collagenase from rheumatoid synovium in tissue culture, J. C/in. Invest. 47:2639-2651. Fambrough, D. M., and Devreotes, P. N., 1978, Newly synthesised acetylcholine receptors are located in the Golgi apparatus, J. Cell Bioi. 76:237-244. Feinstein, G., and Janoff, A., 1975a, A rapid method for purification of human granulocyte cationic neutral proteases: Purification and characterization of human granulocyte chymotrypsin-like enzyme, Biochim. Biophys. Acta 403:477-492. Feinstein, G., and Janoff, A., 1975b, A rapid method of purification of human granulocyte cationic neutral proteases: Purification and further characterization of human granulocyte elastase, Biochim. Biophys. Acta 403:493-505. Gosslau, B., and Barrach, H. J., 1979, Enzyme-linked immunosorbent microassay for quantification of specific antibodies to collagen type I, II, III, J. Immunol. Methods 29:71-77. Harper, E., Bloch, K. J., and Gross, J., 1971, The zymogen of tadpole collagenase, Biochemistry 10:3035-3041. Harris, E. D., Jr., DiBona, D. R., and Krane, S. M., 1970, A mechanism for cartilage destruction in rheumatoid arthritis, Trans. Assoc. Am. Physicians 83:267-276. Harris, E. D., Jr., Glauert, A. M., and Murley, A. H. G., 1977, Intracellular collagen fibres at the pannus-cartilage junction in rheumatoid arthritis, Arthritis Rheum. 20:657-665. Huang, J. S., Huang, S. S., and Tang, J., 1979, Cathepsin D isozymes from porcine spleens. Large scale purification and polypeptide chain arrangements, J. Bioi. Chern. 254:1140511417. Imawari, M., and Goodman, D. S., 1977, Immunological and immunoassay studies of the binding protein for vitamin D and its metabolites in human serum, J. C/in. Invest. 59:432-442. Janoff, A., Feinstein, G., Malemud, C. J., and Elias, J. M., 1976, Degradation of cartilage proteoglycan by human leukocyte granule neutral proteases-A model of joint injury. I. Penetration of enzyme into rabbit articular cartilage and release of J5SO.-labeled material from the tissue, J. C/in. Invest. 57:615-624. Janoff, A., Sloan, B., Wienbaum, G., Damiano, C., Sandhaus, R. A., Elias, J., and Kimbel, P., 1977, Experimental emphysema induced with purified human neutrophil elastase: Tissue localization of the instilled protease, Am. Rev. Respir. Dis. 115:461.-478.

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Kato, K., Fukui, H., Hamaguchi, Y., and Ishikawa, E., 1976, Enzyme-linked immunoassay: Conjugation of the Fab' fragment of rabbit IgG with {3-D-galactosidase from E. coli and its use for immunoassay, J. Immunol. 116: 1554-1560. Keiser, H., Greenwald, R. A., Feinstein, G., and Janoff, A., 1976, Degradation of cartilage proteoglycan by human leucocyte granule neutral proteases-A model of joint injury. II. Degradation of isolated bovine nasal cartilage proteoglycan, J. Clin. Invest 57:625-632. Kirschke, H., Langner, J., Wiederanders, B., Ansorge, S., Bohley, P., and Hanson, H., 1977, Cathepsin H: An endoaminopeptidase from rat liver Iysosomes, Acta Bioi. Med. Ger. 36: 185-199. Kohler, G., and Milstein, c., 1975, Continuous cultures of fused cells secreting antibody of a predefined specifity, Nature (London) 256:495-497. Linsenmayer, T. F., Hendrix, M J. c., and Little, C. D., 1979, Production and characterization of a monoclonal antibody to chicken type I collagen, Proc. Natl. Acad. Sci. USA 76:37033707. Luben, R. A., Mohler, M. A., and Nedwin, G. E., 1979, Production of hybridomas secreting monoclonal antibodies against the lymphokine osteoclast activating factor, J. Clin. Invest. 64:337-341. MacGregor, R. R., Hamilton, J. W., Shofstall, R. E., and Cohn, D. V., 1979, Isolation and characterization of porcine parathyroid cathepsin B, J. Bioi. Chern 254:4423-4427. McLean, I., and Nakane, P. K., 1974, Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy, J. Histochem. Cytochem. 22:1077-1083. Maroudas, A., 1976, Transport of solutes through cartilage: Permeability to large molecules, J.

Anat. 122:335-347. Montford, I., and Perez-Tamayo, R., 1975a, The distribution of collagenase in normal rat tissues, J. Histochem. Cytochem. 23:910-920. Montford, I., and Perez-Tamayo, R., 1975b, The distribution of collagenase in the rat uterus during postpartum involution. An immunohistochemical study, Connect. Tissue Res. 3:245252. Morrison, R. I. G., Barrett, A. J., Dingle, J. T., and Prior, D., 1973, Cathepsin BI and D action on human cartilage proteoglycans, Biochim. Biophys. Acta 302:411-419. Mort, J. S., Recklies, A. D., and Poole, A. R., 1980, Characterization of a thiol proteinase secreted by malignant human breast tumours, Biochim. Biophys. Acta 614:134-143. Mort, J. S., Poole, A. R., and Decker, R. S., 1981, Immunofluorescent localization of cathepsins Band D in human fibroblasts, J. Histochem. Cytochem. 29:649-657. Morton, D. B., 1975, Acrosomal enzymes: Immunochemicallocalization of acrosin and hyaluronidase in ram spermatozoa, J. Reprod. Fertil. 45:375-378. Morton, D. B., 1977, The occurrence and function of proteolytic enzymes in the reproductive tract of mammals, in: Proteinases in Mammalian Cells and Tissues (A. J. Barrett, Ed.), pp. 445-500, Elsevier/North-Holland, Amsterdam. Noguchi, T., and Kandatsu, M., 1971, Purification and properties of a new alkaline protease of rat skeletal muscle, Agric. Bioi. Chern. 35: I 092-11 00. Nolan, c., Hall, L. S., Barlow, G. H., and Tribby, F. I. E., 1977, Plasminogen activator from human embryonic kidney cell cultures. Evidence for a proactivator, Biochim. Biophys. Acta 496:384-400. Ogunro, E. A., Lanman, R. B., Spencer, J. R., Ferguson, A. G., and Lesch, M., 1979, Degradation of canine cardiac myosin and actin by cathepsin D isolated from homologous tissue,

Cardiovasc. Res. 13:621-629. Ohlsson, K., and Olsson, I., 1973, The neutral proteases of human granulocytes. Isolation and partial characterization of two granulocyte collagenases, Eur. J. Biochem. 36:473-481.

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Ohlsson, K., and Olsson, I., 1974, The neutral proteases of human granulocytes. Isolation and partial characterization of granulocyte elastases, Eur. J. Biochem. 42:519-527. Park, D. C, Parson, M. E., and Pennington, R. J. T., 1973, Evidence for mast cell origin of proteinases in skeletal muscle homogenates, Biochem. Soc. Trans. 1:730-733. Paul, D. C., Bobbit, J. L., Williams, D. C, and Hull, R. N., 1979, Immunocytochemical localization of plasminogen activator on porcine kidney cell strain: LLC-PK (LP lOo ), J. Histachem. Cytochem. 27:1035-1040. Pierart-Gallois, M., Trouet, A., and Tulkens, P., 1977, Production of rabbit antibodies against active rat cathepsin B, Acta Bioi. Med. Ger. 36:1887-1891. Poole, A. R., J 974, Immunological methods for the study of the cellular localizations of proteins and their secretion, in: Methodological Developments in Biochemistry, Vol. 4, Subcellular Studies (E. Reid, ed.), pp. 1-12, Longman Group Limited, London. Poole, A. R., 1975, Immunocytochemical studies of the secretion of a proteolytic enzyme, cathepsin D, in relation to cartilage breakdown, in: Dynamics of Connective Tissue M acromolecules (P. M. C Burleigh and A. R. Poole, Eds.), pp. 357-379, North-Holland, Amsterdam. Poole, A. R., 1977, Antibodies to enzymes and their uses, with particular reference to lysosomal enzymes, in: Lysosomes, A Laboratory Handbook (1. T. Dingle, ed), 2nd ed., pp. 245-312, North-Holland, Amsterdam. Poole, A. R., Dingle, J. T., and Barrett, A. J., 1972, The immunocytochemical demonstration of cathepsin D, J. Histochem. Cytochem. 20:261-265. Poole, A. R., Barratt, M. E. J., and Fell, H. B., 1973a, The role of soft connective tissue in the breakdown of pig articular cartilage cultivated in the presence of complement-sufficient antiserum to pig erythrocytes. II. Distribution of immunoglobulin G(IgG), Int. Arch. Allergy Appl. Immunol. 44:469-488. Poole, A. R., Hembry, R. M., and Dingle, J. T., 1973b, Extracellular localization of cathepsin D in ossifying cartilage, Calcif. Tissue Res. 12:313-321. Poole, A. R., Hembry, R. M., and Dingle, J. T., 1974, Cathepsin D in cartilage: The immunohistochemical demonstration of extracellular enzyme in normal and pathological conditions, J. Cell Sci. 14:139-161. Poole, A. R., Hembry, R. M., Dingle, J. T., Pinder, I., Ring, E. F. J., and Cosh, J., 1976, Secretion and localization of cathepsin D in synovial tissue removed from rheumatoid and traumatized joints, Arthritis Rheum. 19: 1295-1307. Poole, A. R., Tiltman, K. J., Recklies, A. D., and Stoker, T. A. M., 1978, Differences in the secretion of the proteinase cathepsin B from the invading edges of mammary carcinomas and fibroadenomas, Nature (London) 273:545-547. Poole, A. R., Recklies, A. D., and Mort, J. S., 1980, Secretion of proteinases from human breast tumours: Excessive release from carcinomas of a thiol proteinase, in: Proteinases and Tumour Invasion (P. Strauli, A. J. Barrett, and A. Baici, eds.), pp. 81-93, Raven Press, New York. Poole, A. R., Pidoux, I., Reiner, A., and Rosenberg, L., 198 I, Ultrastructural organization of proteoglycan monomer and link protein in articular cartilage, Semin. Arthritis Rheum. l1(Suppl. 1):26-28. Radola, B. J., 1974, Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing, Biochim. Biophys. Acta 386: 18 I - I 95. Recklies, A. D., Tiltman, K. J., Stoker, A. M., and Poole, A. R., 1980, Secretion of proteinases from malignant and non-malignant human breast tissue, Cancer Res. 40:550-556. Reddick, M. E., Bauer, E. A., and Eisen, A. Z., 1974, Immunocytochemical localization of collagenase in human skin and fibroblasts in monolayer culture, J. Invest. Dermatol 62:361366.

Immunology of Tissue Proteinases

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Roughley, P. J., and Barrett, A. J., 1977, The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan structure and its susceptibility to proteolysis, Biochem. J. 167:629-637. Sakamoto, S., Sakamoto, M. Goldhaber, P., and Glimcher, M. H., 1978, Mouse bone collagenase. Purification of the enzyme by heparin-substituted Sepharose 4B affinity chromatography and preparation of specific antibody to the enzyme, Arch. Biochem. Biophys. 188:438449. Schwarz, A. N., and Bird, J. W. C., 1977, Degradation of myofibrillar proteins by cathepsins B and D, Biochem. J. 167:811-820. Sellers, A., Cartwright, E., Murphy, G., and Reynolds, J. J., 1977. Evidence that latent collagenases are enzyme-inhibitor complexes, Biochem. J. 163:303-307. Seppa, H. E. J., 1978, Rat skin main neutral protease: Immunohistochemical localization, J. Invest. Dermatol. 71:3 \1-315. Seppa, H. E. J., and Jarvinen, M., 1978, Rat skin main neutral protease: Purification and properties, J. Invest. Dermatol. 70:84-89. Shakespeare, M., and Wolf, P., 1979, The demonstration of urokinase antigen in whole blood, Thromb. Res. 14:825-835. Sharon, J., Morrison, S. L., and Kabat, E. A., 1979, Detection of specific hybridoma clones by replica immunoadsorption of their secreted antibodies, Proc. Natl. Acad. Sci. USA 76:1420-1424. Starkey, P. M., 1977, Elastase and cathepsin G; the serine proteinases of human neutrophil leucocytes and spleen, in: Proteinases in Mammalian Cells and Tissues (A. J. Barrett, ed.), pp. 57-89, Elsevier/North-Holland, Amsterdam. Starkey, P. M., and Barrett, A. J., 1976a, Neutral proteinases of human spleen. Purification and criteria for homogeneity of elastase and cathepsin G, Biochem. J. 155:255-263. Starkey, P. M., and Barrett, A. J., 1976b, Human lysosomal elastase. Catalytic and immunological properties, Biochem. J. 155:265-271. Starkey, P. M., and Barrett, A. J., 1976c, Human cathepsin G. Catalytic and immunological properties, Biochem. J. 155:273-278. Starkey, P. M., Barrett, A. J., and Burleigh, M. c., 1977, The degradation of articular collagen by neutrophil proteinases, Biochim. Biophys. Acta 483:386-397. Sternberger, L. A., Hardy, P. H., Jr., Cuclulis, J. J., and Meyer, H. G., 1970, The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-anti horseradish peroxidase) and its use in identification of spirochetes, J. Histochem. Cytochem. 18:315-333. Vaes, G., 1972, The release of collagenase as an inactive proenzyme by bone explants in culture, Biochem. J. 126:275-289. Warren, B. A., and Khan, S., 1974, The ultrastructure of the lysis of fibrin by endothelium in vitro, Br. J. Exp. Pathol. 55:138-148. Werb, Z., and Burleigh, M. c., 1974, Specific collagenase from rabbit fibroblasts in monolayer culture, Biochem. J. 137:373-385. Werb, Z., and Reynolds, J. J., 1975a, Immunochemical studies with a specific antiserum to rabbit fibroblast collagenase, Biochem. J. 151:655-663. Werb, Z., and Reynolds, J. J., 1975b, Rabbit collagenase. Immunological identity of the enzymes released from cells and tissues in normal and pathological conditions, Biochem. J. 151:665669. Weston, P. D., 1969, A specific antiserum to lysosomal cathepsin D, Immunology 17:421-428. Weston, P. D., and Poole, A. R., 1973, Antibodies to enzymes and their uses with specific reference to cathepsin D and other lysosomal enzymes, in: Lysosomes in Biology and Pathology (1. T. Dingle, ed.), Vol. 3, pp. 425-464, North-Holland, Amsterdam.

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Whitaker, J. N., 1980, Immunochemical characterization of bovine brain cathepsin D, 1. Neurochem. 34:284-292. Wildenthal, K., Poole, A. R., and Dingle, J. T., 1975, Influence of starvation on the activities and localization of cathepsin D and other lysosomal enzymes in hearts of rabbits and mice, 1. Mol. Cell. Cardiol. 7:841-855. Wildenthal, K., Decker, R. S., Poole, A. R., Griffin, E. E., and Dingle, J. T., 1978, Sequential lysosomal alterations during cardiac ischemia. I. Biochemical and immunohistochemical changes, Lab. Invest. 38:656-661. Woodbury, R. G., Everitt, M., Sanada, Y., Katunuma, N., Lagunoff, D., and Neurath, H., 1978, A major serine protease in rat skeletal muscle: Evidence for mast cell origin, Proc. Natl. Acad. Sci. USA 75:5311-5313. Woolley, D. E., Crossley, M. J., and Evanson, J. M., 1976, Antibody to rheumatoid synovial collagenase. Its characterization, specificity and immunological cross-reactivity, Eur. 1. Biochem. 69:421-428. Woolley, D. E., Crossley, M. J., and Evanson, J. M., 1977, Collagenase at sites of cartilage erosion in the rheumatoid joint, Arthritis Rheum. 20:1231-1239. Woolley, D. E., Harris, E. D., Jr., Mainardi, C. L., and Brinckerhoff, C. E., 1978, Collagenase immunolocalization in cultures of rheumatoid synovial cells, Science 200:773-775. Woolley, D. E., Brinckerhoff, C. E., Mainardi, C. L., Vater, C. A., Evanson, J. M., and Harris, E. D., Jr., 1979, Collagenase production by rheumatoid synovial cells: Morphological and immunohistochemical studies of the dendritic cell, Arthritis Rheum. Dis. 38:262-270. Zaneveld, L. J. D., Schumacher, G. F. B., and Travis, J., 1973, Human sperm acrosomal proteinase: Antibody inhibition and immunologic dissimilarity to human pancreatic trypsin, Fertil. Steril. 24:479-484.

Chapter 8

Amino Acids from the Moon: Notes on Meteorites Sidney W. Fox and Kaoru Harada* Institute for Molecular and Cellular Evolution University of Miami Coral Gables, Florida 33124

and P. E. Hare Geophysical Laboratory Carnegie Institution Washington, D.C. 20008

1. INTRODUCTION Our understanding of cosmochemistry and the origin of life is profitably discussed in the two stages represented by that phrase. Our surest information on cosmochemical products relates to sets of amino acids that originated from the simplest forms of matter in the cosmos. This study is necessarily one of analytical chemistry. The second stage is the evolution to living cells from those amino acids (Fox, 1972; Kvenvolden, 1974). Amino acids are seen as having been chemical precursors successively to protein and to the ubiquitous biological cells whose structure and function depend mainly upon protein. In obtaining this kind of information, a constructionistic approach has been uniquely informative (Fox, 1977). This approach consists of experiments in assembly followed by selection from the products; constructionism is in the style of evolution itself. Analytical studies alone are inadequate (Fox, 1981). *Present address: Department of Chemistry, University of Tsukuba, Nihari-Gun, Ibaraki-Ken, Japan. 357

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For the first stage, it has been possible to analyze material from two extraterrestrial sources, our moon and meteorites. Inferences on the nature of sets of amino acids available in the primitive environment have been obtained primarily from these sources, but also from simulation experiments in synthesis (Fox and Dose, 1977). Of special interest is the concordance between results from the lunar surface and results from meteorites, when the two are normalized for carbon content (Fox and Dose, 1977; Harada and Hare, 1980). Since the lunar samples are much more assuredly free of contamination, the results of assays of proteinaceous amino acids obtained from meteorites can be said to confirm those obtained from the lunar fines. The technology of meteoritic analysis has been guided by lunar analyses, much as that was earlier developed on samples of terrestrial lava (Fox et aI., 1972a). [The paper by Harada and Hare (1980) may be used as an introduction to the modern literature on amino acids from meteorites.] Drilling into the interior and stepwise analysis indicated that the Allende meteorite is contaminated on the surface, as apposed to the interior. This meteorite releases amino acids at a level of about 1.0% that of the most-studied Murchison and Murray meteorites. When normalized for carbon content, the identities and amounts of amino acids in the Allende meteorite agree well with both the lunar and the other meteoritic results.

2.

HISTORY

The decade of the 1970s yielded the first bank of data on the availability of amino acids from two extraterrestrial sources. Although data were accumulated earlier on amino acids from meteorites (Degens and Bajor, 1962), such information was open to the possibility that it was the result of terrestrial contamination (Degens and Bajor, 1962; Kvenvolden, 1974). This contamination can result from organic matter in the atmosphere collected during the meteor's fall and later from the soil in which the meteorite rests. Potentially avoidable forms of contamination are those from human fingerprints (Hamilton, 1965; cf. Oro and Skewes, 1965; Hayes, 1967) and those from museumrelated events (Fox, 1966), i.e., during the collection and maintenance of samples. Studies carried out decades ago showed the earth's upper atmosphere to be laden with microbes, pollen, and dust (see Proctor, 1935, and references therein). In testing possible contamination in the soil, Imshenitski (1966) reported that a heat-sterilized meteorite placed in contact with surface soil in the Moscow region for 4 days was infected even in its center by soil microorganisms (cf. also Imshenitski et al., 1966).

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Although it has often been argued that amino acids from non biological (i.e., chemical) sources are identified in meteorites when the amino acids are found to be racemic (e.g., Kvenvolden, 1974), it must be recalled that the anterior surface of a meteor is hot due to friction with the atmosphere (fireball effect). The anterior surface is also the part of the meteor that would collect, for example, microbial protein and protein fragments from the atmosphere and would also rapidly racemize amino acids. The latter are, under some conditions studied, most susceptible to racemization when they are in peptide linkage (Dakin, 1912-1913; Dakin and Dudley, 1913), being significant even at 25°C. The carbonaceous chondrites would possess the added possibility of suffering contamination internally when material from the surface is driven into the interior by the airstream. Additional uncertainties about the chemical history of amino acids in meteorites arise from the possibility that both generation and thermal change of amino acids can occur from impact of the meteorite with the earth's surface (Bunch and Chang, 1980). The process of producing amino acids from various simple compounds by high temperature due to impact has been shown to occur through both hydrous and anhydrous mechanisms (Bar-Nun et al., 1971; BarNun and Tauber, 1972; Barak and Bar-Nun, 1975). There have been no such a priori reasons to indict the biological or chemical history of the lunar samples, but the possibility of contamination has nevertheless been extensively examined. This probable absence of contamination is the reason a number of investigators eagerly anticipated the return of Apollo lunar samples (e.g., Flory and Simoneit, 1972). In the chronology of the studies carried out on lunar samples, confusion was first generated by disagreement between, on one hand, the positive reports for Apollo 11 (and later missions) for amino acids in lunar fines properly extracted, hydrolyzed, and examined by ion-exchange chromatography (1EC) and, on the other hand, negative results by gas-liquid chromatography (GLC) on volatile derivatives of amino acids from Apollo 11 and 12 samples (Gehrke et aI., 19'/2). In an omnibus report on organic matter in Apollo 11 samples (Ponnamperuma et al., 1970), the statement is repeated that there "were no detectable amounts of extractable ... amino acids .... " The analysis included direct HCl hydrolyzates of nearly the entire bulk of lunar fines, which had been extracted with benzene and methanol. To the contrary, two other lunar analysis teams found amino acids by IEC on extracts in the first returned samples. One team found them to be detectable and measurable in free form in direct extracts (Murphy et al., 1970), and one found detectable and measurable amino acids both in direct extracts and in HCl hydrolyzates of extracts (Fox et al., 1970; Hare et al., 1970).

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Each of these latter two teams found amino acids in hydrolyzates of Apollo 12 samples, but essentially none in direct extracts (Nagy et ai., 1971; Fox et ai., 1972a). When the GLC results (Gehrke et ai., 1972; Oro et ai., 1970) disagreed with those of the two teams using IEC on samples from Apollo 11 and 12, a side-by-side analysis was arranged on an especially carefully collected Apollo 14 SESC (special environmental sample container) sample. The results were reported in a lunar analysis conference (Fox et ai., 1972b; Gehrke et ai., 1972; Ponnamperuma, 1972). As a consequence of the collaboration, the IEC and GLC methods were both found to be positive for amino acids. Crucial details are explained later in this review. A summary NASA publication (EP-101, Simmons, 1972) refers to the finding as an "extremely small amount of amino acids." When normalized by carbon content and viewed in an appropriate cosmochemical context, the concordance between the amounts and species in both lunar samples and meteorites suggests connotations that may, however, be extremely significant.

2.1. Analyses of Lunar Samples and Meteorites Some of the primary questions about the agreements that emerged from what were at first disagreements on data can be considered in Figure 1. The results of the IEC analysis are shown in Figure 1B and those of the GLC analysis are shown in Figure 1D. The IEC method quantitates amino acids directly and, due to chromogenic use of ninhydrin, is highly specific for free a amino acids (MacFadyen and Fowler, 1950). The GLC method involves the conversion of amino acids to the volatile t-butyl or other esters of the N-trifluoracetylated bodies, which are then subjected to chromatography. Compounds other than a amino acids, e.g., oxalic acid, also respond. Although the GLC method has been useful in analyses of proteins from agricultural sources and other materials, it determines acids other than amino acids; it thus demonstrated a low order of reliability in analysis of lunar samples. This is illustrated especially by Figure 1D, in the original of which can be counted more than 20 peaks. These are largely unknowns, excepting the five amino acids earlier revealed by IEC (Fox et ai., 1972b). Obscuration of some peaks by others in the GLC chromatograms has been acknowledged by those using GLC. The five amino acids from the lunar samples are glycine, alanine, serine, glutamic acid, and aspartic acid. The IEC method has quantitated these five amino acids in 12 collections from Apollo missions (Table I), in rather close to the same proportions. In some of the IEC analyses (Figure 1B), a sixth amino acid, threonine, was found as a shoulder on the serine peak. Glycine was the most abundant amino acid by IEC, as it seemed to be by GLC. Threonine was the least abundant in all analyses. For the IEC analyses, much care was taken

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Amino Acids from the Moon

A IEC

8 IEC

c

D

IEC,__--__ｾ＠

r-----

__

GLC

FIGURE 1. Ion-exchange chromatography (1EC) of hydrolysate of extract of soil from special collection on Apollo 14, and gas-liquid chromatography (GLC) of volatile derivatives of hydrolysate of extract. (A) Human fingerprint; hydrolysate is similar. (8) Pattern of amino acids of hydrolysate of Apollo II extract. (C) Pattern from hydrolyzed water sample control. (D) Pattern of trifluoroacetyl amino acid (-butyl esters from amino acids in hydrolysate of Apollo 14 extract.

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Table I Analyses of Hydrolysates of Hot Aqueous Extracts of Lunar Samples in the MiamiWashington Series (Molar Ratios") Total

amino Mission

Sample No.

11 12 14 14 14 14 15 15 16 17 17

10086 12033 14003 14163 14240' 14298 15012" 15013' 66041 70011 • 72501 d

Apollo

Glutamic Glycine

Alanine

acid

50 49 62 47 63 57 61

25 16 20 26 15

9 27 12 20

Aspartic acid

11 13 16

Serine

Threonine

4 6 4 10

1 0

Other (unknowns)

12 11

19 0

1 0

45 19 19 30

0 0 0

ca. 5

0 0

37 7 12 12 30 10

73

56 83 70

acids (ng/g)

'All calculations exclude ammonia and basic amino acids (the latter are essentially absent). 'specially collected (SESC) sample. CDistal from the descent engine. dproximal to the descent engine.

to prepare reagents of low contamination. Since total freedom from contamination is not feasible, the amount was estimated in each case. In Figure 1C, the contamination in "pure" water is seen and can be compared with the sample analysis directly above (B). In this case, contamination is negligible. In all samples, the amounts of amino acids were many to several hundred times those in the reagents. The production of clean samples is a matter of some care: in collection on the moon, in the lunar receiving laboratory, and in the analytical laboratory, which can potentially introduce contamination through reagents. In the officially sponsored collaborative analysis, one skillful analyst was assigned the preparation of the "pure" water of Figure 1C (Fox et al., 1972a). The comparisons in Figure 1 provide typical data that explain that the GLC results were confirmatory of the results obtained on lunar samples by IEC. The greater number of unknowns than of knowns and their greater size in the GLC chromatogram explain why those analysts relying on GLC had been unable to make a primary statement on the presence of amino acids in hydrolyzed extracts of Apollo 11 samples, although they could confirm the IEC results. Except for glycine and perhaps alanine, the small size of N-trifluoroacetyl amino acid t-butyl ester peaks relative to the unknowns would understandably predispose to such negative judgment at first glance. The first negative reports for Apollo 11 and 12 samples by the GLC method in the literature are thus explained as due to the ineffectiveness and unreliability of that method for the analysis of extracts of the lunar samples, as well as by preparation of samples by means of direct hydrolysis (Ponnamperuma et al., 1970; Oro et aI., 1970; cf. Fox and Dose, 1977, for related examples).

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363

While the above considerations are primary in evaluating lunar or meteoritic samples, other details are relevant and are discussed below.

2.2. Preparation of Samples In the pre-Apollo era, meteorites were examined for amino acids (Kvenvolden, 1974) following direct Hel hydrolysis. This kind of sample preparation did appear to some to be susceptible to error, mainly because of the high ratio of inorganic mineral to organic matter (peptides). Accordingly, one team prepared for Apollo samples by interposing an aqueous extraction step in sample preparation (Fox et al., 1972a). For this purpose, hot water was found to be much more efficient than cold water (Fox et al., 1972a). In one experiment, hot water extracted 299 j.Lmol of recoverable amino acids, whereas cold water extracted 16 j.Lmol under what were otherwise the same conditions. An absence of amino acids following direct hydrolysis was first observed in pilot experiments with lava in this laboratory (Fox, 1964; Fox et al., 1972a), while they were found in hydrolyzed aqueous extracts. Such methodology was verified on extracted lunar fines. Due to the limited amount of material available, lunar fines remaining after extraction for primary amino acid analysis were used for a controlled test. Into such a batch were introduced amino acid precursors by reaction of formaldehyde with ammonia (Fox and Windsor, 1970). This fortification suitably provided amino acid precursors in lunar fines without further destruction of precious Apollo samples. Such precursor-fortified material was divided into aliquots. One aliquot was hydrolyzed directly by the old method, while another was extracted by hot water and the aqueous extract was hydrolyzed. The results are shown in Table II.

Table II Recovery of Amino Acids· from Fortified Water-Extracted Lunar Fines (in ILmol) Indirect hydrolysis

Direct hydrolysis

0.04 0.35 1.12 0.35 0.43 0.27 0.78

0.00 0.00 0.00 0.00 0.00 0.00 0.28

Aspartic acid Glutamic acid Glycine + alanine Valine Isoleucine Leucine Unknowns "As judged by retention time.

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Direct hydrolysis of the fortified lunar sample did not detect any proteinaceous amino acid, whereas the Apollo 11 extraction-hydrolysis procedure yielded 2550 nmol of total amino acid. This result alone fails to support the preclusive interpretation of negative figures obtained by direct hydrolysis. The explanations offered for the loss of amino acids or amino acid precursors in direct hydrolysis are (1) oxidation by hot H Cl in the presence of mineral catalysts and (2) loss of organic matter in the large amount of salt formed in the necessary desalting of material obtained by direct hydrolysis.

2.3.

Method of Analysis

The main features of the methods of analysis have been discussed. What may be said further is that both IEC and GLC may be used for confirmation in future studies and that samples of meteorites may be prepared in the same manner as lunar samples. The method of extraction by hot water followed by HCl hydrolysis of the extract has in fact been transposed to meteorites (Fox et al., 1972b; Oro, 1972, p. 536; Kvenvolden, 1972; Lawless et aI., 1972; Harada and Hare, 1980). Because the amounts of sample available to analysts from the Apollo 1417 missions were limited, a choice was made between analyzing for amino acids and analyzing for amino acid precursors. After the experience with the Apollo 12 samples, the method of extraction and hydrolysis of the entire extract was chosen.

2.4. Contamination in Lunar Fines The total amount of amino acid present upon hydrolysis, as reported by one team (Fox et al., 1974), was 7-45 ng/g. The question of contamination was considered. As already described, contamination can be of two kinds, biological and chemical. The reasons for discounting the hypothesis of biological contamination of the lunar fines are severalfold: (1) the results from analyses of 11 collections are concordant; (2) the IEC chromatograms show an absence of the "spiking" characteristic of biological contamination; (3) the samples contain 30-200 times as much recoverable amino acid as the blank reagents carried through the analysis; and (4) most importantly, the profile of the 5-6 "known" amino acids differs from that of the 17-19 amino acids found either free or after hydrolysis in contaminating human material (Hamilton, 1965). The evaluations of the 1970s are summarized by Hamilton and Nagy (1975): "if amino acids are present in lunar samples the majority of them do not have the distribution pattern, type, or variety usually associated with biological origin, i.e., they are not contaminations from terrestrial biological sources."

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365

The Miami-Washington lunar amino acid analysis team also has concluded that the results cannot be explained as owing to human contamination (Fox et aI., 1976; Harada and Hare, 1980). The suite of five or six amino acids observed in analyses of 11 collections is less suggestive of a biological than of a chemical history. The possibility of chemical contamination has been examined by analysis of proximal and distal samples (i.e., those closest to and farthest from the descent engine) obtained from Apollo 15 and 17. The results have been compared with amino acids expected by hydrolysis of cyanide measured in the exhaust gases of the corresponding Apollo descent vehicles (Flory et al., 1972). The results obtained in conversion in laboratory studies were closely matched by those obtained on the moon itself (Freeman et aI., 1972). The results indicate that the total amount of amino acid recovered in the distal sample of Apollo 15 is five or more orders of magnitude larger than can be accounted for by conversion of the exhaust gases (Fox et al., 1976). This figure is based on the assumptions that no significant amount of exhaust gas was lost to outer space and that it or conversion products were all implanted in the lunar surface. These assumptions, of course, are extremely conservative relative to the inference drawn. While the two assumptions are attained by different kinds of reasoning, the evaluation of little or no chemical contamination parallels that of little or no biological contamination. The results are a tribute to the care exercised in the collections, although in principle total freedom from contamination cannot be inferred for all samples. The Apollo 11 samples were most suspected of contamination even though the contamination found appears to have been other than amino acids or precursors (Flory and Simoneit, 1972; Murphy, 1972).

2.5. Sources of Amino Acids from the Moon and Meteorites The consensus of lunar investigators appears to be that amino acid precursors are not long-term indigenous components of the lunar surface, for decomposition would occur rapidly in the absence of a protective atmosphere. There seems to be little or no disagreement with this inference. Gehrke et al. (1975) have "particularly" referred to comments of Sagan (1972) on an experimental evaluation of the breakdown of amino acids under conditions designed to simulate the lunar surface. For this literature review, no papers describing such experiments have been found under any of the four names cited for the unpublished paper in Sagan's 1972 bibliography. In a search of the literature from 1972 to 1980, we have not found data that support the claims of Gehrke et al. in citation of Sagan. In addition, Sagan referred to testing the stability of amino acids in a simulated lunar environment; amino acids could not, of course, be expected to serve as a suitable model for amino acid precursors.

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The fact that criticisms imputing contamination appear to be unsupported does not, of course, in itself establish that materials assayed are not contaminated. That inference relies on definitive data. The postulated possibilities for contamination are many; the lunar fines have received major attention in this context (Simoneit et aI., 1969; Flory and Simoneit, 1972; Flory et ai., 1972; Gibert et ai., 1971; Gehrke, 1972; Gehrke et ai., 1970; Hamilton and Nagy, 1972, 1975; Hare, 1972; Fox et ai., 1974, 1976). The prospects and early data for clean meteorites are much less favorable, as indicated earlier; the fact that results on meteorites conform in the main with lunar analyses is a principal basis of support for the former. Informative as comparison of GLC results with IEC results has been, comparison with results from pyrolysis-gas chromatography-mass spectrometry (Preti et ai., 1971) is also revealing. The finding of these workers that Apollo 12 core samples contained more organic material than rocket exhaust material is a result that itself suggests little or no contamination by rocket exhaust (Preti et ai., 1971). Also, pyrolysis-mass spectrometry results indicate that Apollo 12 samples had greater general cleanliness than those from Apollo 11, in agreement with other observations. Another consideration from examination of the pyrolytic products is the summary statement (Preti et ai., 1971) that organic material in Apollo 12 lunar samples is less than 10 ppb, a statement that is qualified by irreproducibility of yields in analytical pyrolyses of lunar materials. One may ask if a figure of 10 ppb will accommodate the amounts of amino acids determined by IEC. The amount found by IEC is 19 ppb (Table I). However, when HCN is converted to glycine by hydrolysis (Fox et ai., 1976), the increase in molecular weight, resulting from addition of elements of water, is almost threefold. The spread is sufficient to accommodate the amounts of amino acid found, if one accepts the 10 ppb figure from mass spectrometry as accurate. Virtually all investigators have constructed analyses on the model that amino acids from the moon are in the form of precursors (probably cyanide oligomers), which do not have the instability of free amino acids. The free amino acids have carboxyl, and especially the reactive primary amino, groups (Fox and Foster, 1957). Moreover, Sagan claimed that his unpublished laboratory results on breakdown signified a half-life of 104 years for free amino acids (identity unspecified). A half-life of 104 years could comfortably be accommodated by a steady-state model involving implantation of C, H, and N from the sun as replenishment for those amino acid precursors suffering destruction. This argument about half-life, while cogent, is nonetheless superfluous because of the chemical none qui valence of amino acids and their precursors and the absence of supportive data for breakdown. One of the sources suggested for amino acid precursors is that of meteorites impacting the moon (Gros et at., 1976; Chang et at., 1971). Other

367

Amino Acids from the Moon

sources include interstellar matter (Snyder and Buhl, 1970), indigenous material (Sagan, 1972), comets (Oro, 1961; Sagan, 1973), and the solar wind (cf. Kaplan et aI., 1970; Moore et al., 1970; Cadogan et al., 1972; cf. Hayes, 1972). The indigenous matter would have to have been sheltered under the surface or under other overlying protection (Sagan, 1972); this stated requirement is consistent with the consensus of the destructibility of organic matter in the absence of such protection. By the time samples through Apollo 16 had been analyzed for carbon and sulfur, Moore et al. (1973) preferred a steady state from solar wind addition (and sputtering), whereas meteoritic carbon flux would require a similar flux of sulfur. The sulfur flux was not observed. For such reasons, the solar wind is here favored as a source. Continuous implantation from the solar wind, which seems to be quantitatively adequate for the amount of precursor found, would replenish material destroyed by radiation, thereby constituting a steady state. Relevant to the solar wind hypothesis is the finding of concordant analyses in various sites on the moon. The failure to find much organic matter on the moon and, subsequently, a failure to recognize small amounts were both surprising. The failure to find the "bonanza of organic molecules" (Ponnamperuma, 1972) prepared for by an elaborate scheme (Kvenvolden, 1972) has been explained as due to unwarranted extrapolation to the moon's surface of experiments in closed flasks (Fox, 1978) plus failure to allow for unhindered destruction by solar radiation. The failure of some (Oro et al., 1970; Gehrke et al., 1972) to find any amino acids at first has been explained in this paper, and elsewhere (Fox and Dose, 1977; Harada and Hare, 1980), as due primarily to inappropriate methods of sample preparation and analysis.

2.6.

The Chemical Nature of Amino Acid Precursors

Amino acids were sought by hydrolysis partly because of the fact that polymers arise easily from amino acids in arid environments (cf. Rohlfing, 1976). However, the probabilities pointed early in the Apollo program to the precursors being cyanide compounds, such as cyanide oligomers (Harada, 1967). Although the precursors are still not positively identified, experience (rather than controlled experiments) has suggested that they are cyanides because of the rapid hydrolyzability of the compounds, a property not observed in proteins (Fox and Foster, 1957) or in thermal copolyamino acids (Fox et aI., 1963). It follows that extraction at temperatures below 100 C in the presence of minerals might itself have caused substantial hydrolysis in some cases. This is one likely explanation for the free amino acids found in the Apollo 11 samples (Nagy et al., 1970) and in various meteorites (Kvenvolden, 1974), since the latter contain water and are heated at the anterior surface during fall and on impact. It is also true that, since free amino acids exist in meteorites at some 0

368

Sidney W. Fox et al.

point, they are subject to conversion from proteinaceous amino acids like glutamic acid to nonproteinaceous ones like a- and 'Y-aminobutyric acids, during heating in the atmosphere and upon impact. In discussions in some papers, the distinction between amino acid precursors and free amino acids is not drawn consistently (Gehrke et al., 1975). Conclusions about amino acids do not apply to their precursors and vice versa in each case. For example, although amino acid precursors were found in Apollo 12 samples, amino acids were not, perhaps because the stabilities of the two kinds of compound differ. Perhaps the first worker to suggest in print that the precursors in lunar fines are, for example, cyanides and aldehydes or cyanide oligomers was Biemann (1972). Mechanisms for the formation of sets of amino acids from cyanide oligomers have been proposed by Harada (1967) and earlier investigators (cf. Harada, 1967), while mechanisms involving cyanohydrin intermediates have been proposed by Miller (1957).

3. SUMMARY AND PROSPECT The amino acid analyses of hydrolysates of extracts of 11 collections from the six Apollo missions are presented in Table I (Fox et al., 1974). Twelve collections were analyzed, but one sample was known to have suffered contamination by outside air during analytical processing. Since the results in that case were atypical, showing contents of tyrosine and phenylalanine for example, some ability to discern contamination was thereby verified. A number of questions can be formulated from the background of the studies touched upon in this review. The results can be said to support some working hypotheses strongly and others weakly or not at all. In common with other pioneering scientific inquiries, "conclusions" are not easily attained. Further reconfirmation of some inferences, regarded by some as established, is nonetheless desired by many viewers of the scene. In this category may be placed the testing of released amino acids for optical activity as a further check on whether the amino acids or precursors are contaminants. What is the chemical nature of the precursors? This line of investigation has been hampered by the unavailability of quantities sufficient for direct determination. This difficulty may be surmounted by indirect identification (e.g., comparison of rates of hydrolysis with standard materials) followed by selected direct determinations. If organic matter exists on Mars (cf. Biemann et al., 1977; Young, 1978; Klein, 1979), how does it compare with that from the moon?

369

Amino Acids from the Moon

The relationships to compounds in meteorites deserve attention. Why is there such good concordance between the two sources, in identity and amounts relative to carbon, with respect to proteinaceous amino acids, while only the meteorites contain significant amounts of nonproteinaceous amino acids? What is the quantitative contribution of friction heat, of heat of impact, and of the presence of water in carbonaceous chondrites to the presence of nonproteinaceous amino acids? How pervasive in the solar system are cyanide oligomers as evolutionary intermediates? Cyanide is found in the Urey-Miller synthesis (Miller, 1957); in the thermal reaction of methane and ammonia (Harada and Fox, 1965; Ferris, 1979), and in the heating of hexamethylenetetramine from formaldehyde and ammonia (Fox and Windsor, 1970; Khare and Sagan, 1979), and the same set of five or six proteinaceous amino acids appears to arise from lunar fines, meteoritic samples, and other simple intermediates (e.g., Kamaluddin et al.. 1979). Metal cyanides may thus have been a nexus at the beginning of organic evolution, with compounds of C, H, and N having been converted to HCN (Khare and Sagan, 1979) and HCN oligomers, from which sets of amino acids could have arisen. The full set could then have been determined by the minerals in which the synthesis or hydrolysis occurred (Harada and Fox, 1965; Aragon de la Cruz and Barbolla, 1979). The essential idea is depicted in Figure 2. Protocells

1 H2 0

Proteinoids

I