MOLECULAR NEUROBIOLOGY - Springer978-1-4615-7488-0/1.pdf · La Jolla, California 92037 JOHN CONNOLLY Molecular Neurobiology Laboratory The Salk Institute ... at present there is no

MOLECULAR NEUROBIOLOGY

Recombinant DNA Approaches

CURRENT TOPICS IN NEUROBIOLOGY

Series Editor

Samuel H. Barondes Professor and Chairman, Department of Psychiatry and Director, Langley Porter Psychiatric Institute University of California, San Francisco San Francisco, California

Cell Culture in the Neurosciences Edited by Jane E. BoHenstein and Gordon Sato

Molecular Neurobiology: Recombinant DNA Approaches Edited by Steve Heinemann and James Patrick

Neuroimmunology Edited by Jeremy Brockes

Neuronal Development Edited by Nicholas C. Spitzer

Neuronal Recognition Edited by Samuel H. Barondes

Pep tides in Neurobiology Edited by Harold Gainer

Tissue Culture of the Nervous System Edited by Gordon Sa to

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 informa· tion please contact the publisher.

MOLECULAR NEUROBIOLOGY

Recombinant DNA Approaches

Edited by Steve Heinemann

and James Patrick

The Salk Institute San Diego, California

PLENUM PRESS. NEW YORK AND LONDON

Library of Congress Cataloging in Publication Data

Molecular neurobiology.

(Current topics in neurobiology) Includes bibliography and index. 1. Molecular neurobiology. 2. Recombinant DNA. I. Heinemann, Steve. II. Patrick,

James, 1942- . III. Series. [DNLM: 1. Cloning, Molecular. 2. DNA, Recombinant. 3. Gene Expression Regulation. 4. Molecular Biology. 5. Nervous System. 6. Neurobiology. WL 100 M7188] QP356.2.M65 1987 599'.0188 87-2512

ISBN 978-1-4615-7490-3 ISBN 978-1-4615-7488-0 (eBook) DOl 10.1007/978-1-4615-7488-0 0

© 1987 Plenum Press, New York Softcover reprint of the hardcover 15t edition 1987 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

GIGI ASOULINE Molecular Neurobiology Laboratory The Salk Institute La Jolla, California 92037

MARC BALLIVET Molecular Neurobiology Laboratory The Salk Institute La Jolla, California 92037

JIM BOULTER Molecular Neurobiology Laboratory The Salk Institute La Jolla, California 92037

JOHN CONNOLLY Molecular Neurobiology Laboratory The Salk Institute La Jolla, California 92037

EVAN DENERIS Molecular Neurobiology Laboratory The Salk Institute La Jolla, California 92037

NICK J. DIBB MRC Laboratory of Molecular Biology University Postgraduate Medical School Cambridge CB2 2QH, England

JANET RETTIG EMANUEL Department of Cell Biology Yale University School of Medicine New Haven, Connecticut 06510

v

vi

GLEN A. EVANS

KAREN EVANS

SYLVIA EVANS

LLOYD D. FRICKER

JOHN FORREST

PAUL GARDNER

SUSAN GARETZ

ORA GOLDBERG

DAN GOLDMAN

MARK GRIMES

Cancer Biology Laboratory The Salk Institute San Diego, California 92138

Contributors

Molecular Neurobiology Laboratory The Salk Institute La Jolla, California 92037


Molecular Pharmacology Department Albert Einstein College of Medicine Bronx, New York 10461



Department of Cell Biology Yale University School of Medicine New Haven, Connecticut 06510

Department of Organic Chemistry The Weizmann Institute of Science Rehovot 76100, Israel


Institute for Advanced Biomedical Research

The Oregon Health Sciences University Portland, Oregon 97201

Contributors vii

STEVE HEINEMANN Molecular Neurobiology Laboratory The Salk Institute La Jolla, California 92037

EDWARD HERBERT Institute for Advanced Biomedical Research


JONATHAN KARN MRC Laboratory of Molecular Biology University Postgraduate Medical School Cambridge CB2 2QH, England

ABHA KOCHHAR Molecular Neurobiology Laboratory The Salk Institute La Jolla, California 92037

GREG LEMKE Molecular Neurobiology Laboratory The Salk Institute for Biological Studies La Jolla, California 92037

ROBERT LEVENSON Department of Cell Biology Yale University School of Medicine New Haven, Connecticut 06510

DANE LISTON Institute for Advanced Biomedical Research


WALTER LUYTEN Molecular Neurobiology Laboratory The Salk Institute La Jolla, California 92037

ANNE C. MAHON Department of Biological Sciences Stanford University Stanford, California 94305

PAM MASON Molecular Neurobiology Laboratory The Salk Institute La Jolla, California 92037

viii

DAVID M. MILLER

E. JANE MITCHELL

JIM PATRICK

CATHERINE PRODY

RICHARD H. SCHELLER

JAY W. SCHNEIDER

HERMONA SOREQ

DINA ZEVIN-SONKIN

DOUG TRECO

KEIJI WADA

Contributors

MRC Laboratory of Molecular Biology University Postgraduate Medical School Cambridge CB2 2QH, England

MRC Laboratory of Molecular Biology University Postgraduate Medical School Cambridge CB2 2QH, England


Department of Neurobiology The Weizmann Institute of Science Rehovot 76100, Israel

Department of Biological Sciences Stanford University Stanford, California 94305

Department of Cell Biology Yale University School of Medicine New Haven, Connecticut 06510





Preface

This book is a collection of papers describing some of the first attempts to apply the techniques of recombinant DNA and molecular biology to studies of the nervous system. We believe this is an important new direction for brain research that will eventually lead to insights not possible with more traditional approaches. At first glance, the marriage of molecular biology to brain research seems an unlikely one because of the tremendous disparity in the histories of these two disciplines and the problems they face. Molecular biology is by nature a reductionist approach to biology. Molecular biologists have always tried to attack central questions in the most direct approach possible, usually in the most simple system available: a bacterium or a bacterial virus. Important experiments can usually be repeated quickly and cheaply, in many cases by the latest group of graduate students entering the field. The success of molecular biology has been so profound because the result of each important experiment has made the next critical question obvious, and usually answerable, in short order.

Studies of the nervous system have a very different history. First, the human brain is what really interests us and it is the most complex structure that we know in biology. The central question is clear: How do we carry out higher functions such as learning and thinking? However, at present there is no widely accepted and testable theory of learning and no clear path to such a theory. Numerous attempts have been made to find simple brains with interesting properties and, while some insights have been gained from these studies, the search for simple systems has justified the investment of scarce talent and resources into the study of many different species. Thus many experiments are not replicated quickly; sometimes they are forgotten before it is possible to know whether they are true or not. In many cases, the experiments

ix

x Preface

require expensive specialized equipment as well as expertise and are hard to replicate. Thus brain research has not always had the quick feedback it needs to make steady progress.

Despite these problems, however, there have been some great triumphs in neurobiology. The working out of the basis of the action potential and synaptic transmission are impressive examples. More recently progress in understanding the visual system has been encouraging. This progress has induced a new group of investigators trained in cell biology and molecular biology to enter the neurosciences. At first glance, there is reason to be skeptical that the reductionist approach of molecular biology can make important contributions to the study of a system as complex as the brain. But there are some encouraging developments that make this a pessimistic view. Molecular biologists have recently moved into three very different areas involving complex systems: cancer, immunology, and the general problem of development. It is already clear that knowledge of the structure of the genes involved in these complex systems has dramatically changed the way we think about these problems. We believe that molecular biology will make this same kind of contribution to the study of the brain. For example, many plausible theories of brain function postulate that the stimulation of nerve networks results in a stable change in the efficiency of synaptic transmission at individual synapses. This must involve a change in some molecule associated with the synapse. Since we know little about the detailed structure of the molecules present at synapses in the brain, it is difficult to test these ideas. It seems to us that the best J:tope for studying the molecules important for brain function is through the use of gene cloning and the techniques of molecular biology. The chapters in this book represent a beginning.

Steve Heinemann Jim Patrick

Contents

1. The Molecular Biology of the Na,K-ATPase and Other Genes Involved in the Ouabain-Resistant Phenotype ROBERT LEVENSON, JANET RETTIG EMANUEL, SUSAN GARETZ,

AND JAY W. SCHNEIDER

1. Introduction....................................... 1 2. Molecular Cloning of the Na,K-ATPase Catalytic

Subunit........................................... 3 3. Organization and Expression of the Rat Sodium

Pump a-Subunit Gene ............................. 3 4. Organization and Expression of the a-Subunit Gene

in Ouabain-Resistant Cell Lines . . . . . . . . . . . . . . . . . . . . . 4 5. Isolation and Characterization of a Ouabain-Resistance

Gene. . . . .. ... . . . . .. . .. . . ... . . . . . . . . . . .. . . .. . . . . . . 10 6. Transfer of the Sodium Pump a-Subunit Gene

Confers Ouabain Resistance to Ouabain-Sensitive Cells ............................................. 12

7. Isolation of Genes Related to the Sodium Pump and Their Expression in a Ouabain-Resistant Cell Line. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

8. Conclusions and Future Prospects. . . . . . . . . . . . . . . . . . . 16 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2. Molecular Biology of the Genes Encoding the Major Myelin Proteins GREG LEMKE

1. Introduction....................................... 21 2. Formation and Structure of the Myelin Sheath. . . . . . . . 22

xi

xii Contents

3. Myelin-Specific Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.1. Myelin Basic Protein. . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.2. Po............................................ 29 3.3. Proteolipid Protein. . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.4. Other Myelin Proteins . . . . . . . . . . . . . . . . . . . . . . . . . 37

4. Conclusion........................................ 38 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3. Molecular Biology of the Neural and Muscle Nicotinic Acetylcholine Receptors STEVE HEINEMANN, GIGI ASOULINE, MARC BALLIVET, JIM

BOULTER, JOHN CONNOLLY, EVAN DENERIS, KAREN EVANS,

SYLVIA EVANS, JOHN FORREST, PAUL GARDNER, DAN GOLDMAN,

ABHA KOCHHAR, WALTER LUYTEN, PAM MASON, DOUG

TRECO, KEIJI WADA, AND JIM PATRICK

1. Introduction....................................... 45 2. Isolation of cDNA Clones Coding for the Acetylcholine

Receptor Expressed in the Torpedo Electric Organ. . . . . 48 2.1. Torpedo -y-Subunit ............................. 50 2.2. Torpedo a-Subunit ............................. 51 2.3. Torpedo Clone 3C1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

3. Isolation of cDNA Clones Coding for Mouse Skeletal Muscle Acetylcholine Receptor. . . . . . . . . . . . . . . . . . . . . . 57 3.1. Mouse a-Subunit. . . . . . . . . . . ... . . .... . . . .. . .. . . 58 3.2. Mouse ~-Subunit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 3.3. Mouse -y-Subunit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 3.4. Mouse 5-Subunit . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 59 3.5. Expression of Mouse Receptor in Oocytes ....... 62

4. Structure of the Acetylcholine Receptor. . . . . . . . . . . . . . 63 4.1. Primary Structure ............................. 63 4.2. Folding through the Membrane. ... . . .. . . . ... ... 63 4.3. Acetylcholine-Binding Site ..................... 75

5. Expression of the Acetylcholine Receptor Genes in Skeletal Muscle ................................. 76 5.1. Skeletal Muscle Denervation. . . . . . . . . . . . . . . . . . . . 76 5.2. Synaptic versus Extrasynaptic Acetylcholine

Receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 6. Brain Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

6.1. Neuronal a-Subunit Clone ..................... 82 6.2. Structure of the Neuronal a-Subunit ............ 82

Contents xiii

6.3. Expression of the Neural a-Subunit RNA. . . ... . . 84 6.4. Evidence for a Gene Family . . . . . . . . . . . . . . . . . . . . 89

7. Conclusion........................................ 89 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

4. Molecular Biology of Muscle Development: The Myosin Gene Family of Caenorhabditis elegans JONATHAN KARN, NICK J. DIBB, DAVID M. MILLER, AND E. JANE MITCHELL

1. Introduction....................................... 97 2. Genetics of the unc-54 Locus. . . . . . . . . . . . . . . . . . . . . . . . 99

2.1. Selection of unc-54 Mutations. . . . . . . . . . . . . . . . . .. 101 2.2. Deletions and Duplications of the unc-54 Locus .. 101 2.3. Orientation of the unc-54 Gene. . . . . . . . . . . . . . . .. 102 2.4. In Situ Hybridization to Chromosomes. . . . . . . . .. 102 2.5. Interactions with Other Genes. . . . . . . . . . . . . . . . .. 104

3. Immunological Identification and Localization of Myosin Isoforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 107 3.1. Production of Four Sarcomeric Myosin Heavy-

Chain Isoforms by Caenorhabditis elegans ......... 107 3.2. Monoclonal Antibodies Specific for Different

Myosin Isoforms .............................. 109 3.3. Two Myosins Required to Make the Body Wall

Thick Filament . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 110 4. Molecular Cloning of the unc-54 and myo-1,2,3

MHC Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 114 4.1. Cell-Free Translation of MHC mONA . . . . . . . . . .. 114 4.2. unc-54 cDNA Clones. . . . . . ... . . ...... .... . ..... 114 4.3. Cloning the unc-54 Gene. . . . . . . . . . . . . . . . . . . . . .. 117 4.4. Identification of the myo-1,2,3 MHC Genes by

Homology to unc-54 .. ... . . . . . . . . . . . .. . . ... . . .. 119 5. Immunological Identification of the Products of the

myo-1,2,3 MHC Genes ............................. 119 6. Structural Organization of the Nematode MHC

Genes ............................................ 124 6.1. Highly Conserved Head Sequences and

Variable Rod Sequences. . . . . . . . . . . . . . . . . . . . . . .. 124 6.2. Selective Loss of Introns from the Nematode

MHC Genes .................................. 125

xiv Contents

6.3. Unusual Features at Intron Junctions. . . . . . . . . . .. 127 6.4. Terminal Sequences ........................... 133

7. Molecular Anatomy of the Myosin Molecule. .. ... . .. 134 7.1. Topography of the Head. . . . . . .... . . . . . . . . ... .. 136 7.2. Rod Sequences. . . ... .. .. . . . ... .. . . . . . . ... .. . .. 139

8. Sequences and Molecular Interpretation of unc-54 Mutations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 148 8.1. Rapid Cloning and Sequencing of Mutations. . . .. 148 8.2. Correlation of Physical and Genetic Fine-

Structure Map ................................ 149 8.3. Deletions in the Rod .......................... , 150 8.4. Nonsense Mutations and Suppressors. . . . . . . . . .. 152 8.5. Mutations Affecting the Synthesis of MHC ...... 154 8.6. A Dominant Assembly-Defective Missense

Mutation ............................ . . . . . . . .. 154 8.7. Mutations in the Head. . . . . . . . . . . . . . . . . . . . . . . .. 155 8.8. Mechanisms of Mutagenesis in the Nematode. . .. 156

9. Myosin Protein Expression and Mutagenesis in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 157 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 162

5. Small Cardioactive Peptides in A and B: Chemical Messengers in the Aplysia Nervous System ANNE C. MAHON AND RICHARD H. SCHELLER

1. Introduction....................................... 173 2. The Distribution of SCP-Immunoreactive Neurons

in the CNS .................. " . . . . . .... . .. ... . . . .. 175 3. The SCP Gene and Precursor Protein. . . . . . . . . . . . . . .. 178

3.1. Cloning and Characterization of the SCP Gene. . . . . . . . . . . . . . . . . . . . . . . .. ... . . . . . . . . . . ... 178

3.2. The SCP Precursor Protein . . . . . . . . . . . . . . . . . . . .. 180 4. SCP Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 181 5. Subcellular Localization ............................ 181 6. Coexistence of Multiple Transmitters ................ 183

6.1. SCP A and SCPB .. .. .... .. .. ...... .. .. .. .. ..... 183 6.2. Acetylcholine in Neuron B2 .................... 183

7. Physiological Activities. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 183 8. Conclusions....................................... 188

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 189

Contents

6. Molecular Biology Approach to the Expression and Properties of Mammalian Cholinesterases HERMONA SOREQ, DINA ZEVIN-SONKIN, ORA GOLDBERG, AND

CATHERINE PRODY

1. Introduction: Expression of Cholinesterases as a Research Subject-Scientific Significance, Advantages, and Difficulties ........................ 1.1. Detection of Enzymatic Activity ................. 1.2. Polymorphism of Cholinesterases ............... 1.3. Tissue and Cell-Type Specificity ................ 1.4. Putative Biological Role(s) ...................... 1.5. Genetic Evidence for Allelic Polymorphism ...... 1.6. Molecular Approach to Cholinesterases .......... 1.7. General Research Strategy: Simultaneous

Experiments Approaching Various Levels of Gene Expression ............................

2. Expression of Cholinesterase mRNAs in Microinjected Xenopus Oocytes ......................

3. Identification of Drosophila DNA Fragment that Hybridizes with Cholinesterase mRNA .............. 3.1. Assignment of Drosophila Transcripts that

Hybridize with DroS ........................... 3.2. Hybrid Selection of Cholinesterase-Inducing

mRNA by DroSR .............................. 3.3. RNA Blot Hybridization Reveals Homology

between DroSR and mRNA from Human Cholinesterase-Expressing Tissues ..............

4. Isolation and Partial Characterization of Human DNA Fragments Homologous to DroSR .............. 4.1 Isolation and Characterization of a Human

Genomic Fragment: Huache1 .................... 4.2. Hybridization of Huache1R DNA with Poly(A)+

RNA Species from Fetal Human Brain ........... 4.3. Hybrid Selection of Acetylcholinesterase-

Inducing mRNA with Huache1R DNA .......... 4.4. Immunoprecipitation of Acetylcholinesterase

Polypeptides from Oocytes Injected with Hybrid-Selected mRNA ........................

4.5. Preliminary Characterization of Huache1R DNA .. 4.6. Isolation of Huache1R-Homologous Genomic

DNA Fragments ...............................

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191 192 193 194 195 196 196

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199

201

202

203

203

204

205

205

206

206 207

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xvi Contents

4.7. Preparation of Huache1R-Homologous Fetal cDNA Clones ................................. 208

4.8. Conclusions .................................. 209 5. Preparation of Synthetic Oligonucleotide Probes

According to the Consensus Sequence at the Organophosphate-Binding Site ...................... 209 5.1. Strategy for Preparation and Selection of the

Correct Mixture of Synthetic Oligonucleotides .... 210 5.2. Probing of Selected DNA Fragments with the

Synthetic Oligonucleotide ...................... 211 5.3. Screening for OPSYN-Containing cDNA

Sequences from cDNA Libraries in Agt Vectors ... 212 6. Preliminary Characterization of Neuroche

cDNA Clones ..................................... 214 6.1. Blot Hybridizations with Restricted Genomic

DNA (Human and Mouse) and with mRNA ..... 214 6.2. Identification of Acetylcholinesterase-

Immunoreactive Fusion Protein by Crossed Immunoelectrophoresis and Immunoprecipitation .......................... 217

7. Summary and Conclusions ......................... 219 References ........................................ 220

7. Genes and Gene Families Related to Immunoglobulin Genes GLEN A. EVANS

1. Introduction....................................... 225 2. Immunoglobulin Genes and the Immunoglobulin

Domain ........................................... 227 3. The T-Lymphocyte Cell-Surface Receptor

for Antigen ....................................... 233 4. Class I MHC Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 235

4.1. H-2 K, D, and L. . . . . . . .. . . . . . .... . . . . . . . . ... .. 235 4.2. QafTLa Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 239 4.3. ~z-Microglobulin .............................. 241

5. Class II MHC Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 241 6. Cell-Surface Receptors for Transepithelial Transport

of Immunoglobulin ................................ 242 7. The T-Cell Accessory Molecules T4 and T8 . . . . . . . . . .. 243 8. Related Members of the Immunoglobulin Supergene

Family Expressed in the Nervous System. . . . . . . . . . .. 246

Contents xvii

8.1. The Thy-1 Glycoprotein.. . . . . ........... . . .. . .. 246 8.2. The OX-2 Antigen. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 248

9. Conclusion: The Evolution of the Immunoglobulin Superfamily.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 249 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 253

8. Specificity of Prohormone Processing: The Promise of Molecular Biology LLOYD D. FRICKER, DANE LISTON, MARK GRIMES, AND

EDWARD HERBERT

1. Introduction....................................... 259 1.1. Tissue-Specific Processing of Opioid Peptides . . .. 260 1.2. Limitations of Classic Approaches to the Study

of Prohormone Processing ..................... 263 2. Gene-Transfer Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 263

2.1. Transfer of Proenkephalin into Mouse Pituitary Cells .................................... '" .. 264

2.2. Other Gene-Transfer Studies. . . . . . . . . . . . . . . . . .. 267 2.3. Reduction of Enzyme Activity with Antisense

RNA ......................................... 271 3. Enzymatic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 272

3.1. Trypsinlike Processing Enzymes . . . . . . . . . . . . . . .. 273 3.2. Carboxypeptidase E (Enkephalin Convertase) . . .. 275

4. The Internal Environment of Secretory Granules. . . . .. 279 5. Perspectives....................................... 282

References ....................................... , 284

Index ...................................................... 293

Documents

MOLECULAR NEUROBIOLOGY - Springer978-1-4615-7488-0/1.pdf · La Jolla, California 92037 JOHN CONNOLLY Molecular Neurobiology Laboratory The Salk Institute ... at present there is no