Protocols in Molecular Neurobiology

Methods in Molecular Biology

John M. Walker, SERIES EDITOR

1. Proteins, edited by John M. Walker, 1984 2 . Nucleic Acids, edited by John M. Walker, 1984 3. New Protein Techniques, edited by

John M. Walker, 1988 4. New Nucleic Acid Techniques, edited by

John M. Walker, 1988 5. Animal Cell Culture, edited by J e m W. P o U d

and John M. WaUzer, 1990 6. Plant Cell and Tissue Culture, edited by

Jeffiey W. Pollard and John M. Walker, 1990 7. Gene Transfer and Expression Protocols,

edited by E. J. Murray, 1991 8. Practical Molecular Virology, edited by

Mary K. L. Collins, 1991 9. Protocols in Human Molecular Genetics,

edited by Christopher G. Mathew, 1991 10. Immunochemical Protocols , edited by

Margaret M. Manson, 1992 11. Practical Protein Chromatography, edited by

Andrew Kenney and Susan Fowell, 1992 12. Pulsed-Field Gel Electrophoresis:

Protocols, Methods, and Theories, edited by Margit Burmeister and Levy ~ v s k y , 1992

13. Protocols in Molecular Neurobiology, edited by Alan Longstaff and Patricia Revest, 1992

Protocols in Molecular Neurobiology

Edited by

Alan Longstaff University of Hertfordshire, Hatfield, UK

Patricia Revest King's College, University of London, UK

Humana Press Totowa, New Jersey

Q 1992 The Humana Press Inc. 999 Riverview Drive Totowa, New Jersey 0751 2

MI rights resewed

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form o r by any means, electronic, mechanical, photocopying, microfilming, recording, o r otherwise without written permission from the Publisher.

Printed in the United States of America. 9 8 7 6 5 4 3 2 1

Library of Congress Cataloging in Publication Data Main entry under title:

Methods in molecular biology.

Protocols in molecular neurobiology / edited by Alan Longstaff, Patricia Revest

p. cm. - (Methods in molecular biology ; 13) Includes index. ISBN 0-89603-199-3 1. Molecular neurobiology-Methodology. I. Longstaff, Alan.

11. Revest, Patricia. 111. Series: Methods in molecular biology (Totowa, N.J.) ; 13. QP356.2.P76 1992 599'.0186-dc20 92-30701

CIP

Preface

Neurobiologists are bound to differ in their perceptions of what the discipline of molecular neurobiology should encompass. We have taken the view that molecular neurobiology should cover any aspect of brain science that uses the techniques of modern molecular biology, though we accept the fact that classification of a technique as a "biochemical* or "molecular biological* one is in itself somewhat arbitrary.

Each chapter of this volume sets out to identify a clear problem in neurobiology and to place it in its context within the literature- i.e., indicating how the solution of the problem will advance knowledge in the field. The core of the chapter then details the approaches taken to solve the problem, in sufficient detail that the reader can appreci- ate exactly why a specific strategy was adopted and how it was executed. Each chapter also includes detailed protocols providing all the infor- mation necessary to reproduce the technique and its results in any appropriately equipped laboratory.

Moving in the general direction of the central dogma, the vol- ume starts with chapters on manipulation of DNA. It seems likely that many genomic regions of special interest to neuroscientists will turn out to be quite large, as, for example, the dystrophin gene. Large DNA molecules pose particular problems for the experimenter, and Shaw in the first chapter of this volume gives an account of how DNA frag- ments greater than 30 kilobases, which cannot be successfully resolved using conventional agarose gel electrophoresis, can be separated by using pulsed-field gel electrophoresis. With its precisely timed inver- sions of the electric field direction, the technique separates smaller from larger molecules, larger ones take longer to reorient within the electric field vector than do smaller molecules, and thus migrate more slowly.

A standard and widely used strategy of molecular biologists inter- ested in isolating and sequencing DNA coding for molecules of inter- est is to isolate total mRNA from the appropriate tissue and from this

vi Preface

to generate complementary DNA (cDNA) . Vreugdenhil and Darlison point out that this approach may cause difficulties for mRNAs of low abundance (frequently the case for molecules of interest to neurobi- ologists, such as receptors) and that the alternative use of genomic libraries is particularly applicable for invertebrates with-when com- pared to vertebrates-relatively small genomes. Although preparation ofvertebrate DNA is simple and widely described, isolation of genomic DNA from invertebrates can be problematic. The continuing impor- tance of invertebrate neurobiology guarantees the wide utility of this procedure.

Often the amount of DNA available for study is limited. This is the case for mitochondria1 DNA (mtDNA), mutations of which are associated with various neurological disorders, including Parkinson's disease. The characterization of small amounts of DNA is made pos sible by iterative procedures that successively increase the amount of specified DNA fragments-the polymerase chain reaction (PCR) . PCR allows millions of copies of a specific DNA fragment to be synthesized in vitro from, theoretically, only a single copy of the original sequence. Two oligonucleotide primers complementary to regions flanking the DNA fragment of interest are chosen, one for each strand of the double- stranded DNA. Repeated cycles of denaturation of the double-stranded DNA, annealing of the primers to their complementary DNA, and replication of the complementa~y strand by DNA polymerase allow rapid production of copies of the region between the primers. Since the DNA produced in each cycle acts as the template for subsequent cycles, the growth in the number of copies is exponential. The uses of different varieties of PCR, in relation to mtDNA, is described in detail by Tanaka and Ozawa.

A key approach to isolating a specific fragment of DNA from het- erogeneous fragments distributed among a large number of recom- binant DNA clones requires the synthesis of oligonucleotide probes, which are predicted to be complementary to, and thus will hybridize with, only the DNA of interest. Two chapters, one from Bateson and Darlison and another from Webb and Bateson, deal with the use of these probes. The first chapter provides crucial theoretical insights into the design of suitable oligonucleotides-including PCR primer- using many examples from neurobiology as illustration. The second chapter gives a detailed example, describing precisely how the design of degenerate oligonucleotide primers allows the isolation of Gpro- teincoupled receptors using PCR amplification.

Preface vii

There are a large number of proteins present in cells that are thought to be involved in specialized functions and a first step in their identification is the isolation of tissue-specific mRNk The use of labeled cDNA to probe a retinal cDNA library is described by Kuo, who provides a detailed account of differential colony hybridization in the analysis of photoreceptorspecific molecules implicated in visual transduction. The generation of the retinal-specific probes is accomplished by the hybridization of retinal and brain cDNA with the subsequent removal of both the hybridized doubleatranded DNA and the brain-specific cDNA, leaving only the retinalspecific cDNA.

There are now many documented instances of molecules that occur in a variety of isoforms arising either by differential mutation of copies of an ancestral gene or by alternative splicing of heterologous RNA transcripts. These isoforms, though displaying high homology with each other, often exhibit significant differences in cellular compartmentation, tissue by localization, developmental expression, and pharmacological properties, all of which strongly implies func- tional differences. Clearly, antibodies that are specific for particular isoforms could prove invaluable in investigating the roles of these isoforms. How can specific antibodies be generated if the proteins are highly homologous or when sufficiently pure preparations of the pr* tein are unavailable? This is the question tackled by Shyjan and Levenson in their study of isoforms of Na/K-ATPase in brain. The strategy they report is to ligate cDNA fragments derived from the dis- tinct isoforms to an E. colireporter gene using a plasmid vector. The E. coli clones generate fusion proteins that are then used as antigens to produce polyclonal antisera. The fusion proteins that are monospecific for a particular isoform can then be purified by immunoabsorption.

The expression of brain-specific proteins is regulated at least in part by modulation of the mechanisms responsible for initiating transcription of RNA by RNA polymerase 11. The study of these pr* cesses is obviously advanced by a knowledge of initiation sites, which can be deduced by use of either the S, nuclease digestion assay or primer extension. This is discussed by Weisinger, DeCristofBro, and La Gamma. The essence of the S, assay is that a labeled oligonucle- otide probe is constructed that will hybridize to the putative 5' end of the mRNA under investigation. This 5' end is now protected-being doublestranded-from digestion by S, nuclease, which removes DNA probe material upstream of the RNA start site and downstream RNA not recognized by the probe. The labeled fragments may now be frac-

... ULLL Preface

tionated by gel electrophoresis and visualized by autoradiography. Primer extension is useful if a labeled deoxyribonucleotide probe can be constructed that is complementary to a sequence of RNA close to the start site. The probe is hybridized to the RNA and reverse tran- scriptase catalyzes the extension of the probe up to the start of the transcript.

Modulation of transcription by nuclear transcription factors that bind to DNA is of considerable physiological importance. Weisinger and La Gamma provide detailed protocols for making nuclear extracts and for methylation interference footprinting, which enables the identification of the DNA binding sites that recognize and bind tran- scription factors. In the technique of methylation interference assays, the end-labeled DNA fragment of interest is randomly methylated and then incubated with the putative DNA binding factor(s). The regula- tory proteins can only bind to nonmethylated DNA and both free and bound fractions can be separated by gel-retardation assays. The dif- ferent fractions can then be analyzed in sequencing gels. The DNA is cleaved at the methylated residues by piperidine and the different sized fragments separated on a gel. The band corresponding to the region of protein binding will be missing in the bound fraction because of the interference of protein binding by the methylation of the binding site. This can be seen as a gap or footprint in the gel that corresponds to a band in the free fraction gel from which the sequence of the protein binding region can be deduced.

Low provides a detailed account of how transgenic mice can be produced by microinjection of cloned DNA into fertilized oocytes. The motivation for this procedure is the study of how neuropeptide genes are regulated. The central idea is the construction of reporter fusion genes, made from a reporter gene-the expression of which can be readily distinguished-linked to the upstream regulatory elements of interest. It is these fusion genes that are used to generate the transgenic animals. Thus, the regulation of the neuropeptide gene can be moni- tored in the transgenic animals by the concomitant expression of the reporter gene.

The key technique of in situ hybridization histochemist~y, directed at particular mRNAs, is discussed in two chapters. Pasinetti describes the use of [95S]-labeled antisense complementary RNA to localize RNAs in either paraffin or frozen brain sections by hybridization and autora- diography. These protocols have been devised with a view to execut- ing combined in situ hybridization and immunocytochemistry, an

Preface ix

approach that may become increasingly important in determining the efficiency with which specific mRNA molecules are translated; a low transcript abundance does not necessarily imply low levels of expres- sion of the coded protein. Subsequently, Kiyama, Emson, and Tohyama consider in situ hybridization using probes coupled to a reporter molecule-specifically alkaline phosphatase-rather than a radiola- bel. This strategy is speedier than autoradiography and can be par- ticularly useful when hunting for mRNA molecules present only in low concentrations.

Once a particular DNA molecule has been cloned, it is necessary to confirm whether it codes for the specific protein of interest. The transcription of a cloned DNA molecule and the subsequent translation of the RNA transcript in a suitable expression vector followed by its identification by an appropriate assay, pharmacological response, or electrophysiological signature is the ultimate test of what is coded by the DNA. Alternatively, faced with mRNA extracted from brain, the RNA can be fractionated by size and each fraction examined using the expression vector to determine which fraction contains the transcript of interest. An iterative procedure using cycles of RNA fractionation may permit identification of the required mRNA. Indeed, this was exactly the method used in the recent cloning of the NMDA gluta- mate receptor subtype. The most widely used expression vector is the Xenopls oocyte, which will translate many (but not all) mRNA mol- ecules injected into it. The chapter by Dascal and Lotan provides protocols for several total RNA extraction methods, affinity chroma- tography to purify mRNA and for preparation, injection, and care of the oocytes. Additionally, these authors show how the use of antisense oligodeoxynucleotides directed at a specific mRNA, in this case cod- ing for a voltagedependent calcium channel, can selectively inhibit its expression in the oocyte.

At this point the emphasis of the volume changes from techniques applicable to nucleic acid research to those applicable to proteins. Indeed the next five chapters are effectively case studies illustrating how particular techniques have been used to reveal the molecular biology of proteins with importance for neuroscientists. Although the meth- odologies were designed for specific experimental models, all are widely applicable. Adamo and his colleagues begin the section by providing a battery of protocols they use for the study of insulin and insulin-like growth factor I receptors. There are methods for the preparation and primary culture of neurons and astrocytes, receptor binding studies,

x Preface

receptor solubilization, and partial purification using wheat germ agglutinin. There is also a description of receptor phosphorylation methods for both cell-free and intactcell systems, and of the methods by which phosphorylation sites may be determined. Nakata, using a selective antagonist as an immobilizing ligand, describes the purifica- tion by affinity chromatography of an adenosine receptor. The strategy involves two affinity chromatographic separations with an intervening hydroxyapatite column chromatography to achieve a 50,000-fold purification of the receptor.

The regulation of calcium fluxes into the cytosol is of consider- able importance to nervous system function and two chapters exam- ine how molecular biology has revealed the structure of two of the channels involved. Schneider et al. label the voltagedependent, L type calcium channel with a dihydropyridine having a high binding affinity for the channel, so that they can track it throughout a purifi- cation procedure that uses affinity chromatography (with wheat germ agglutinin), ion exchange chromatography, and sucrose density gra- dients. Lai and Meissner turn their attention to the ryanodine recep tor, an ion channel in the sarcoplasmic reticulum ofvertebrate skeletal and heart muscle through which calcium ions flood into the cytosol on depolarization. Interestingly, ryanodine receptors are present in brain and they are homologous with the inositol trisphosphate-sensi- tive calcium channels in the endoplasmic reticulum. These authors provide protocols for the purification and characterization of ryanodine receptors using sucrose density gradients and sodium dodecyl sulfate polyacrylamide gels. They also describe how the molecules can be reconstituted into planar lipid bilayers for further functional studies.

The nicotinic acetylcholine receptor (nAChR) was the first recep tor to be completely sequenced and as such has been the most exten- sively studied. One important approach in identifying the functional sites on the receptor is photoafinity labeling, in which a radiolabeled ligand with an &nity for specific regions of a receptor or ion channel- an agonist binding site, for example--can be covalently bonded to the receptor by a brief pulse of ultraviolet light. The complex between the ligand and the protein is now stable, and standard protein chemistry techniques can be used to identifjl the ligand binding site. Hucho reports the use of a channel blocker to elucidate those portions of the nAChR that form the ion channel wall.

An introduction to the seminal role being played by patch clamp ing needs no apology since it involves measuring current flow through

Preface xi

single ion channels and may thus be regarded as molecular electro- physiology. Patch clamping may be used as an "assayn that allows char- acterization of the exact nature of a molecule coded for by cloned DNA or its mRNA transcript when expressed in an appropriate vector.

It is no exaggeration to claim that the techniques of patch clamp ing have revolutionized our understanding of cell membrane hnc- tion. Indeed, in recognition of this, Sakmann and Neher, who invented the original methods, received the Nobel prize for physiology or medicine in 1991. Standen and Stanfield provide a theoretical back- ground that illustrates how these powerful techniques can be appllied to the analysis of channel function. Using methods applicable to mammalian cell lines, Xenopls oocytes that have been injected with mRNA or cell lines that can be transfected with DNA, they describe how both macroscopic (whole-cell) and microscopic (singlechannel) currents can be analyzed to derive models of the kinetic behavior of channels. Scott and Dolphin explain their use of wholecell patch clamping to study the regulation of voltagedependent calcium chan- nels by Gproteins. They use a variety of GTP/GDP analogs, and of particular note is their use of caged compounds. These may be esters of substrates of interest-for example, GTP or inositol trisphosphate, which can be rapidly cleaved by brief illumination, thus enabling the time course of processes involving the substrates to be studied accu- rately without problems of diffusion, or substrate instability. They also indicate some of the problems involved in single channel recording of calcium channels caused by channel rundown.

Although it has to be accepted that there is no better way of learn- ing a new technique than working in a laboratory where it is used on a regular basis, we believe this volume provides a viable alternative. The format of detailed protocols, coupled with the "insider'sn tips on cru- cial points, will enable a greater understanding of the experiments than is possible with the level of detail available from a standard research paper. We hope that this book will give some ideas both to newcomers to the diverse methods of molecular neurobiology and to those who are already familiar with some of the techniques detailed here.

Contents Preface .................................................................................................................. v Contributors ...................................................................................................... xu

Separation of Large DNA Molecules by Pulsed-Field Gel Electrophoresis,

D u r n J. Shuw ........................................................................... 1 The Isolation of Genomic DNA from Invertebrates,

Erno V q d e n h i l and Mark G. Darlison ...................................... 15 Analysis of Mitochondria1 DNA Mutations,

M d i Tanaka and Takayuki Oxuwa ........................................ 25 The Design and Use of Oligonucleotides,

Alan N. Bateson and Mark G. Darlison ........................................ 55 The Use of Degenerate Oligonucleotides for Polymerase Chain-

Reaction-Based Isolation of Related DNA Sequences, Tcania E. Webb and Alan N. Bateson ....................................... . 6 7

Isolation of Photoreceptor Cell-Specific MEKA cDNA by Differential Hybridization,

............................................................................. &-Hui Kuo 7 9 Generation of Isoform-Specific Antisera from Cloned cDNAs:

Aj$licatim to Multipk F m of the Na/K-ATPase Exp'essed in Rat Brain,

Andrew W. Shyjan and Robert Lamson ....................................... 93 Determination of Transcriptional Initiation Sites and Their

Usage in the Nervous System, Gary Weisinget-, Joseph D. DeOisto&ro,

and Edmund F. La Gumma ............................................... 1 1 5 Mapping of Trans-Acting Factor Binding in the Ne~vous System,

Gary Weisinget- and Edmund F. La Gamma ................................ 139 In Situ Hybridization to Brain Tissue Sections Using Labeled

Single-Strand Complementary RNA Probes, Giulio M. Pasineni ................................................................... 155

In Situ Hybridization Histochemistry Using Alkaline Phosphatase-Labeled Oligodeoxynucleotide Probe,

Hiroshi Kiyama, Piers C. Emon, and Masaya Tohyama .............. 167

... XZZZ

xiv Contents

The Identification of Neuropeptide Gene Regulatory Elements in Transgenic Mice,

....................................................................... Malaolm J. Low 181 Expression of Exogenous Ion Channels and Neurotransmitter

Receptors in RNA-Injected Xenopus Oocytes, ................................................. Nathan Dasaal and llana Lotan 205

Analysis of Insulin and Insulin-Like Growth Factor4 Receptors in Neural Tissues,

Martin L. Adamo, Mohan K Rtaida, Joshua Jlremer, Akim Ota, Colin Sumners, John Olson, and Derek LeRoidr ....................... 227

Use of Affinity Chromatography in Purification of A, Adenosine Receptors from Rat Brain Membranes,

..................................................................... Hiroyasu Nakata 261 Purification and Structure of L-Type Calcium Channels, Tom' Schneider, Stefan Regulla, Wolfgang Nastaincryk,

.......................................................... and Fmnr Hofmann 2 73 Purification and Reconstitution of the Ryanodine-Sensitive

Ca2+ Release Channel Complex from Muscle Sarcoplasmic Reticulum,

........................................ I;. Anthony LM' and Gerhard Meismer 287 Identification of a Ligand-Gated Ion Channel by Photoaffinity

Labeling and Microsequencing, .................................................................... Ferdinand Hudao 307

Voltage-Gated Ion Channels: Electrophysiological Approaches, ................................. Nichoh B. Standen and Peter R Stanfield 325

An Electrophysiolo~cal Approach to the Regulation of Neuronal Voltage-Activated Calcium Channels by Guanine Nucleotide Binding Proteins,

................................... Roderick H. Scott and Annette C. Dolphin 34 7

Index ................................................................................................................ 367

Contributors

MARTIN L. ADAM0 Diabetes Branch, National Institutes $Diabetes, and Digestive and Kidnqr fimes, National Institutes ofHBalth, Bethda, MD

ALAN N. BATESON MRC Molecular Biology Unit, Cambridge, UK (Present address: Department of Pharmacology, University of Alberta, Edmonton, Albmta, Canada)

MARK G. DARLISON Institul f ir Zellbiochie und Klinische Neurobiologie, Universitats-Krankenhaus E@end$ Hamburg Gennany

NATHAN DASCAL Sackh School of Medicine, Division of Physiology and Pharmacology, Tel Aviv University, Tel Aviv, Israel

JOSEPH D. DECRISTOFARO Department of Pediatrics and Neurobiology, State University o f N m York ad Stony Brook, NY

ANNETIE C. DOLPHIN Department of Pharmacology, Royal Free Hospital School of Mediane, London, UK

mERs C. EMSON A4RC Group, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, UK

FRANZ HOW Institut fur Pharmakologie und Toxikologie der Technische Universitat Munchen, M u n c h , Germuny

FERDINAND HUMO Fachbereich Chemie, Institut fur Biochie, Freie Universitat Berlin, Berlin, Germuny

HIROSHI KIM Department of Anatomy and Neuroscience, Osaka University Medical School, Osaka, Japan

CHE-HUI KUO Department of Pharmacology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan

EDMUND F. LA GAMMA Department of Pediatrics and Neurobiology, State University of New York at Stony Brook, NY

F. ANTHONY LAI Division of Physical Biochemistry, National Institute for Medical Research, London, UK

DEREK LEROITH Diabetes Branch, National Institutes of Diabetes, and D i g a h and K i d 9 fimes, National Institutes of H d h , Bethda, MLI

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

xvi Contributors

MALCOLM J. LOW Vollum Institute for Advanced Bimdical Research, Oregon Health Sciences University, Portland, OR

ILANA LOTAN Sacklm School of Medicine, Division of Physiology and Pharmacology, Tel Aviv University, Tel Aviv, Israel

GERHARD MEISSNER Department of Biochemistry, University ofNorth Carolina, Chapel Hill, NC

HIROW NAKATA Department of Neurochemistry, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan

WOLFGANG NASTAINCZYK Medizinische Biochie , Medizinische Fakultat der Universitat des Saarlundes, Hmber/Saar, Gennany

JOHN OLSON Department of Physiology, University OfFlorida, College of Medicine, Gain~ville, FL

ANRA OTA Diabetes Branch, National Institutes OfDiabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD

TAKAWKI OZAWA Dqbartment o fBiodica l Chemistry, Faculty of Medicine, University of Nagoya, Nagoya, Japan

GIUUO M. P m m Ethel Percy Andrus Gerontology Center, University of Southern Califmia, Los Angeles, C4

MOHAN K. RAIZADA Department of Physiology, University of Florida, College of Medicine, Gainesville, FL

STEFAN REGULLA Institut fur Phamnakologie und Toxikologie der Technischen Universitat M u n c h , Munchen, Gennany

TONI SCHNEIDER Institut fur Phannakologie und Toxikologie der Technischen Universitat M u n c h , Munchen, Gennany

RODERICK H. SCOTI. Department ofPhysiology, St. Gewge's Hospital Medical School, London, UK

DUNCAN J. S w Department of Medical Genetics, University of Wales College of Medicine, Card$ UK

JOSHUA SHEMER Diabetes Branch, National Institutes c i a b e t a , and Digestive and Kidnq Dismes, National Institutes ofHaalth, Bet- MD

ANDREW W. S ~ A N Department of Cell Biology, Yale University School of Medicine, New Haven, CT

NICHOLM B. STANDEN Department of Physiology, University of L.eices&, UK l'li-n~ R. STANFIELD Department of Physiology, University of kcester, UK COLIN SUMNERS Department of Physiology, University ofFlononda, College of

Medicine, Gainesville, FL -HI TANAKA Department of Biomedical Chemistry, Faculty of

Medicine, University ofNagoy4 Japan

Contributors xvii

MAsAw TO- Department of Anatamy and Neuroscience, Osaka University Medical School, Osaka, Japan

ERNO VREUGDENHIL Biochemisch Labmatmiurn, Vrije Universih't, Amsterdam, The Netherlands

TANIA E. WEBB MRC Molecular NmbiolOgy Unit, Cambridge, UK GARY WEISINGER Department of Pediatrics and Neurobiology,

State University of New York at Stony Brook, NY