Molecular Neurobiological Techniques

306 Pages·1990·22.19 MB·English

by  David R. Colman (auth.), Alan A. Boulton, Glen B. Baker, Anthony T. Campagnoni (eds.)

NEUROMETHODS E~ 16
Molecular Neurobiological Techniques
NEUROMETHODS
Program Editors: Alan A. Boulton and Glen B. Baker
1 General Neurochemical Techniques
Edited by Alan A. Boulton and Glen B. Baker, 1985
2. Amines and Their Metabolites
Edlted by Alan A. Boulton, Glen B. Baker, and Judith M. Baker, 1985
3 Amino Acids
Edited by Alan A. Boulton, Glen B. Baker, and James D. Wood, 1985
4 Receptor Binding Techniques
Edited by Alan A. Boulton, Glen B. Baker, and Paoel D. Hrdlna, 1986
5 Neurotransmitter Enzymes
Edlted by Alan ,4. Boulton, Glen B. Baker, and Peter H. Yu, 1986
6 PepUdes
Edlted by Alan A. Boulton, Glen B. Baker, and Quentin Pittman, 1987
7. Upids and Related Compounds
Edlted by Alan A. Boulton, Glen B. Baker, and Lloyd A. Horrocks, 1988
8 Imaging and Correlative Physicochemical Techniques
Edited by Alan A. Boulton, Glen B. Baker, and Donald P. Boisvert, 1988
9 The Neuronal MJcroenvironment
Edited by Alan A. Boulton, Glen B. Baker, and Wolfgang Walz, 1988
10 Analysis of Psychiatric Drugs
Edited by Alan A. Boulton, Glen B. Baker, and Ronald T. Coutts, 1988
11 Carbohydrates and Energy Metabolism
Edited by Alan .4. Boulton, Glen B. Baker, and Roger F. Burrerworth, 1989
12 Drugs as Tools in Neurotransmitter Research
Edited by Alan A. Boulton, Glen B. Baker, and Augu.sto V. Juorio 1989
13 Psychopharmacology
Edited by Alan ,4. Boulton, Glen B. Baker, and Andrew J. Greenshaw, 1989
14 Neurophysiology
Edited by Alan A. Boulton, Glen B. Baker, and Case H. Vanderwolf, 1990
15 Neuropsychology
Edited by Alan A. Boulton, Glen B. Baker, and Merrll Hiscock, 1990
16 Molecular Neurobiological Techniques
Edited by Alan A. Boulton, Glen B. Baker, and Anthony T. Campagnoni, 1990
NEUROMETHODS
Program Editors: Alan A. Boulton and Glen B. Baker
NEUROMETHODS [-7 16
Molecular Neurobiological
Techniques
Edited by
Alan A. Boulton
Unwerslty of Saskatchewan, Saskatoon, Canada
Glen B. Baker
Umverslty of Alberta, Edmonton, Canada
and
Anthony T. Campagnoni
Universzty of Cahfomia, Los Angeles, Cahfornia
Humana Press • Clifton, New Jersey
Library of Congress Cataloging in Publication Data
Mare entry under title
Molecular neuroblologlcal Techniques / edited by Alan A Boulton, Glen B Baker, and
Anthony T Campagnom
p cm -- (Neuromethods , 16)
Includes bibhographles and mdex
ISBN 0-89603-140-3
1 Molecular neurobmology--Methodology 2 Nucleic acid
hydrmdlzatlon 3. DNA probes I Boulton, A A (Alan A )
II Baker, Glen B, 1947- III Campagnonl, Anthony T
IV Series
[DNLM 1 Genetics, Biochemical 2 Neuroblology W1 NE337G v
16 / WL 102 M7185]
QP356 2 M643 1989
599' 0188---dc19
DNLM/DLC
for Library of Congress 89-2053
CIP
© 1990 The Humana Press Inc
Crescent Manor
PO Box 2148
Chfton, NJ 07015
All rights reserved
No part of this book may be reproduced, stored m a retrieval system, or transmitted m any
form or by any means, electronic, mechamcal, photocopying, microfilming, recordmg, or
otherwise without written permission from the Publisher
Printed in the Umted States of America
Preface to the Series
When the President of Humana Press first suggested that a series
on methods in the neurosciences might be useful, one of us (AAB)
was quite skeptical; only after discussions with GBB and some
searching both of memory and library shelves did it seem that
perhaps the publisher was right. Although some excellent
methods books have recently appeared, notably in neuroanatomy,
it is a fact that there is a dearth in this particular field, a fact attested
to by the alacrity and enthusiasm with which most of the con-
tributors to this series accepted our invitations and suggested
additional topics and areas. After a somewhat hesitant start, es-
sentially in the neurochemistry section, the series has grown and
will encompass neurochemistry, neuropsychiatry, neurology,
neuropathology, neurogenetics, neuroethology, molecular
neurobiology, animal models of nervous disease, and no doubt
many more "neuros." Although we have tried to include adequate
methodological detail and in many cases detailed protocols, we
have also tried to include wherever possible a short introductory
review of the methods and/or related substances, comparisons
with other methods, and the relationship of the substances being
analyzed to neurological and psychiatric disorders. Recognizing
our own limitations, we have invited a guest editor to join with us
on most volumes in order to ensure complete coverage of the field.
These editors will add their specialized knowledge and competen-
cies. We anticipate that this series will fill a gap; we can only hope
that it will be filled appropriately and with the right amount of
expertise with respect to each method, substance or group of
substances, and area treated.
Alan A. Boulton
Glen B. Baker
Preface
It goes without saying that the principles and techniques of
molecular biology are having and will continue to have a major
impact on investigations into nervous system structure and func-
tion. It is becoming increasingly apparent to neuroscientists in all
subdisciplines that a working knowledge of the language,
approaches, and techniques of molecular biology is indispensable
for their work. For these reasons, the editors have decided to
devote this volume of Neuromethods to the techniques of molecular
biology and their application to neural systems. There currently
exist a number of excellent reference technical manuals that de-
scribe molecular neurobiological techniques in great detail, and
many of these are cited within the chapters included in this
volume. It was not the intention of the editors or authors of this
volume to duplicate these efforts. Rather, our intention was to
present to the neuroscientist who is relatively unfamiliar with
these methodologies an understanding of how specific techniques
are used to approach major molecular neurobiological problems as
well as a set of techniques that work in the laboratories of the
individuals writing the chapters. In some cases, there are duplica-
tions of techniques--these have been retained to illustrate the
range of variability of the technique and/or the flexibility of the
method to study different types of problems. We hope that the
chapters will provide the reader with an understanding of the
methods and their applicability to neurobiological problems; and,
perhaps, suggest new directions for the reader's research efforts.
Anthony T. Campagnoni
vii
Contents
Preface to the Series ............................................................ v
Preface ................................................................................ vii
List of Contributors ............................................................ xvii
ANALYSIS OF BRAIN mRNAs BY TRANSLATION IN VITRO
David R. Colman
1. Introduction .................................................... 1
2. Choice of Translation Systems ............................ 1
3. Selecting the Starting Material ............................ 2
4. Wheat Germ Extract Preparation ......................... 3
5. Amino-Acid Mixture ......................................... 4
6. Master (Energy) Mix ......................................... 5
7. Preparation of Dog Pancreas Microsomes ............. 6
7.1. Dog Pancreas Membrane Preparation ............ 6
7.2. Stock Solutions .......................................... 6
7.3. Solutions ................................................... 7
7.4. Procedure ................................................. 7
8. Outline of mRNA Preparation ............................ 8
9. Translation ...................................................... 8
9.1. Assembly of the Translation Mixture ............. 8
10. Processing of Translation Mixtures ..................... 9
10.1. TCA Precipitation (for 3SS Labeled
Polypeptides) ............................................ 10
10.2. TCA Pre4ipitation (3H Label) ....................... 10
11. Immuneprecipitation ........................................ 10
References ....................................................... 12
/x
x Contents
PREPARATION OF cDNA LIBRARIES AND ISOLATION AND
ANALYSIS OF SPECIFIC CLONES
Sammye Newman and Anthony T. Campagnoni
1. Introduction ..................................................... 13
2. Background ..................................................... 14
2.1. First Strand Synthesis .................................. 14
2.2. Second Strand Synthesis .............................. 15
2.3. Ligation to Vector ....................................... 16
2.4. Introduction of Recombinant Molecules
into a Bacterial Host .................................... 18
2.5. Vector-Host Systems ................................... 18
2.6. Protocols for Screening the Completed Li-
brary ......................................................... 21
2.7. Analysis of Specific Clones ........................... 22
3. Methods .......................................................... 24
3.1. Preparation of a cDNA Library in Lambda
gt11 .......................................................... 25
3.2. Amplification and Screening of the Com-
pleted Library ............................................ 35
3.3. Characterization of Positive Clones ................ 37
References ....................................................... 43
PREPARATION AND USE OF SUBTRACTIVE cDNA
HYBRIDIZATION PROBES FOR cDNA CLONING
Gabriel H. Travis, Robert J. Milner, and J. Gregor Sutcliffe
1. Introduction ..................................................... 49
2. The Arithmetic of mRNA ................................... 50
2.1. Total RNA ................................................. 50
2.2. Polyadenylated RNA ................................... 50
2.3. mRNA Complexity ...................................... 51
3. General Principles of Cloning ............................. 52
3.1. cDNA Libraries .......................................... 53
3.2. Clone Isolation Requires Information ............. 54
4. Clones of Tissue-Specific mRNAs ........................ 58
4.1. Brute-Force Isolation ................................... 58
4.2. Plus-Minus Screening .................................. 58
Contents xi
5. Subtractive Hybridization ................................... 60
5.1. Standard Procedure ..................................... 60
5.2. Phenol Emulsion-Enhanced DNA-Driven
Subtractive Hybridization ............................. 62
6. Future Applications ........................................... 75
References ....................................................... 76
ANALYSIS OF BRAIN-SPECIFIC GENE PRODUCTS
Robert J. Milner and J. Gregor Sutcliffe
1. Introduction ..................................................... 79
2. Patterns of mRNA Expression ............................. 80
2.1. Northern Blotting ....................................... 80
2.2. In Situ Hybridization ................................... 82
3. Interpretation of Nucleotide and Amino-Acid
Sequences ........................................................ 83
3.1. Definition of Open Reading Frames ............... 84
3.2. Consensus Sequences .................................. 85
4. Sequence Comparisons ...................................... 92
4.1. Searching the Databases .............................. 92
4.2. Assessment of "Homology" .......................... 93
5. Preparation of Antibodies to Encoded Proteins ...... 97
5.1. Antibodies to Synthetic Peptides ................... 98
5.2. Antibodies to Expressed Proteins ................ 103
6. Characterization of Encoded Proteins ................. 105
6.1. Criteria for Protein Identification ................. 105
6.2. Biochemical Analysis ................................. 106
6.3. Immunocytochemical Analysis .................... 109
7. Summary: From Structure to Function ............... 110
References ..................................................... 110
ISOLATION AND STRUCTURE DETERMINATION OF GENES
Wendy B. Macklin and Celia W. Campagnoni
1. Introduction ................................................... 117
2. Generation of Genomic Libraries ....................... 119
2.1. Preparation of High Mol Wt Genomic DNA .... 119
2.2. Preparation of Genomic Library .................. 120