Protocols in Molecular Neurobiology

377 Pages·1993·28.04 MB·English

by  Duncan J. Shaw (auth.)

Protocols in Molecular Neurobiology
Methods in Molecular Biology
John M. Walker, SERIEESD ITOR
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 mW . 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 y19,92
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
or by any means, electronic, mechanical, photocopying, microfilming, recording, or 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~yst rand 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-
...
Preface
ULLL
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~yd, irected
at particular mRNAs, is discussed in two chapters. Pasinetti describes
the use of [95S]-labeleda ntisense 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-
odologiesw ere 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,