Identification of Transcribed Sequences

298 Pages·1994·12.608 MB·English

by  Miles B. Brennan (auth.), Ute Hochgeschwender, Katheleen Gardiner (eds.)

Identification of
Transcribed Sequences
Identification of
Transcribed Sequences
Edited by
Ute Hochgeschwender
National Institute of Mental Health
Bethesda, Maryland
and
Katheleen Gardiner
Eleanor Roosevelt Institute for Cancer Research
Denver, Colorado
Springer Science+Business Media, LLC
Librar y o f Congress Cata\oging-1n-Publicatio n Data
Identification of transcribed sequences / edited by Ute
Hochgeschwender and Katheleen Gardiner,
p. cm.
"Proceedings of the third International Workshop on the
Identification of Transcribed Sequences, held October 2-4, 1993, in
New Orleans, LDUIs1 ana"--T.p. verso.
Includes bibliographical references and Index.
ISBN 978-1-4613-6094-0 ISBN 978-1-4615-2562-2 (eBook)
DOI 10.1007/978-1-4615-2562-2
1. Human gene mapping—Congresses. 2. Genetic transcriptIon-
-Cangresses. I. Hochgeschwender, Ute. II. Gardiner, Katheleen
Jane. III. International Workshop on the Identification of
Transcribed Sequences (3rd : 1993 : New Orleans)
QH445.2.I34 1994
573.2'12—dc20 94-36835
CIP
Proceedings of the Third International Workshop on the Identification of Transcribed Sequences,
held October 2-4, 1993, in New Orleans, Louisiana
ISBN 978-1-4613-6094-0
©1994 Springer Science+Business Media New York
Softcover reprint of the hardcover 1 st edition 1994
Originally published by Plenum Press in 1994
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
PREFACE
The Human Genome Project, an endeavor to map and sequence the entire human
genome, has been in existence for almost seven years. One result of this effort has been
the development of increasingly detailed genetic and physical maps spanning large regions
of virtually every chromosome. Paralleling this has been the increasingly high resolution
mapping of many &wnetic diseases. Together, these developments have facilitated the
isolation of specific disease genes and are now motivating the construction of
comprehensive transcriptional maps. This latter endeavor represents a new facet of the
genome project, and as such requires the recognition and solution of a new set of problems,
with the attendant development and application of a new set of techniques.
The First International Workshop on the Identification of Transcribed Sequences in
the Human Genome was held in 1991 and was attended by 23 investigators. Discussions
at this meeting were largely devoted to defining the magnitude of the problem and
describing the available techniques. A small number of laboratories reported the
development of new techniques (at that time, for example, exon trapping, cDNA hybrid
selection, direct cDNA screening, use of splice junction conserved sequences,etc.), but data
were too limited to permit comparisons of their relative efficiencies.
As expected, interest in the problem of transcription mapping has grown - the third
workshop, held in 1993, was attended by 58 investigators. With more laboratories having
experience in transcriptional mapping, an important focus of this meeting was discussion
of the relative strengths and weaknesses of the more popular techniques and potential
complementaries among them. This has permitted some direct determination of the
robustness and portability of particular methods, as well as some evaluation of their relative
efficiencies for mapping projects of different scales. Variations and improvements on these
techniques, as well as the development of alternative approaches also provided important
topics for discussion.
With an increasing number of laboratories interested in transcribed sequence
identification, it seemed timely to make the presentations and discussions of this latest
meeting accessible to a wider audience.The purpose of these proceedings is four-fold: 1)
to realistically assess the magnitude of the problem of transcribed sequence identification
in the human genome; 2) to give an overview of current approaches to this problem; 3) to
assist researchers in choosing method(s) appropriate to their projects and resources, through
reports from other laboratories; and 4) to summarize both the general problems of
transcribed sequences identification and the specific concerns about specific methods.
There are many ways to classify methods for transcriptional mapping, but for most
purposes, a useful division is into two types of methods: 1) those which take genomic
clones of known location and attempt to isolate transcribed sequences contained within
them, and 2) those which start with transcribed sequences (cDNA clones) and attempt to
characterize and map them to chromosomal regions.
v
This volume opens with a discussion of the scope of the problem facing a
transcriptional mapper - how many genes are there and what is the complexity of the
transcribed sequences in any tissue? This is followed by descriptions of some classical
approaches that include use of CpG islands, evolutionary conserved sequences and whole
cosmid clones to screen cDNA libraries for transcribed sequences from a defined
chromosomal region. Such approaches remain effective and, depending on the
circumstances, may still be the method of choice.
Approaches based on hybridization between cDNAs and genomic sequences include
cDNA hybrid selection applied to YAC clones and cosmid contigs. Several groups
describe experiences with this approach, making it currently one of the most popular
methods. Several variations on cDNA hybrid selection are also described which make use
of RNA intermediates, metaphase chromosomes, etc. Another hybridization based method,
that of direct cDNA screening of arrayed genomic clones, is discussed in two reports.
Four reports discuss exon trapping, a technique that relies on retention within
mRNA of spliced exons from cloned genomic fragments. Experience with its application
to cosmids, cosmid pools and YA C clones is reported.
Computer based analyses include GRAIL, which uses a neural network based
approach to rate the potential exon content of genomic sequences. A discussion of the
GRAIL program and use is presented elsewhere, but results of its application are included
in several reports. It has been particularly effective in conjunction with direct cDNA
screening. Also presented is a discussion of the efficiency of direct genomic sequencing
for transcribed sequence identification.
The foregoing start with genomic clones and attempt to identify the transcribed
sequences contained within them. An alternative set of methodologies approaches
transcriptional mapping from the opposite direction - starting with transcribed sequences
and collecting mapping and sequencing data for them. While generally less well suited for
disease gene isolation, these approaches may provide data useful for large scale (e.g. whole
genome, whole chromosome) transcriptional map construction. Two groups report on
methods for, and results of, mapping numerous cDNAs from several random cDNA
sequencing efforts. Two other groups report on the use of oligonucleotide hybridization
to derive sequence information from arrayed cDNA libraries, and one group reports
experiences using a sophisticated mRNA display methodology.
The last chapter summarizes formal and informal discussions addressing problems
important to the transcriptional mapping field. These problems include defining a
transcriptional map, and setting criteria for the inclusion of a novel DNA in a
transcriptional map. The chapter also summarizes problematic aspects of particular
methods.
Reports in this volume present an overview of current techniques in use for
transcriptional mapping; they also illustrate the infant stage at which transcriptional
mapping currently lies. Equally clear, however, is that transcriptional mapping will rapidly
grow beyond this stage. This volume in no way represents the last word in transcriptional
mapping. Rather, it presents a collection of first words in what will be a long and
entertaining novel.
Katheleen Gardiner and Ute Hochgeschwender
vi
ACKNOWLEDGMENTS
We wish to thank the Department of Energy, the National Center for Human
Genome Research, and Amgen, Inc., whose support made this meeting possible. We also
wish to thank the session chairs, and particularly, the discussion group leaders whose
efforts and input made it a more valuable experience for all who attended. Special thanks
are due Nan Matthews whose patience, organizational skills and attention to detail made
both the meeting itself and these proceedings possible.
vii
CONTENTS
Introduction: Seven Blind Men and An Elephant ....................... 1
M.B. Brennan
FROM GENOMIC DNA TO TRANSCRIBED SEQUENCES
Classical Approaches
Identification of Genes and Construction of a Transcriptional Map in Xq28 .. , 5
C. Tribioli, E. Maestrini, S. Bione, F. Tamanini, M. Mancini, C. Sala,
G. Torri, S. Rivella, and D. Toniolo
Use of cDNA Selection and Evolutionarily Conserved Sequences to Isolate
Transcribed Sequences from Region Xp11.21 . . . . . . . . . . . . . . . . .. 11
E.N. Burright, N.G. Pasteris, M.D. Bialecki, and lL. Gorski
Identification of cDNAs by Direct Hybridization Using Cosmid Probes. . . . .. 23
G.G. Lennon and K. Lieuallen
Hybridization Based Approaches
Hybridization Selection:
Locus Specific Identification of Transcribed Sequences Using YACs and
Whole Yeast Genomic DNA ............................. , 29
S.R. Patanjali, H.x. Xu, S. Parimoo and S. Weissman
Towards a Transcriptional Map of Human Chromosome 21 .............. 37
K. Gardiner, H. Xu, W. Bonds, F. Tassone, S. Parimoo, R.
Sivakamasundari, F. Hisama, A. Rynditch, and S. Weissman
Isolation of Expressed Sequences from the Chromosome 17q21 BRCA1
Region by Magnetic Bead Capture . . . . . . . . . . . . . . . . . . . . . . . . .. 51
F.J. Couch, B.L. Weber, F.S. Collins and D.A. Tagle
ix
Towards a Transcriptional Map of the q21-q22 Region of Chromosome 7 . . .. 65
1.M. Rommens, L. Mar, 1. McArthur, L.-e. Tsui, and S.W. Scherer
Direct cDNA Selection Using Human and Mouse cDNAs:Application to
Xq13.3 Chromosomal Region .............................. 81
J. Gecz, L. Villard, AM. Lossi, and M. Fontes
Variations on Hybridization Selection:
A Sandwich-Hybridization Method for Specific and Efficient Selection of
cDNA Clones from Genomic Regions ....................... 91
D. Yan and A Swaroop
Novel Strategy for Isolating Unknown Coding Sequences from Genomic
DNA by Generating Genomic-cDNA Chimeras ................ 101
P. Jagadeeswaran, M.W. Odom, and E.J. Boland
Identifying and Directly Purifying Transcribed Elements by Coincident
Sequence Cloning ...................................... 111
AJ. Brookes
Finding Candidate Genes by Preparative in situ Hybridization ............. 123
J.C. Hozier, L.M. Davis, P.D. Siebert, K Dietrich, and M.e. Paterson
Direct cDNA Screening:
Direct cDNA Screening of Genomic Reference Libraries -A Rapid Method
for the Identification of Transcribed Sequences in Large
Genomic Regions ...................................... 139
W. Schwabe, B.J. Lawrence, AS. Robb, RM. Hopfinger, U.
Hochgeschwender, and M.B. Brennan
Identification of Expressed Sequences on Human Chromosome 9q32-34 ..... 157
J.D. Falk, H. Usui, and J.G. Sutcliffe
Exon Trapping
An Exon Trapping System Providing Size Selection of Spliced Clones and
Facilitating Direct Cloning ................................ 169
N.A Datson, G.M. Duyk, L.AI Blonden, G.-IR Van Ommen, and
J.T. Den Dunnen
Isolation of Gene Sequences from the BRCA1 Region of Chromosome
17q21 by Exon Amplification .............................. 183
KJ. Abel, L.H. Castilla, AJ. Buckler, FJ. Couch, P. Ho, I. Schaefer,
S.C. Chandrasekharappa, F.S. Collins, and RL. Weber
Isolation of Coding Sequence from Cosmids and YACs by Exon
Amplification .......................................... 199
M. North, F. Gibson, S. Brown, R Griffiths, E. Solomon, and H. Lehrach
x
Integrated Transcriptional Maps of Large DNA Regions: Towards a
Transcriptional Map of Human Chromosome 21 ............... 213
M.-L. Yaspo, P. Sanseau, D. Nizetic, B. Korn, A Poustka, and H. Lehrach
Computer Based Approaches
Shallow Shotgun Sequencing as a Strategy for Finding Coding Exons ....... 229
J.-M. Claverie
FROM TRANSCRIBED SEQUENCES TO GENOMIC LOCALIZATION
Generating and Sequencing cDNAs
Requirements in Screening cDNA Libraries for New Genes and Solutions
Offered by SBH Technology .............................. 239
R. Drmanac, S. Drmanac, I. Labat, and N. Stavropoulos
Establishing Catalogues of Expressed Sequences by Oligonucleotide
Fingerprinting of cDNA Libraries .......................... 253
S. Meier-Ewert, J. Rothe, R. Mott, and H. Lehrach
PCR-Based Technologies to Study Differential Gene Expression in
Rat Brain ............................................ 261
M.G. Erlander, A Dopazo, P.E. Foye, and lG. Sutcliffe
Mapping cDNAs
259 Human Brain Expressed Sequence Tags (ESTs): Chromosome
Localization, Subregional Assignment, and Sequence Analysis ..... 273
D.R. Maglott, AS. Durkin, and W.e. Nierman
Mapping cDNAs by Hybridization to Gridded Arrays of DNA from
Y AC Clones .......................................... 289
D.R. Moir, R. Lundstrom, P. Richterich, X. Wang, M. Atkinson, K. Falls,
J. Mao, D.R. Smith, and G.F. Vovis
Discussion ................................................... 299
K. Gardiner and U. Hochgeschwender
Index ....................................................... 303
xi
INTRODUCTION: Seven Blind Men and an Elephant
Miles B. Brennan
Unit on Genomics, NIMH
9000 Rockville Pike, Bethesda, MD 20892, USA
An oft-stated goal of the Human Genome Project is the cataloging of all human
genes. To know the magnitude of this undertaking, an enumeration of the genes in man
would be necessary. While the question of gene number has interested geneticists at least
since Muller (1), it has also influenced thought on evolution and development. Indeed, the
C-value paradox was important because of the implication that "simpler" organisms had
more genetic complexity than mammals. Now the number of human genes has become a
question of practical relevance, affecting how we pursue comprehensive transcriptional
mapping. I present here a short, critical review of papers representing the major approaches
which have been used to address this question.
The first approach derived from the theory of genetic load (2). Briefly, at steady
state, the rate of new, deleterious mutations entering a population is considered to be off-set
by the rate of their loss through their phenotypic effects. It follows that no more than one
recessive or 0.5 dominant "lethal" mutations can arise per haploid genome per generation.
(Dominant deleterious alleles result in the genetic death of two genomes, one of which does
not carry the mutation; for recessive alleles, both genomes contain the deleterious allele.)
The concept of a lethal mutation here is quite different from its currently common usage.
In evolutionary terms, whether a mutation leads to the immediate death of an organism or
to the death of one progeny after 10 generations is irrelevant: selection will act with equal
rigor in either case. There is no evolutionary reason to attribute a special significance to
"essential" genes, those with fully penetrant lethal phenotypes.
Thus, the rate of new lethal mutations must equal the rate of their loss. Assuming
that the upper bound for genetically determined death is 1, Muller concluded that the
average rate of new lethal mutations must be less than 0.5 per haploid genome per
generation. An upper limit to the number of genes would be given by the inverse of the
average per locus rate of deleterious mutations. Unfortunately, little data existed on the
frequency of deleterious mutations in man. Assuming that most deleterious mutations are
dominant and that the average per locus rate for deleterious mutations is 1 in 50,000,
Muller concluded that there are a maximum of 20,000 genes in man.
Following this line of reasoning, Ohta and Kimura (3) in 1971 attempted to define
the intrinsic mutation rate for mammals as well as the fraction of mutations in mammalian
cytochrome c which are deleterious using recently obtained protein sequence data. They
estimated the intrinsic mutation rate in mammals at 8.3xlO-9 per amino acid codon per year.
Identification of Transcribed Sequences, Edited by
U_ Hochgeschwender and K. Gardiner, Plenwn Press, New York, 1994