Table Of ContentFunctional
Analysis of the
Hutnan Genotne
The HUMAN MOLECULAR GENETICS series
Series Advisors
D.N. Cooper, Charter Molecular Genetics Laboratory, Thrombosis Research
Institute, University of London, UK
S.E. Humphries, Division ofCardiovascular Genetics, University College
London Medical School, London, UK
T. Strachan, Department of Human Genetics, University of
Newcast/e-upon-Tyne, Newcast/e-upon-Tyne, UK
Human Gene Mutation
From Genotype to Phenotype
Functional Analysis of the Human Genome
Forthcoming titles
Molecular Genetics of Cancer
Environmental Mutagenesis
Human Genome Evolution
Functional
Analysis of the
Human Genome
Farzin Farzaneh
Department ofMolecular Medicine, King's College School of
Medicine and Dentistry, London, UK
David N. Cooper
Charter Molecular Genetics Laboratory, Thrombosis Research Institute,
London, UK
@
Taylor & Francis
~ Taylor &Francis Group
LONDON AND NEW YORK
© Taylor & Francis, 1995
First published 1995
All rights reserved. No part of this book may be reproduced or transmitted,
in any form or by any means, without permission.
A CIP catalogue record for this book is available from the British Library.
ISBN 1 872748465
Published byTaylor & Francis
2 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN
270 Madison Ave, NewYork NY 10016
Transferred to Digital Printing 2006
TO ALl, BERYL AND NEIL
Typeset by Touchpaper, Abingdon, UK.
Publisher's Note
The publisher has gone to great lengths to ensure the quality ofthis reprint
but points out that some imperfections in the original may be apparent
Contents
Contributors xi
Abbreviations xiii
Preface xvii
Foreword. V.A. McKusick xix
1 Structure and function in the human genome. D.N. Cooper 1
Introduction 1
Chromatin structure and transcription 2
Chromatin structure 2
Nuc1eosome positioning 3
Transcriptional domains 4
Chiasmata, recombination and recombination hotspots 4
Scaffold attachment regions 5
Centromeres 6
Telomeres 6
Origins of DNA replication 7
~~ 7
Gene structure and organization 7
Pseudogenes 8
Functional organization of human genes 9
Repetitive sequence elements 11
Tandem repeats 12
Alu sequences 12
LINE elements 13
Endogenous retroviral sequences 14
Transcriptional regulation 14
Promoter elements 14
Enhancers 15
Negative regulatory elements 16
Locus control regions 17
Boundary elements 19
Trans-acting protein factors 20
Sequences involved in transcriptional termination 21
mRNA splicing and processing 21
Sequences involved in determining mRNA stability 22
Role of sequences in 5' untranslated regions 23
DNA methylation 23
Distribution of 5-methylcytosine 23
Replication of the methylation pattern and de novo methylation 24
Role of DNA methylation in the regulation of transcription 25
Role of DNA methylation in X-inactivation 25
Changes in DNA methylation during embryogenesis 26
DNA methylation and imprinting 27
References 28
v
CONTENTS
2 Mapping the hUll1an genOll1e. D.N. Cooper 43
Introduction 43
~arkers 45
Gene sequences 45
DNA polymorphisms 46
D-segments 47
Sequence-tagged sites 47
Inter-Alu PCR probes 48
Allele-specific oligonucleotides 49
Cytogenetic mapping 49
Somatic cell hybrid analysis 49
Radiation hybrid mapping 49
Fluorescence in situ hybridization 50
In situ PCR 50
High-resolution physical mapping 51
Yeast artificial chromosome cloning 51
Contig assembly 52
Pulsed-field gel electrophoresis and CpG island mapping 52
Chromosome jumping/linking libraries 53
DNA sequencing 54
Progress in physical mapping 54
Genetic mapping 54
Transcription map of the human genome 57
Comparative gene mapping 58
References 59
3 Cloning the transcribed portion ofthe genOll1e. P. Towner 69
Introduction 69
Gene detection 70
Preparation of target material 71
Isolation of total RNA 71
Isolation of mRNA 72
Preparation of cDNA 72
Selection of specific genes 73
Library-based cDNA cloning strategies 73
Construction of a cDNA library 74
Screening cDNA libraries 74
~anipulation of identified cDNA sequences 76
PCR-based isolation of genes from cDNA 76
Primer design 77
~ixed-pool or redundant oligonucleotide primers 78
Primary PCR reaction 78
Isolation of the 3' end of a cDNA 79
Isolation of the 5' end of a cDNA 79
Gene identification by differential display 81
Expression systems 81
Expression using E. coli 82
Eukaryotic expression systems 83
References 83
vi
CONTENTS
4 Retroviral insertional tnutagenesis. F. Farzaneh, J. Giiken
s.-u.
and Gan 87
Introduction 87
The retroviral life cycle 88
Host range 91
Replication-defective retroviral vectors 92
Packaging cell lines 93
Conditions required for efficient mutagenesis 95
Mechanisms involved in retroviral insertional mutagenesis 96
Mutation frequency 98
Multiplicity of infection 99
Mutant selection procedures 99
Cloning of the sites of provirus integration 99
Construction of genomic libraries 100
PCR-mediated amplification 100
Identification of the gene of interest 100
Identification of common sites of provirus integration 101
Library screening by nuclear run-on probes 102
References 103
5 Gene entraptnent. H. von Melchner and H.E. Ruley 109
Introduction 109
Gene trap vectors 110
Cloning and analysis of flanking sequences 116
Isolation and use of promoter-tagged sites 116
Insertional mutagenesis in cultured cells 118
Insertional mutagenesis in mice 119
Identification of regulated genes 121
References 124
6 Gene transfer studies. D. Darling and M. Kuiper 131
Introduction 131
What is transfection? 131
What form should the DNA be in? 132
Generalized requirements for eukaryotic gene transcription 133
Eukaryotic gene transcription 133
SV40-based plasmids 134
Specialized eukaryotic host cells 135
Specialized plasmids 136
Double insert plasmids 136
Inducible expression 137
Epstein-Barr virus-based plasmids 138
Shuttle vectors 140
Multifunctional plasmids 140
Transfection procedures 140
Calcium phosphate co-precipitation 141
DEAE-dextran 142
Electroporation 143
Liposomes and lipid-based transfection 143
VII
CONTENTS
Adenovirus and POlY-L-lysine-conjugated complexes 144
Alternative transfection procedures 145
Assays for new protein synthesis 146
Dominant selectable marker genes 146
Reporter genes 147
Analysis of cloned genes 148
Identification of ligands for novel receptors 148
Identification of transcription factors 149
References 151
7 Foreign DNA integration and DNA methylation patterns.
W Doeifler 155
Introduction 155
The adenovirus system as a model 156
Site selection in the integration of adenovirus DNA 157
Modes of adenovirus DNA integration - a synopsis of data 159
On the mechanism of integrative recombination 163
Insertion of foreign DNA by a versatile mechanism 163
Studies on the mechanism of integrative recombination
in a cell-free system 165
De novo DNA methylation of integrated foreign DNA 167
De novo methylation of integrated foreign DNA: a cellular
defence mechanism? 167
Initiation of de novo methylation in mammalian cells is not
predominantly dependent upon the nucleotide sequence
of foreign DNA 169
Methylation of triplet repeat amplifications in the human
genome: manifestation of the cellular defence mechanism 171
Alterations in patterns of cellular DNA methylation and
gene expression as consequences of foreign DNA insertions
into mammalian genomes 172
DNA methylation and gene activity 173
A fully 5'-CG-3' but not a 5'-CCGG-3' methylated late
FV3 promoter retains activity 173
Topology of the promoter of RNA polymerase Il
and Ill-transcribed genes is modified by the methylation
of5'-CG-3' dinucleotides 174
Impact of 5'-CG-3' methylation on the activity of
different eukaryotic promoters: a comparison 175
Uptake of foreign DNA through the gastrointestinal tract 175
A concept of oncogenesis - implications for gene therapy
and research on transgenic organisms 177
References 178
8 Transgenic animals in human gene analysis. F. Theuring 185
Introduction 185
Methodology 187
Transgenes to study gene regulation 189
Transgenes to study gene function 193
viii
CONTENTS
Functional analysis: gain-of-function 194
Functional analysis: loss-of-function 197
Conclusions 199
References 200
9 HOlnologous recOlnbination. A. Mansouri 207
Introduction 207
Embryonic stem cells 208
Principles of homologous recombination in mammalian cells 209
Targeting vectors 209
Promoterless constructs 212
Positive-negative selection procedure 212
Hit-and-run and in-out targeting strategies 213
Potential of homologous recombination in embryonic stem cells 213
Developmental biology 213
Animal models of human disease 214
Homologous recombination and gene therapy 216
Future perspectives: ere-LoxP mediated gene targeting 216
References 217
10 COInplementation analysis. A. Patel 221
Introduction 221
Principles of somatic cell hybridization 222
De novo and salvage pathways of nucleotide synthesis 223
Purine nucleotide synthesis 223
The HPRT gene 224
HPRT variants 224
Pyrimidine nucleotide synthesis 225
Metabolic cooperation 225
The HAT selection system 226
Selection procedures for the isolation of hybrid cells 226
Identification of complementation groups and topological relationships 227
Extinction and activation 227
Assignment of complementation groups in clinical diseases 228
Assignment of complementation groups in senescence 229
Assignment of complementation groups in biochemical pathways 230
Assignment of complementation groups in cytokine activity 230
Identification of the dominant/recessive nature of genetic lesions 231
Chromosome segregation 231
Dominant and recessive genetic changes involved in senescence 232
Dominant and recessive nature of viral genes 232
Dominant and recessive events in tumour progression 232
Dominant nature of multi-drug resistance genes 233
Dominant and recessive events involved in the immunological
process 233
Dominant and recessive developmentally regulated genes 233
Microcell fusion: principles and application to the chromosomal
localization of genes 234
Introduction to microcell fusion 234
ix
