List of Contributors xv
Abbreviations xvii
Evolutionary biology of the nuclear receptor 1 (28)
Superfamily
H. Escriva
M. Robinson
V. Laudet
Introduction 1 (2)
Molecular Phylogeny of nuclear receptors 3 (16)
General overview 3 (1)
Sequence alignments 4 (3)
Tree reconstruction methods 7 (6)
Interpretation of sequence trees 13 (6)
Cloning of new receptor genes from 19 (10)
divergent animal species
Overview of the Strategy 19 (1)
Designing PCR primers 20 (2)
PCR isolation of nuclear receptor gene 22 (5)
fragments
RT-PCR with degenerate oligonucleotides 27 (1)
References ...28 (1)
Initial characterization of new orphan 29 (42)
receptors
R. Sladek
V. Giguere
Introduction 29 (2)
Sources of receptor protein for DNA binding 31 (9)
studies
Protein expression in bacteria 32 (3)
Protein expression in vitro 35 (1)
Protein expression in eukaryotic cells 36 (4)
Identifying the DNA binding mode: monomers 40 (5)
and dimers
GST-pulldown 40 (3)
Mammalian two-hybrid system 43 (2)
Identifying hormone-response elements 45 (18)
Characterizing DNA binding proteins using 45 (7)
the electrophoretic mobility shift assay
Identifying response elements using 52 (1)
consensus binding sites
Using EMSA to detect RXR heterodimers and 53 (1)
receptor homodimers
Determining the polarity of RXR 54 (3)
heterodimers using chimeric receptors
Identifying response elements using SAAB 57 (3)
selection
Identifying DNA-protein contacts using 60 (3)
the methylation interference assay
Transcriptional activation assays: 63 (8)
transient and stabletransfection
References 68 (3)
Adopting orphans: finding ligands 71 (24)
S. Kersten
W. Wahli
Introduction 71 (1)
First step: guessing ligand classes 71 (1)
Tissue fractionation 72 (2)
Transactivation assays 74 (3)
Standard transactivation assay 74 (2)
Gal4-LBD fusion transactivation system 76 (1)
Quantitation of transactivation assays 77 (1)
Ligand binding assays 77 (3)
Competition assay 77 (3)
Monitoring changes in receptor conformation 80 (3)
Protease sensitivity 80 (2)
Non-denaturing gel electrophoresis 82 (1)
Co-activator-dependent receptor ligand 83 (3)
assay (CARLA)
Fluorescence spectroscopy 86 (5)
Principles and utility of fluorescence 87 (2)
spectroscopy
Emission scans 89 (1)
Fluorescence titrations 90 (1)
Conclusions 91 (4)
References 92 (3)
Kinetic analysis of nuclear receptor 95 (24)
interactions
B.J. Cheskis
L.P. Freedman
Introduction 95 (3)
Analysis of nuclear receptor functions 98 (18)
using surface plasmon resonance (SPR)
technology
The basic principle of interaction 98 (1)
analysis with BIAcore
Overexpression and purification of 99 (4)
nuclear receptors for kinetic and
equilibrium analyses
Analysis of protein-protein interactions 103 (3)
Analysis of protein-DNA interactions 106 (8)
Kinetic analysis 114 (2)
Conclusion 116 (3)
References 116 (3)
Functional characterization of co-activators 119 (18)
using mammalian cell microinjection
D.W. Rose
T.-M. Mullen
M.G. Rosenfeld
C.K. Glass
Introduction 119 (1)
Why microinjection? 119 (1)
Equipment and materials 120 (9)
Equipment 120 (1)
Antibodies 120 (4)
Choice of cells 124 (1)
Preparation of cells 125 (1)
Preparation of Samples to be injected 126 (1)
Choice of DNA to inject 126 (3)
Microinjection technique 129 (8)
Basic microinjection technique 129 (2)
Monitoring expression 131 (3)
Analysis of results 134 (1)
References 135 (2)
Analysis of steroid/nuclear receptor 137 (30)
phosphorylation
B.G. Rowan
N.L. Weigel
Introduction 137 (1)
Phosphorylation of the nuclear receptor 137 (1)
superfamily
General guidelines for studying nuclear 138 (2)
receptor phosphorylation
Identification of phosphorylation sites in 140 (18)
nuclear receptors
Approaches to identify phosphorylation 140 (1)
sites in nuclear receptors and other
proteins
Conventional method 141 (12)
Approach for use with low protein levels 153 (4)
Additional experiments to provide more 157 (1)
sequence information
Indirect method 158 (1)
Identifying candidate kinases 158 (4)
Introduction 158 (1)
In vitro phosphorylation of nuclear 159 (3)
receptors
Assaying the functional significance of 162 (5)
selected phosphorylation sites
Introduction 162 (1)
Site-directed mutagenesis of nuclear 163 (1)
receptors
References 163 (4)
Ligand-and cofactor-regulated transcription 167 (24)
with chromatin templates
W.L. Kraus
J.T. Kadonaga
Introduction 167 (1)
Synthesis and purification of recombinant 167 (6)
nuclear hormone receptors and cofactors
Choice of expression systems 167 (1)
Purification of recombinant receptors and 168 (5)
cofactors
Functional analyses of purified receptors 173 (1)
and cofactors
In vitro chromatin assembly 173 (6)
Chromatin assembly systems 173 (1)
Assembly of plasmid DNA templates into 173 (3)
chromatin
Analysis of chromatin templates assembled 176 (1)
in vitro
Mock chromatin assembly of plasmid DNA 177 (2)
templates
In vitro transcription of chromatin 179 (9)
templates
In vitro transcription systemes 179 (1)
Ligand-and cofactor-regulated in vitro 179 (6)
transcription with chromatin templates
Chromatin versus non-chromatin 185 (1)
transcription experiments
Single versus multiple round 186 (2)
transcription experiments
Exploiting ligand-and cofactor-regulated in 188 (3)
vitro transcription systems
Acknowledgements 188 (1)
References 188 (3)
Hormone-resistance syndromes 191 (22)
M. Gurnell
V. K. K. Chatterjee
Introduction 191 (1)
Receptor analysis 192 (9)
Gene sequencing 192 (2)
cDNA sequencing 194 (3)
Genotyping by restriction analysis 197 (1)
Haplotype analysis of kindreds with 198 (3)
identical mutations
Ligand binding and DNA binding studies 201 (4)
Site-directed mutagenesis to generate 201 (1)
mutated receptor cDNA
Synthesis of mutant receptor protein and 201 (3)
ligand binding assays
DNA binding properties of mutant receptors 204 (1)
Assaying mutant receptor function by 205 (4)
transient transfection
T3-dependent transactivation 205 (1)
Dominant negative inhibition by mutant 206 (3)
receptors
Interaction of mutant receptors with 209 (4)
cofactors
References 210 (3)
In vitro assembly of steroid receptor-Hsp90 213 (20)
complexes
D. F. Smith
Introduction 213 (4)
Background 213 (1)
Proteins associating with unactivated 214 (1)
steroid receptors
Basic pathway for assembly of steroid 214 (3)
receptor complexs
Receptor isolation 217 (2)
Immunoaffinity purification 217 (2)
In vitro expression to produce 219 (1)
radiolabelled receptor
Assembly reactions in RL 219 (3)
Lysate sources and considerations 219 (1)
Standard assembly conditions and further 220 (2)
considerations
Analysis of assembly reactions 222 (5)
Identification of assembly components 222 (3)
Analysis of assembly dynamics 225 (1)
Inhibition of specific assembly steps and 225 (2)
chaperone components
Assembly of receptor complexes in a 227 (6)
purified system
References 229 (4)
Yeast as a system for the study of nuclear 233 (28)
receptor function
M. J. Garabedian
J. A. Iniguez-Lluhi
A. Kralli
Introduction 233 (1)
Why study nuclear receptors in yeast? 233 (2)
Expression of nuclear receptors in yeast 235 (1)
Reporter genes 236 (7)
The colorimetric reporter, LACZ 237 (5)
Reporter genes for positive and negative 242 (1)
selection
Yeast host strains 243 (1)
Mutagenesis and screens for receptor 244 (3)
mutations
High density random mutagenesis using 245 (2)
error-prone PCR
Strategies for identifying cellular factors 247 (7)
that affect nuclear receptor function
Identification of gene products affecting 247 (2)
NR function using yeast knockout strains
Identifying components in NR signal 249 (3)
transduction through random mutagenesis
of the yeast genome
Identifying components in NR signal 252 (2)
transduction through dosage suppression
analysis
Rescuing plasmids from yeast 254 (1)
Preparation of protein extracts from yeast 255 (6)
for Western blotting
Acknowledgements 256 (1)
References 256 (5)
Regulation of heterologous proteins by fusion 261 (14)
to a hormone binding domain
D. Picard
Introduction 261 (1)
Key features 262 (5)
Mechanism 266 (1)
Practical implications of the proposed 266 (1)
mechanism
Problems and troubleshooting 267 (1)
Potential complications 267 (1)
Unexpected behaviour 268 (1)
Troubleshooting 268 (1)
Application to yeast 268 (7)
Acknowledgements 271 (1)
References 271 (4)
Appendix I 275 (2)
Appendix II 277 (6)
Index 283