Transcription factors: a practical approach
Gespeichert in:
| Format: | Buch |
|---|---|
| Sprache: | English |
| Veröffentlicht: |
Oxford [u.a.]
Oxford Univ. Pr.
1999
|
| Ausgabe: | 2. ed. |
| Schriftenreihe: | The practical approach series
201 |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XXII, 303 S. Ill., graph. Darst. |
| ISBN: | 0199636974 0199636966 |
Internformat
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| 245 | 1 | 0 | |a Transcription factors |b a practical approach |c ed. by David S. Latchman |
| 250 | |a 2. ed. | ||
| 264 | 1 | |a Oxford [u.a.] |b Oxford Univ. Pr. |c 1999 | |
| 300 | |a XXII, 303 S. |b Ill., graph. Darst. | ||
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| 490 | 1 | |a The practical approach series |v 201 | |
| 650 | 4 | |a Facteurs de transcription - Manuels de laboratoire | |
| 650 | 7 | |a Facteurs de transcription - Manuels de laboratoire |2 ram | |
| 650 | 4 | |a Transcription génétique - Régulation - Manuels de laboratoire | |
| 650 | 4 | |a Transcription Factors |x physiology | |
| 650 | 4 | |a Transcription factors |x Laboratory manuals | |
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Datensatz im Suchindex
| _version_ | 1805072859388706816 |
|---|---|
| adam_text |
Contents
List of contributors Xix
Abbreviations XXi
1. The DNA mobility shift assay 1
C. L. Dent, M. D. Smith, and D. S. Latchman
1. Introduction 1
2. Detection of DNA binding proteins 2
Applications of the DNA mobility shift assay 2
Selection of DNA probe 6
Preparation of labelled oligonucleotide probes for retardation assay 7
Labelling of fragment probes 8
Preparation of protein extracts 9
The binding reaction 10
Preparation of mini extracts 13
3. Other sources of protein for use in the binding assay 14
Expression of DNA binding proteins in bacteria 14
Expression of proteins in mammalian cells 15
Expression by in vitro transcription and translation 16
Expression of cloned transcription factor in baculovirus 17
Purification of transcription factor protein 17
4. Investigation of DNA binding specificity 17
5. Characterization of DNA binding proteins 19
Addition of antibodies 19
Addition of potential ligands 21
Proteolytic clipping band shift assay 21
6. Study of protein protein interactions 23
7. Concluding comments 25
References 25
2. Footprint analysis of DNA protein
complexes in vitro and in vivo 27
Craig Spiro and Cynthia T. McMurray
1. Overview 27
2. In vitro footprinting 29
Contents
Analysis at base resolution of binding sites on 32P end labelled
fragments 29
Analysis of binding on closed, circular plasmid 41
3. In vivo footprinting 47
Modification of DNA in vivo 48
Nested gene specific primers 55
Linker for LMPCR 55
Visualization to nucleotide resolution by LMPCR 56
References 61
3. In vitro transcription and characterization
of transcription 63
Austin J. Cooney, Sophia Y. Tsai, and Ming Jer Tsai
1. Introduction 63
2. In vitro transcription assays 63
3. Determination of the molecular weight of native
transcription factors 69
Molecular weight determination using gel filtration chromatography 69
Molecular weight determination using glycerol gradient centrifugation 73
Determination of molecular weight of native factors using non
denaturing gradient gel electrophoresis 75
4. Determination of the molecular weight of denatured
transcription factors 76
UV cross linking of transcription factors to DNA 77
UV cross linking with a bromodeoxyuridine substituted DNA 77
UV cross linking of transcription factors to non substituted probes 80
Renaturation of transcription factors from SDS polyacrylamide gels 82
5. Analysis of the monomer/dimer structure of the DNA binding
forms of a transcription factor 84
Analysis of transcription factors by DNA mobility shift assay 84
Analysis of transcription factor subunits by chemical cross linking 85
Analysis of cooperative binding between dimers bound to adjacent
response elements 86
6. Identification and initial characterization of non DNA
binding transcription factors 88
Assay of direct interactions between DNA binding and non DNA
binding transcription factors 90
'Supershift' gel mobility shift analysis 90
Analysis of the dissociation of transcription factor DNA complexes
in the presence and absence of the non DNA binding
transcription factor 91
Analysis of direct interactions by co immunoprecipitation 92
References 95
xii
Contents
4. Purification and cloning of DNA binding
transcription factors 97
R. H. Nicolas, G. Hynes, and G. H. Goodwin
1. Introduction 97
2. Buffers and solutions 99
3. Preparation of nuclear extract 99
4. DNA cellulose chromatography 103
5. DNA affinity chromatography 104
6. Reverse phase chromatography 111
7. Production and isolation of peptides 114
8. Design of oligonucleotides for cDNA isolation 118
Acknowledgements 121
References 121
5. Cloning transcription factors from a cDNA
expression library 123
Ian G. Cowell and Helen C. Hurst
1. Introduction 123
2. Handling bacteriophage expression libraries 124
Library selection 124
Library plating for screening 125
Plaque purification 126
3. Screening methods 126
Introduction 126
Screening with DNA binding site probes 127
Immunological screening 134
Other approaches to library screening and their relative merits 137
4. Proving the identity of the factor 139
References 142
6. Cloning transcription factors by sequence
similarity 145
Alan Ashworth
1. Introduction 145
xiii
Contents
2. Methods for cloning related transcription factors 145
Low stringency hybridization 145
Polymerase chain reaction approaches 148
In silico approaches to identifying novel transcription factors 160
3. Some examples of the cloning of transcription factors by
homology 163
Acknowledgements 163
References 163
7. Identification of target genes for a
transcription factor by genomic binding
site cloning 165
Satoshi Inoue, Shigeru Kondo, and Masami Muramatsu
1. Introduction 165
2. Method for genomic binding site (GBS) cloning 165
Preparation of a transcription factor protein 167
Source of DNA 169
Selection procedure 170
GBS cloning procedure 172
GBS cloning from CpG islands 173
3. Molecular cloning of target genes using the DNA fragments
obtained by GBS cloning 174
Zoo blotting 174
Northern blot analysis 175
Sequencing and DNA database searches 175
Binding activity of the genomic fragment 176
Enhancer activity of the genomic fragment 176
Molecular cloning of target genes using the DNA fragment obtained
by cloning 177
4. Concluding remarks 177
References 178
8. Analysis of cloned factors i8i
Roger White and Malcolm Parker
1. Introduction 181
Identification of conserved domains 182
2. Mapping and analysis of domains by deletion and point
mutagenesis 182
Preparation of deletion mutants using PCR 182
Point mutagenesis 185
xiv
Ik
Contents
3. Expression systems 187
The use of in vitro translation systems and overexpression systems
to analyse transcription factor function 187
Expression in bacteria 189
Expression in mammalian cells 192
Overexpression in mammalian cells 193
Overexpression in yeast and insect cells 194
Comparison of expression systems 195
4. Analysis of the properties of cloned factors 196
Transient transfection in mammalian cells 196
Reporter genes and control plasmids 196
Methods of transfection 198
Assays for reporter genes in transfected cells 200
Identification of transactivation domains using chimeric proteins 202
5. Analysis of protein—DNA interactions 202
Analysis of DNA binding activity using a gel retardation assay 202
6. Analysis of protein protein interactions in vitro 206
Analysis of interactions between proteins using a GST pull down
assay 206
Analysis of protein protein interactions using immunoprecipitation 208
Analysis of protein protein interactions using the gel retardation
assay 209
Analysis of protein protein interactions on DNA using an ABCD
assay 209
Analysis of protein protein interactions in cells using two hybrid
analysis 211
4. Summary 212
References 213
9. Neuronal promoter analysis by in vitro
transcription using nuclear extracts from
rat brain 215
M. L. Schwartz and W. W. Schlaepfer
1. Introduction 215
2. Background 216
3. Applications 216
Comparison to transfection and transgenic mouse studies 216
Studies of the effect of chromatin structure on transcription 217
4. Preparation of nuclear extracts 217
Overview 217
xv
Contents
Purification of nuclei from rat brain 217
Extraction of nuclear proteins 220
5. In vitro transcription 222
Overview of the reaction 222
Typical results of in vitro transcription 224
Notes on in vitro transcription 224
Promoter templates 226
6. Concluding remarks 227
References 227
10. Preparation of chromatin templates for
transcription studies 229
Joan Boyes
1. Introduction 229
2. Experimental approaches using chromatin templates 229
Introduction to chromatin structure 229
Initiation and elongation of transcription on chromatin templates 230
Mononucleosome templates versus nucleosome arrays 230
3. Preparation of chromatin templates 232
Technical considerations 232
Xenopus nuclear extracts 233
Drosphila embryo nuclear extracts 233
Minichromosome preparation 234
Mononucleosome preparation 234
4. Preparation of histones 236
Principle of hydroxyapatite purification 236
5. Reconstitution of mononucleosomes by salt—urea dialysis 243
Considerations for the DNA fragment for reconstitution 243
Amount of DNA needed in the reconstitution reaction 246
Calculation of the amount of histones in the reconstitution reaction 248
Purification of mononucleosome reconstitutes 249
Characterization of the reconstitutes 252
6. Transcription factor binding to nucleosome templates 255
Acknowledgements 257
References 258
11. Analysis of transcription factor
modifications 26i
N. Shaun B. Thomas
1. Introduction 261
xvi
Contents
2. Phosphorylation 261
Gel electrophoresis 263
Dephosphorylation in vitro 266
Radioactive labelling 270
Mapping phosphorylation sites 274
Phosphorylation in vitro 281
Generation and use of phosphorylation site specific antibodies 283
Functional analysis 283
Other methods for analysing phosphorylated proteins 284
3. Other post translational modifications 285
O Linked glycosylation 285
Other modifications 290
4. Conclusions 290
Acknowledgements 291
References 291
Appendix 1 List of suppliers 295
Index 297
xvii |
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| isbn | 0199636974 0199636966 |
| language | English |
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| spelling | Transcription factors a practical approach ed. by David S. Latchman 2. ed. Oxford [u.a.] Oxford Univ. Pr. 1999 XXII, 303 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier The practical approach series 201 Facteurs de transcription - Manuels de laboratoire Facteurs de transcription - Manuels de laboratoire ram Transcription génétique - Régulation - Manuels de laboratoire Transcription Factors physiology Transcription factors Laboratory manuals Methode (DE-588)4038971-6 gnd rswk-swf Transkriptionsfaktor (DE-588)4303350-7 gnd rswk-swf Transkription Genetik (DE-588)4185906-6 gnd rswk-swf Transkription Genetik (DE-588)4185906-6 s Methode (DE-588)4038971-6 s DE-604 Transkriptionsfaktor (DE-588)4303350-7 s Latchman, David S. 1956- Sonstige (DE-588)120416859 oth The practical approach series 201 (DE-604)BV007921321 201 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008562795&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Transcription factors a practical approach The practical approach series Facteurs de transcription - Manuels de laboratoire Facteurs de transcription - Manuels de laboratoire ram Transcription génétique - Régulation - Manuels de laboratoire Transcription Factors physiology Transcription factors Laboratory manuals Methode (DE-588)4038971-6 gnd Transkriptionsfaktor (DE-588)4303350-7 gnd Transkription Genetik (DE-588)4185906-6 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4303350-7 (DE-588)4185906-6 |
| title | Transcription factors a practical approach |
| title_auth | Transcription factors a practical approach |
| title_exact_search | Transcription factors a practical approach |
| title_full | Transcription factors a practical approach ed. by David S. Latchman |
| title_fullStr | Transcription factors a practical approach ed. by David S. Latchman |
| title_full_unstemmed | Transcription factors a practical approach ed. by David S. Latchman |
| title_short | Transcription factors |
| title_sort | transcription factors a practical approach |
| title_sub | a practical approach |
| topic | Facteurs de transcription - Manuels de laboratoire Facteurs de transcription - Manuels de laboratoire ram Transcription génétique - Régulation - Manuels de laboratoire Transcription Factors physiology Transcription factors Laboratory manuals Methode (DE-588)4038971-6 gnd Transkriptionsfaktor (DE-588)4303350-7 gnd Transkription Genetik (DE-588)4185906-6 gnd |
| topic_facet | Facteurs de transcription - Manuels de laboratoire Transcription génétique - Régulation - Manuels de laboratoire Transcription Factors physiology Transcription factors Laboratory manuals Methode Transkriptionsfaktor Transkription Genetik |
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