Transcriptional regulation in eukaryotes: concepts, strategies, and techniques
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| Main Authors: | , , |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
Cold Spring Harbor, N.Y.
Cold Spring Harbor Laboratory Press
2009
|
| Edition: | 2. ed. |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Includes bibliographical references and index |
| Physical Description: | XIX, 620 S. Ill., graph. Darst. |
| ISBN: | 9780879697624 9780879697778 |
Staff View
MARC
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| 020 | |a 9780879697624 |c pbk. : alk. paper |9 978-0-87969-762-4 | ||
| 020 | |a 9780879697778 |c hardcover : alk. paper |9 978-0-87969-777-8 | ||
| 035 | |a (OCoLC)249135580 | ||
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| 100 | 1 | |a Carey, Michael |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Transcriptional regulation in eukaryotes |b concepts, strategies, and techniques |c Michael F. Carey ; Craig L. Peterson ; Stephen T. Smale |
| 250 | |a 2. ed. | ||
| 264 | 1 | |a Cold Spring Harbor, N.Y. |b Cold Spring Harbor Laboratory Press |c 2009 | |
| 300 | |a XIX, 620 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Includes bibliographical references and index | ||
| 650 | 4 | |a Genetic transcription |x Regulation | |
| 650 | 4 | |a Transcription factors | |
| 650 | 4 | |a Genetic transcription |x Regulation |x Research |x Methodology | |
| 650 | 0 | 7 | |a Transkription |g Genetik |0 (DE-588)4185906-6 |2 gnd |9 rswk-swf |
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| 650 | 0 | 7 | |a Eukaryoten |0 (DE-588)4070991-7 |2 gnd |9 rswk-swf |
| 689 | 0 | 0 | |a Eukaryoten |0 (DE-588)4070991-7 |D s |
| 689 | 0 | 1 | |a Genregulation |0 (DE-588)4122166-7 |D s |
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| 689 | 1 | 0 | |a Eukaryoten |0 (DE-588)4070991-7 |D s |
| 689 | 1 | 1 | |a Transkription |g Genetik |0 (DE-588)4185906-6 |D s |
| 689 | 1 | |5 DE-604 | |
| 700 | 1 | |a Peterson, Craig L. |e Verfasser |4 aut | |
| 700 | 1 | |a Smale, Stephen T. |e Verfasser |4 aut | |
| 856 | 4 | 2 | |m Digitalisierung UB Bayreuth |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016994783&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-016994783 | ||
Record in the Search Index
| _version_ | 1804138365095772160 |
|---|---|
| adam_text | Contents
PREFACE,
xi
OVERVIEW, xiii
ABBREVIATIONS AND ACRONYMS,
xvii
1
A PRIMER ON TRANSCRIPTION
AL
REGULATION IN MAMMALIAN CELLS
INTRODUCTION AND OVERVIEW,
1
Brief summary of genome-wide methods,
2
CHROMATIN AND THE GENERAL TRANSCRIPTION MACHINERY,
5
Chromatin structure and organization,
5
Chromatin modification,
8
Chromatin remodeling,
11
The general transcription machinery,
15
Organization of the regulatory region,
15
Basal transcription complex assembly and initiation,
22
Mediator,
25
TFIID and TAFs,
26
ACTIVATION AND REPRESSION,
28
Gene activation,
28
Chromatin modification and remodeling during transcription initiation,
29
A model for recruitment of the general machinery,
29
Initial stages of Pol II elongation,
31
Pol II encounters nucleosomes within the gene,
34
Silencing/repression of transcription,
35
CONCLUDING PERSPECTIVE,
41
VÍ
/ Contents
2 INITIAL
STRATEGIC
ISSUES
INTRODUCTION AND OVERVIEW,
47
EXPERIMENTAL STRATEGIES,
48
Approaches toward the characterization of a new transcription factor,
48
Analyzing the regulation of a new gene,
52
Box
2.1
Nuclear run-on transcription assay,
55
Consider the time commitment and resources needed to reach a defined goal,
56
Defining the project goals,
57
Evaluating the feasibility of the analysis,
57
Initiating a comprehensive transcriptional regulation analysis of a new gene,
60
TECHNIQUES,
63
Protocol
2.1
Nuclear run-on assay,
63
TRANSCRIPTION INITIATION SITE MAPPING
INTRODUCTION AND OVERVIEW,
71
EXPERIMENTAL STRATEGIES,
74
Initial considerations,
74
Rapid amplification of cDNA ends (RACE),
76
Box
3.1
CAP-dependent RACE procedures,
77
Primer extension,
78
Box
3.2
Primer extension,
79
RNase protection,
82
Box
3.3
RNase protection,
83
S1 nuclease analysis,
86
Box
3.4
Nuclease protection,
87
Box
3.5
Effect of
introns
on interpretation of start-site mapping results,
88
TECHNIQUES,
90
Protocol
3.1
Primer extension assay,
90
Protocol
3.2
RNase protection assay,
98
FUNCTIONAL ASSAYS FOR PROMOTER ANALYSIS
INTRODUCTION AND OVERVIEW,
107
EXPERIMENTAL STRATEGIES,
110
Choosing an assay: Advantages and disadvantages of each assay,
110
Transient transfection assays,
118
Box
4.1
Common transfection methods,
119
Box
4.2
Luciferase reporter gene assay,
121
Box
4.3
CAT reporter gene assay,
122
Box
4.4
lacZ, SEAP, and GFP reporter gene assays,
123
Box
4.5
Isolation of transiently transfected cells,
124
Contents
I
VÜ
Stable transfection assays by chromosomal integration,
129
Box
4.6
Replication-competent vectors,
131
TECHNIQUES,
137
Common transfection methods for mammalian cells,
137
Protocol
4.1
Calcium phosphate transfection of 3T3
fibroblasts,
138
Protocol
4.2
DEAE-dextran transfection of lymphocyte cell lines,
141
Protocol
4.3
Transfection by electroporation of RAW264.7
macrophages,
144
Additional considerations for Protocols
4.1-4.3, 146
Common reporter enzyme assays,
148
Protocol
4.4
Luciferase assay,
149
Protocol
4.5
Chloramphenicol acetyltransferase assay,
152
Protocol
4.6 ß-galactosidase
assay,
155
IDENTIFICATION AND ANALYSIS OF DISTANT CONTROL REGIONS
INTRODUCTION AND OVERVIEW,
161
Box
5.1
DNase I hypersensitivity assay,
165
EXPERIMENTAL STRATEGIES,
168
DNase I hypersensitivity,
168
Identification of matrix attachment regions,
171
Box
5.2
Methods for identifying MARs,
172
Functional approaches for the identification of distant control regions,
173
Functional assays for the characterization of distant control regions,
177
IDENTIFYING C/S-ACTING
DNA
ELEMENTS WITHIN A CONTROL REGION
INTRODUCTION AND OVERVIEW,
185
EXPERIMENTAL STRATEGIES,
187
Identification
of control elements by comprehensive mutant analysis,
187
Strategies for a comprehensive analysis,
188
Insights provided by a comprehensive mutant analysis versus a phylogenetic analysis,
199
IDENTIFICATION OF DNA-BINDING PROTEINS AND THEIR GENES
INTRODUCTION AND OVERVIEW,
205
EXPERIMENTAL STRATEGIES FOR THE IDENTIFICATION OF DNA-BINDING PROTEINS,
207
Database methods,
207
Development of a protein-DNA interaction assay for crude cell lysates,
210
Box
7.1
Hypothetical EMSA results,
216
EXPERIMENTAL STRATEGIES FOR CLONING OR IDENTIFYING GENES ENCODING
DNA-BINDING PROTEINS,
223
Box
7.2
Cloning by protein purification,
225
Cloning by protein purification and
peptide
sequence analysis,
227
Alternative cloning methods,
229
viii
/ Contents
8
CONFIRMING THE FUNCTIONAL IMPORTANCE OF
INTRODUCTION AND OVERVIEW,
235
EXPERIMENTAL STRATEGIES,
237
Chromatin immunoprecipitation,
237
Loss-of-function studies by gene disruption or
RNA
interference,
240
Abundance of a protein-DNA complex in vitro,
243
Relative expression patterns of the DNA-binding protein and target gene,
243
Correlation between nucleotides required for protein binding and those required for activity of the
control element,
245
Transactivation of a reporter gene or endogenous gene by overexpression of a DNA-binding
protein,
246
Cooperative binding and synergistic function of proteins bound to adjacent control elements,
247
Comparison of genomic and in vitro footprinting patterns,
249
Relative affinity of a protein-DNA interaction,
250
Dominant-negative mutants,
252
In vitro transcription strategies,
254
Altered-specificity experiments,
256
IN VIVO ANALYSIS OF AN ENDOGENOUS CONTROL REGION
INTRODUCTION AND OVERVIEW,
261
EXPERIMENTAL STRATEGIES,
263
Chromatin immunoprecipitation,
263
DamID,
264
DNase I and DMS genomic footprinting,
266
Box
9.1
Ligation-mediated PCR,
267
Potassium permanganate genomic footprinting,
270
Southern blot analysis of nucleosome presence and positioning,
270
LM-PCR, PCR, and ChIP strategies for monitoring nucleosome presence and positioning,
272
Box
9.2
Low-resolution analysis of nucleosomal positioning by MNase-Southern blot assay,
272
Overview of in vivo methods for analyzing nucleosome remodeling,
276
DNase I hypersensitivity to monitor nucleosome remodeling,
276
MNase methods for monitoring nucleosome remodeling,
277
Restriction enzyme accessibility assays,
278
Box
9.3
Restriction enzyme accessibility-LM-PCR assay,
281
Chromatin conformation capture,
283
DNA methylation, 285
TECHNIQUES,
287
Protocol
9.1
MNase-Southern blot assay,
287
Protocol
9.2
LM-PCR methods,
296
Protocol
9.3
Chromatin immunoprecipitation,
313
Contentss
Ι ¡Χ
1
0
IDENTIFYING AND CHARACTERIZING DOMAINS OF TRANSCRIPTION
REGULATORY FACTORS
INTRODUCTION AND OVERVIEW,
323
EXPERIMENTAL STRATEGIES: DEFINING DOMAINS,
324
Identification of functional domains,
324
Box
10.1
Computational analysis of domains,
325
Basic mutagenesis principles,
327
Box
10.2
Expression systems,
327
Box
10.3
Common purification and detection tags,
329
Domains of a sequence-specific regulatory factor,
333
Separating DNA-binding and activation/repression domains of a sequence-specific regulatory factor,
334
Box
10.4
The VP16 activation domain: A case study,
337
Box
10.5
CAL4: A case study,
338
Box
10.6
The
KRAB
repression domain: A case study,
339
Subdividing
DNA
recognition and oligomerization
subdomains,
341
CONCEPTS AND STRATEGIES: PROTEIN-PROTEIN INTERACTIONS,
342
Isolation and cloning of coactivators and corepressors,
342
Approaches for studying interaction of activation domains with coactivators,
343
Studying interactions between an activation/repression domain and its target by affinity
chromatography,
344
Altered-specificity genetic systems,
346
Structure-function analysis of the general transcriptional machinery,
349
Analysis of coactivators,
352
TECHNIQUES,
356
Protocol
10.1
PCR-mediated site-directed mutagenesis,
356
11 DNA
BINDING BY REGULATORY TRANSCRIPTION FACTORS
INTRODUCTION AND OVERVIEW,
367
EXPERIMENTAL STRATEGIES,
369
General theory and examples of DNA-protein interactions,
369
Box
11.1
Examples of Kd scenarios,
381
Analysis and modeling of DNA-protein interactions,
383
Box
11.2
SAAB analysis,
385
Box
11.3
DNase I and exonuclease footprinting,
388
Box
11.4
Chemical probes for minor groove interactions,
390
Analysis of promoter-specific multicomponent nucleoprotein complexes,
397
TECHNIQUES,
405
Protocol
11.1
DNase I footprinting,
405
Protocol
11.2
Hydroxyl-radical footprinting,
414
Protocol
11.3
Phosphate ethylation interference assay,
418
Protocol
11.4
Methylation interference assay,
422
Protocol
11.5
Electrophoretic mobility-shift assays,
427
Protocol
11.6
Preparation of 32P-end-labeled
DNA
fragments,
431
Χ
/ Contents
12
TRANSCRIPTION
AND PREINITIATION COMPLEX ASSEMBLY IN VITRO
INTRODUCTION AND OVERVIEW,
439
EXPERIMENTAL STRATEGIES,
442
Preparation of extracts,
442
Transcription assays,
445
Box
12.1
Methods for measuring transcription in vitro,
446
Fractionated systems,
451
Box
12.2
Purified transcription factors,
453
Formation of the basal preinitiation complex,
456
Open complex formation, initiation, and promoter escape,
466
Assembly of activated complexes at a promoter,
471
TECHNIQUES,
480
Preparation of nuclear extracts: An overview,
480
Protocol
12.1
The
Dignam
and Roeder nuclear extract,
482
Protocol
12.2
In vitro transcription using HeLa cell extracts and primer extension,
487
Protocol
12.3
G-less cassette in vitro transcription using HeLa cell nuclear extracts,
493
Coactivator purification (Protocols
12.4
and
12.5), 497
Protocol
12.4
Purification of epitope-tagged TFIID,
498
Protocol
12.5
Purification of Mediator from HeLa cell lines expressing a FLAG-tagged Mediator
subunit,
504
Protocol
12.6
Immobilized template assay,
508
Protocol
12.7
Potassium permanganate probing of Pol II open complexes,
518
Protocol
12.8
Magnesi
u
m-agarose EMSA of TFIID binding to
DNA, 523
13
STUDYING CHROMATIN DYNAMICS IN VITRO: CHROMATIN ASSEMBLY,
REMODELING, AND TRANSCRIPTION
INTRODUCTION AND OVERVIEW,
539
EXPERIMENTAL STRATEGIES,
541
Strategies for assembling chromatin,
541
Box
13.1
Effects of
DNA
template and
reconstitution
method on positioning of nucleosomes within
arrays,
543
Source of histones,
546
Biochemical characterization of chromatin
reconstitutions,
549
Box
13.2
MNase analysis of nudeosomal arrays,
554
Box
13.3
EcoRI analysis of nudeosomal arrays,
556
Strategies to test the role of histone modifications in vitro,
557
Analysis of chromatin remodeling/modification enzymes,
561
In vitro transcription with chromatin templates,
569
TECHNIQUES,
573
Protocol
13.1
Chicken erythrocyte histone octamer preparation,
573
Protocol
13.2
Salt gradient dialysis
reconstitution
of nucleosomes,
583
Protocol
13.3
Reconstitution
of nudeosomal arrays using
recombinant
Drosophila
ACF and NAP1,
589
APPENDIX: CAUTIONS,
605
INDEX,
613
|
| adam_txt |
Contents
PREFACE,
xi
OVERVIEW, xiii
ABBREVIATIONS AND ACRONYMS,
xvii
1
A PRIMER ON TRANSCRIPTION
AL
REGULATION IN MAMMALIAN CELLS
INTRODUCTION AND OVERVIEW,
1
Brief summary of genome-wide methods,
2
CHROMATIN AND THE GENERAL TRANSCRIPTION MACHINERY,
5
Chromatin structure and organization,
5
Chromatin modification,
8
Chromatin remodeling,
11
The general transcription machinery,
15
Organization of the regulatory region,
15
Basal transcription complex assembly and initiation,
22
Mediator,
25
TFIID and TAFs,
26
ACTIVATION AND REPRESSION,
28
Gene activation,
28
Chromatin modification and remodeling during transcription initiation,
29
A model for recruitment of the general machinery,
29
Initial stages of Pol II elongation,
31
Pol II encounters nucleosomes within the gene,
34
Silencing/repression of transcription,
35
CONCLUDING PERSPECTIVE,
41
VÍ
/ Contents
2 INITIAL
STRATEGIC
ISSUES
INTRODUCTION AND OVERVIEW,
47
EXPERIMENTAL STRATEGIES,
48
Approaches toward the characterization of a new transcription factor,
48
Analyzing the regulation of a new gene,
52
Box
2.1
Nuclear run-on transcription assay,
55
Consider the time commitment and resources needed to reach a defined goal,
56
Defining the project goals,
57
Evaluating the feasibility of the analysis,
57
Initiating a comprehensive transcriptional regulation analysis of a new gene,
60
TECHNIQUES,
63
Protocol
2.1
Nuclear run-on assay,
63
TRANSCRIPTION INITIATION SITE MAPPING
INTRODUCTION AND OVERVIEW,
71
EXPERIMENTAL STRATEGIES,
74
Initial considerations,
74
Rapid amplification of cDNA ends (RACE),
76
Box
3.1
CAP-dependent RACE procedures,
77
Primer extension,
78
Box
3.2
Primer extension,
79
RNase protection,
82
Box
3.3
RNase protection,
83
S1 nuclease analysis,
86
Box
3.4
Nuclease protection,
87
Box
3.5
Effect of
introns
on interpretation of start-site mapping results,
88
TECHNIQUES,
90
Protocol
3.1
Primer extension assay,
90
Protocol
3.2
RNase protection assay,
98
FUNCTIONAL ASSAYS FOR PROMOTER ANALYSIS
INTRODUCTION AND OVERVIEW,
107
EXPERIMENTAL STRATEGIES,
110
Choosing an assay: Advantages and disadvantages of each assay,
110
Transient transfection assays,
118
Box
4.1
Common transfection methods,
119
Box
4.2
Luciferase reporter gene assay,
121
Box
4.3
CAT reporter gene assay,
122
Box
4.4
lacZ, SEAP, and GFP reporter gene assays,
123
Box
4.5
Isolation of transiently transfected cells,
124
Contents
I
VÜ
Stable transfection assays by chromosomal integration,
129
Box
4.6
Replication-competent vectors,
131
TECHNIQUES,
137
Common transfection methods for mammalian cells,
137
Protocol
4.1
Calcium phosphate transfection of 3T3
fibroblasts,
138
Protocol
4.2
DEAE-dextran transfection of lymphocyte cell lines,
141
Protocol
4.3
Transfection by electroporation of RAW264.7
macrophages,
144
Additional considerations for Protocols
4.1-4.3, 146
Common reporter enzyme assays,
148
Protocol
4.4
Luciferase assay,
149
Protocol
4.5
Chloramphenicol acetyltransferase assay,
152
Protocol
4.6 ß-galactosidase
assay,
155
IDENTIFICATION AND ANALYSIS OF DISTANT CONTROL REGIONS
INTRODUCTION AND OVERVIEW,
161
Box
5.1
DNase I hypersensitivity assay,
165
EXPERIMENTAL STRATEGIES,
168
DNase I hypersensitivity,
168
Identification of matrix attachment regions,
171
Box
5.2
Methods for identifying MARs,
172
Functional approaches for the identification of distant control regions,
173
Functional assays for the characterization of distant control regions,
177
IDENTIFYING C/S-ACTING
DNA
ELEMENTS WITHIN A CONTROL REGION
INTRODUCTION AND OVERVIEW,
185
EXPERIMENTAL STRATEGIES,
187
Identification
of control elements by comprehensive mutant analysis,
187
Strategies for a comprehensive analysis,
188
Insights provided by a comprehensive mutant analysis versus a phylogenetic analysis,
199
IDENTIFICATION OF DNA-BINDING PROTEINS AND THEIR GENES
INTRODUCTION AND OVERVIEW,
205
EXPERIMENTAL STRATEGIES FOR THE IDENTIFICATION OF DNA-BINDING PROTEINS,
207
Database methods,
207
Development of a protein-DNA interaction assay for crude cell lysates,
210
Box
7.1
Hypothetical EMSA results,
216
EXPERIMENTAL STRATEGIES FOR CLONING OR IDENTIFYING GENES ENCODING
DNA-BINDING PROTEINS,
223
Box
7.2
Cloning by protein purification,
225
Cloning by protein purification and
peptide
sequence analysis,
227
Alternative cloning methods,
229
viii
/ Contents
8
CONFIRMING THE FUNCTIONAL IMPORTANCE OF
INTRODUCTION AND OVERVIEW,
235
EXPERIMENTAL STRATEGIES,
237
Chromatin immunoprecipitation,
237
Loss-of-function studies by gene disruption or
RNA
interference,
240
Abundance of a protein-DNA complex in vitro,
243
Relative expression patterns of the DNA-binding protein and target gene,
243
Correlation between nucleotides required for protein binding and those required for activity of the
control element,
245
Transactivation of a reporter gene or endogenous gene by overexpression of a DNA-binding
protein,
246
Cooperative binding and synergistic function of proteins bound to adjacent control elements,
247
Comparison of genomic and in vitro footprinting patterns,
249
Relative affinity of a protein-DNA interaction,
250
Dominant-negative mutants,
252
In vitro transcription strategies,
254
Altered-specificity experiments,
256
IN VIVO ANALYSIS OF AN ENDOGENOUS CONTROL REGION
INTRODUCTION AND OVERVIEW,
261
EXPERIMENTAL STRATEGIES,
263
Chromatin immunoprecipitation,
263
DamID,
264
DNase I and DMS genomic footprinting,
266
Box
9.1
Ligation-mediated PCR,
267
Potassium permanganate genomic footprinting,
270
Southern blot analysis of nucleosome presence and positioning,
270
LM-PCR, PCR, and ChIP strategies for monitoring nucleosome presence and positioning,
272
Box
9.2
Low-resolution analysis of nucleosomal positioning by MNase-Southern blot assay,
272
Overview of in vivo methods for analyzing nucleosome remodeling,
276
DNase I hypersensitivity to monitor nucleosome remodeling,
276
MNase methods for monitoring nucleosome remodeling,
277
Restriction enzyme accessibility assays,
278
Box
9.3
Restriction enzyme accessibility-LM-PCR assay,
281
Chromatin conformation capture,
283
DNA methylation, 285
TECHNIQUES,
287
Protocol
9.1
MNase-Southern blot assay,
287
Protocol
9.2
LM-PCR methods,
296
Protocol
9.3
Chromatin immunoprecipitation,
313
Contentss
Ι ¡Χ
1
0
IDENTIFYING AND CHARACTERIZING DOMAINS OF TRANSCRIPTION
REGULATORY FACTORS
INTRODUCTION AND OVERVIEW,
323
EXPERIMENTAL STRATEGIES: DEFINING DOMAINS,
324
Identification of functional domains,
324
Box
10.1
Computational analysis of domains,
325
Basic mutagenesis principles,
327
Box
10.2
Expression systems,
327
Box
10.3
Common purification and detection tags,
329
Domains of a sequence-specific regulatory factor,
333
Separating DNA-binding and activation/repression domains of a sequence-specific regulatory factor,
334
Box
10.4
The VP16 activation domain: A case study,
337
Box
10.5
CAL4: A case study,
338
Box
10.6
The
KRAB
repression domain: A case study,
339
Subdividing
DNA
recognition and oligomerization
subdomains,
341
CONCEPTS AND STRATEGIES: PROTEIN-PROTEIN INTERACTIONS,
342
Isolation and cloning of coactivators and corepressors,
342
Approaches for studying interaction of activation domains with coactivators,
343
Studying interactions between an activation/repression domain and its target by affinity
chromatography,
344
Altered-specificity genetic systems,
346
Structure-function analysis of the general transcriptional machinery,
349
Analysis of coactivators,
352
TECHNIQUES,
356
Protocol
10.1
PCR-mediated site-directed mutagenesis,
356
11 DNA
BINDING BY REGULATORY TRANSCRIPTION FACTORS
INTRODUCTION AND OVERVIEW,
367
EXPERIMENTAL STRATEGIES,
369
General theory and examples of DNA-protein interactions,
369
Box
11.1
Examples of Kd scenarios,
381
Analysis and modeling of DNA-protein interactions,
383
Box
11.2
SAAB analysis,
385
Box
11.3
DNase I and exonuclease footprinting,
388
Box
11.4
Chemical probes for minor groove interactions,
390
Analysis of promoter-specific multicomponent nucleoprotein complexes,
397
TECHNIQUES,
405
Protocol
11.1
DNase I footprinting,
405
Protocol
11.2
Hydroxyl-radical footprinting,
414
Protocol
11.3
Phosphate ethylation interference assay,
418
Protocol
11.4
Methylation interference assay,
422
Protocol
11.5
Electrophoretic mobility-shift assays,
427
Protocol
11.6
Preparation of 32P-end-labeled
DNA
fragments,
431
Χ
/ Contents
12
TRANSCRIPTION
AND PREINITIATION COMPLEX ASSEMBLY IN VITRO
INTRODUCTION AND OVERVIEW,
439
EXPERIMENTAL STRATEGIES,
442
Preparation of extracts,
442
Transcription assays,
445
Box
12.1
Methods for measuring transcription in vitro,
446
Fractionated systems,
451
Box
12.2
Purified transcription factors,
453
Formation of the basal preinitiation complex,
456
Open complex formation, initiation, and promoter escape,
466
Assembly of activated complexes at a promoter,
471
TECHNIQUES,
480
Preparation of nuclear extracts: An overview,
480
Protocol
12.1
The
Dignam
and Roeder nuclear extract,
482
Protocol
12.2
In vitro transcription using HeLa cell extracts and primer extension,
487
Protocol
12.3
G-less cassette in vitro transcription using HeLa cell nuclear extracts,
493
Coactivator purification (Protocols
12.4
and
12.5), 497
Protocol
12.4
Purification of epitope-tagged TFIID,
498
Protocol
12.5
Purification of Mediator from HeLa cell lines expressing a FLAG-tagged Mediator
subunit,
504
Protocol
12.6
Immobilized template assay,
508
Protocol
12.7
Potassium permanganate probing of Pol II open complexes,
518
Protocol
12.8
Magnesi
u
m-agarose EMSA of TFIID binding to
DNA, 523
13
STUDYING CHROMATIN DYNAMICS IN VITRO: CHROMATIN ASSEMBLY,
REMODELING, AND TRANSCRIPTION
INTRODUCTION AND OVERVIEW,
539
EXPERIMENTAL STRATEGIES,
541
Strategies for assembling chromatin,
541
Box
13.1
Effects of
DNA
template and
reconstitution
method on positioning of nucleosomes within
arrays,
543
Source of histones,
546
Biochemical characterization of chromatin
reconstitutions,
549
Box
13.2
MNase analysis of nudeosomal arrays,
554
Box
13.3
EcoRI analysis of nudeosomal arrays,
556
Strategies to test the role of histone modifications in vitro,
557
Analysis of chromatin remodeling/modification enzymes,
561
In vitro transcription with chromatin templates,
569
TECHNIQUES,
573
Protocol
13.1
Chicken erythrocyte histone octamer preparation,
573
Protocol
13.2
Salt gradient dialysis
reconstitution
of nucleosomes,
583
Protocol
13.3
Reconstitution
of nudeosomal arrays using
recombinant
Drosophila
ACF and NAP1,
589
APPENDIX: CAUTIONS,
605
INDEX,
613 |
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| any_adam_object_boolean | 1 |
| author | Carey, Michael Peterson, Craig L. Smale, Stephen T. |
| author_facet | Carey, Michael Peterson, Craig L. Smale, Stephen T. |
| author_role | aut aut aut |
| author_sort | Carey, Michael |
| author_variant | m c mc c l p cl clp s t s st sts |
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| bvnumber | BV035188124 |
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| callnumber-search | QH450.2 |
| callnumber-sort | QH 3450.2 |
| callnumber-subject | QH - Natural History and Biology |
| classification_rvk | WG 1840 WG 1940 |
| ctrlnum | (OCoLC)249135580 (DE-599)BVBBV035188124 |
| dewey-full | 572.8/845 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.8/845 |
| dewey-search | 572.8/845 |
| dewey-sort | 3572.8 3845 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| discipline_str_mv | Biologie |
| edition | 2. ed. |
| format | Book |
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| id | DE-604.BV035188124 |
| illustrated | Illustrated |
| index_date | 2024-07-02T23:00:43Z |
| indexdate | 2024-07-09T21:27:01Z |
| institution | BVB |
| isbn | 9780879697624 9780879697778 |
| language | English |
| lccn | 2008039755 |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-016994783 |
| oclc_num | 249135580 |
| open_access_boolean | |
| owner | DE-703 DE-20 DE-19 DE-BY-UBM DE-526 |
| owner_facet | DE-703 DE-20 DE-19 DE-BY-UBM DE-526 |
| physical | XIX, 620 S. Ill., graph. Darst. |
| publishDate | 2009 |
| publishDateSearch | 2009 |
| publishDateSort | 2009 |
| publisher | Cold Spring Harbor Laboratory Press |
| record_format | marc |
| spelling | Carey, Michael Verfasser aut Transcriptional regulation in eukaryotes concepts, strategies, and techniques Michael F. Carey ; Craig L. Peterson ; Stephen T. Smale 2. ed. Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press 2009 XIX, 620 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Includes bibliographical references and index Genetic transcription Regulation Transcription factors Genetic transcription Regulation Research Methodology Transkription Genetik (DE-588)4185906-6 gnd rswk-swf Genregulation (DE-588)4122166-7 gnd rswk-swf Eukaryoten (DE-588)4070991-7 gnd rswk-swf Eukaryoten (DE-588)4070991-7 s Genregulation (DE-588)4122166-7 s DE-604 Transkription Genetik (DE-588)4185906-6 s Peterson, Craig L. Verfasser aut Smale, Stephen T. Verfasser aut Digitalisierung UB Bayreuth application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016994783&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Carey, Michael Peterson, Craig L. Smale, Stephen T. Transcriptional regulation in eukaryotes concepts, strategies, and techniques Genetic transcription Regulation Transcription factors Genetic transcription Regulation Research Methodology Transkription Genetik (DE-588)4185906-6 gnd Genregulation (DE-588)4122166-7 gnd Eukaryoten (DE-588)4070991-7 gnd |
| subject_GND | (DE-588)4185906-6 (DE-588)4122166-7 (DE-588)4070991-7 |
| title | Transcriptional regulation in eukaryotes concepts, strategies, and techniques |
| title_auth | Transcriptional regulation in eukaryotes concepts, strategies, and techniques |
| title_exact_search | Transcriptional regulation in eukaryotes concepts, strategies, and techniques |
| title_exact_search_txtP | Transcriptional regulation in eukaryotes concepts, strategies, and techniques |
| title_full | Transcriptional regulation in eukaryotes concepts, strategies, and techniques Michael F. Carey ; Craig L. Peterson ; Stephen T. Smale |
| title_fullStr | Transcriptional regulation in eukaryotes concepts, strategies, and techniques Michael F. Carey ; Craig L. Peterson ; Stephen T. Smale |
| title_full_unstemmed | Transcriptional regulation in eukaryotes concepts, strategies, and techniques Michael F. Carey ; Craig L. Peterson ; Stephen T. Smale |
| title_short | Transcriptional regulation in eukaryotes |
| title_sort | transcriptional regulation in eukaryotes concepts strategies and techniques |
| title_sub | concepts, strategies, and techniques |
| topic | Genetic transcription Regulation Transcription factors Genetic transcription Regulation Research Methodology Transkription Genetik (DE-588)4185906-6 gnd Genregulation (DE-588)4122166-7 gnd Eukaryoten (DE-588)4070991-7 gnd |
| topic_facet | Genetic transcription Regulation Transcription factors Genetic transcription Regulation Research Methodology Transkription Genetik Genregulation Eukaryoten |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016994783&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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