Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
|
| Schlagworte: | |
| Online-Zugang: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-265861 Inhaltsverzeichnis |
| Beschreibung: | 161 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae |c vorgelegt von Stephan Hamperl |
| 264 | 1 | |c 2012 | |
| 300 | |a 161 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
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| 650 | 0 | 7 | |a Chromatin |0 (DE-588)4010152-6 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Histone |0 (DE-588)4159992-5 |2 gnd |9 rswk-swf |
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Datensatz im Suchindex
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| adam_text | IMAGE 1
TABLE OF CONTENTS
1 SUMMARY 1
2 INTRODUCTION 3
2.1 CHROMATIN 3
2.1.1 THE NUCLEOSOME 3
2.1.2 POSTTRANSLATIONAL MODIFICATIONS OF HISTONES 4
2.1.2.1 ACETYLATION OF HISTONE LYSINE RESIDUES 6
2.1.2.2 METHYLATION OF HISTONE LYSINE AND ARGININE RESIDUES 7
2.1.2.3 OTHER HISTONE MODIFICATIONS 8
2.1.3 INCORPORATION OF HISTONE VARIANTS 9
2.1.4 THE POSITIONING OF NUCLEOSOMES 10
2.1.5 THE LINKER HISTONE H1 AND HIGHER ORDER STRUCTURES OF CHROMATIN 11
2.1.6 NON-HISTONE COMPONENTS OF CHROMATIN 12
2.2 COMPOSITIONAL AND STRUCTURAL ANALYSIS OF CHROMATIN 12
2.2.1 RECONSTITUTION OF CHROMATIN IN VITRO 13
2.2.2 ANALYSIS OF DNA-PROTEIN INTERACTIONS IN VIVO 14
2.2.2.1 CHROMATIN IMMUNOPRECIPITATION (CHIP) 14
2.2.2.2 DNA ADENINE METHYLTRANSFERASE IDENTIFICATION (DAMID) 15
2.2.2.3 CHROMATIN ENDOGENOUS CLEAVAGE (CHEC) 16
2.2.3 ISOLATION AND ANALYSIS OF IN VIVO ASSEMBLED CHROMATIN 16
2.2.3.1 ENRICHMENT OF CHROMOSOMAL REGIONS BY FRACTIONATION 16
2.2.3.2 PURIFICATION OF YEAST MINI CHROMOSOMES 17
2.2.3.3 PROTEOMICS OF ISOLATED CHROMATIN SEGMENTS 18
2.2.3.4 PURIFICATION OF DEFINED CHROMATIN DOMAINS BY SITE-SPECIFIC
RECOMBINATION.
19
2.3 CHROMATIN STRUCTURES AT THE ESSENTIAL MULTI-COPY RDNA LOCUS AND THE
NON-ESSENTIAL SINGLE-COPY PH05 LOCUS 21
2.3.1 CHROMATIN STRUCTURE AT THE YEAST RDNA LOCUS 21
2.3.1.1 CELLULAR LOCALIZATION AND CHROMOSOMAL ORGANIZATION OF THE
MULTI-COPY
YEAST RDNA LOCUS 22
HTTP://D-NB.INFO/1028664109
IMAGE 2
2.3.1.2 DISTINCT CHROMATIN STRUCTURES AT THE 35S RRNA GENES 25
2.3.1.3 CHROMATIN STRUCTURE AT THE INTERGENIC SPACER 29
2.3.2 CHROMATIN STRUCTURE AT THE YEAST PH05 LOCUS 31
2.4 OBJECTIVES 32
3 RESULTS 34
3.1 PURIFICATION OF DEFINED CHROMOSOMAL DOMAINS BY SITE-SPECIFIC
RECOMBINATION IN VIVO 34
3.1.1 ESTABLISHMENT OF YEAST STRAINS WITH A MODIFIED RDNA LOCUS
COMPETENT FOR
EXCISION OF DISTINCT RDNA CHROMATIN DOMAINS 35
3.1.1.1 STRATEGY FOR CHROMOSOMAL INTEGRATION AND EXPANSION OF
GENETICALLY
MODIFIED RDNA REPEATS 35
3.1.1.2 35S RRNA GENE CHROMATIN STATES ARE ESTABLISHED AFTER EXPANSION
OF THE
GENETICALLY MODIFIED RDNA REPEATS 37
3.1.2 ESTABLISHMENT OF A SINGLE STEP PURIFICATION TECHNIQUE FOR SELECTED
CHROMOSOMAL DOMAINS 40
3.1.2.1 SINGLE-STEP AFFINITY PURIFICATION WITH IGG COUPLED MAGNETIC
BEADS ALLOWS
EFFICIENT ENRICHMENT OF RDNA CHROMATIN DOMAINS 40
3.1.2.2 LEXA-TAP EXPRESSION LEVEL INFLUENCE THE SPECIFIC ENRICHMENT OF
THE
TARGETED DOMAINS 42
3.1.2.3 CHROMOSOMAL INTEGRATION OF THE R RECOMBINASE AND LEXA-TAP
EXPRESSION CASSETTE ALLOWS CELL GROWTH IN COMPLEX MEDIUM 44
3.1.2.4 DISTINCT DOMAINS OF THE RDNA LOCUS CAN BE PURIFIED FROM THE
YEAST
CHROMOSOME 45
3.2 COMPOSITIONAL ANALYSIS REVEALS DISTINCT PROTEOMES FOR INDIVIDUAL
RDNA
CHROMATIN DOMAINS 46
3.2.1 COVALENTLY MODIFIED HISTONES ARE SELECTIVELY ENRICHED IN
PURIFICATIONS OF
DISTINCT RDNA DOMAINS 46
3.2.2 SPECIFIC NON-HISTONE CHROMATIN COMPONENTS ARE SELECTIVELY ENRICHED
IN
PURIFICATIONS OF DISTINCT RDNA DOMAINS 50
3.2.3 COMPARATIVE MASS SPECTROMETRY REVEALS DISTINCT PROTEOMES FOR
INDIVIDUAL
RDNA CHROMATIN DOMAINS 52
3.2.3.1 STRATEGY FOR SEMIQUANTITATIVE COMPARATIVE ANALYSIS OF RDNA
CHROMATIN
COMPOSITION USING THE ITRAQ TECHNOLOGY 52
IMAGE 3
3.2.3.2 COMPARATIVE ANALYSIS OF PROTEINS CO-PURIFYING WITH LEXA-TAP FROM
STRAINS
WITH AND WITHOUT RECOMBINED RDNA CHROMATIN DOMAINS 53
3.3 SELECTED COMPLEXES AND FACTORS IDENTIFIED BY THE PROTEOME ANALYSIS
INTERACT WITH RDNA CHROMATIN IN VIVO 58
3.4 IMPORTANT STRUCTURAL AND CONFORMATIONAL CHROMATIN FEATURES OF
SPECIFIC
RDNA CHROMATIN DOMAINS ARE CONSERVED UPON ISOLATION 62
3.4.1 GEL FILTRATION ANALYSIS OF THE CIRCULAR 5S RDNA AND E-PRO REGION
SUGGESTS
STRUCTURAL DIFFERENCES 62
3.4.2 SINGLE MOLECULE ELECTRON MICROSCOPIC ANALYSIS OF 5S RRNA GENE
CIRCLES
SUGGESTS A HETEROGENEOUS POPULATION OF DIFFERENT CHROMATIN STATES 64
3.4.3 RESTRICTION ENDONUCLEASE ACCESSIBILITY ANALYSIS OF 5S RRNA GENE
CHROMATIN CONFIRMS THE RESULTS OF THE SINGLE MOLECULE APPROACH 67
3.5 CHROMATIN DOMAINS OF SINGLE COPY GENES CAN BE ENRICHED IN SUFFICIENT
AMOUNTS AND PURITY TO PERFORM MASS SPECTROMETRIC ANALYSIS 69
DISCUSSION 72
4.1 A SINGLE-STEP PURIFICATION STRATEGY ALLOWS ROBUST ENRICHMENT OF
NATIVE
RDNA CHROMATIN 72
4.1.1 YIELD AND SPECIFICITY OF RDNA CHROMATIN ISOLATION PROCEDURE
COMPARE WELL
WITH ALTERNATIVE CHROMATIN PURIFICATION STRATEGIES 72
4.1.2 POL I ASSOCIATED CHROMATIN PURIFIED AFTER FORMALDEHYDE
CROSSLINKING IN VIVO
SHOWS SIMILARITIES AND DIFFERENCES WHEN COMPARED TO NATIVE 35S RRNA GENE
CHROMATIN 75
4.2 THE PURIFICATION APPROACH ALLOWS THE UNBIASED IDENTIFICATION OF NEW
PROTEIN COMPONENTS OF CHROMATIN IN VIVO 78
4.3 THE NATIVE PURIFICATION STRATEGY IS COMPATIBLE WITH DOWNSTREAM
STRUCTURAL AND BIOCHEMICAL ANALYSIS OF THE ISOLATED MATERIAL 81
4.4 OUTLOOK 82
MATERIAL AND METHODS 83
5.1 MATERIAL 83
5.1.1 CHEMICALS 83
5.1.2 BUFFERS AND MEDIA 83
5.1.3 NUCLEIC ACIDS 88
5.1.4 ENZYMES AND POLYPEPTIDES 106
IMAGE 4
5.1.5 ANTIBODIES 107
5.1.6 ORGANISMS 107
5.1.7 EQUIPMENT 113
5.1.8 CONSUMABLES 114
5.1.9 SOFTWARE 115
5.2 METHODS 115
5.2.1 ENZYMATIC MANIPULATION OF DNA 115
5.2.2 PURIFICATION OF NUCLEIC ACIDS 116
5.2.3 QUANTITATIVE AND QUALITATIVE ANALYSIS OF NUCLEIC ACIDS 118
5.2.4 MANIPULATION OF ESCHERICHIA COLI 121
5.2.5 MANIPULATION OF SACCHARAMYCES CEREV/SAE 121
5.2.6 FORMALDEHYDE CROSSLINKING (FA-X) OF YEAST CULTURES 123
5.2.7 PREPARATION OF NUCLEI 124
5.2.8 CHROMATIN ENDOGENOUS CLEAVAGE (CHEC) 124
5.2.9 DNA WORKUP OF CHEC SAMPLES 125
5.2.10 RESTRICTION DIGEST AND AGAROSE GEL ELECTROPHORESIS OF CHEC
SAMPLES. 125
5.2.11 CHROMATIN IMMUNO PRECIPITATION (CHIP) 126
5.2.12 PURIFICATION OF SPECIFIC CHROMATIN CIRCLES FROM S. CEREVISIAE 127
5.2.13 ENDONUCLEASE DIGESTION ANALYSIS OF PURIFIED CHROMATIN DOMAINS 129
5.2.14 MICROCOCCUS NUCLEASE DIGESTION OF PURIFIED CHROMATIN DOMAINS 129
5.2.15 GEL FILTRATION CHROMATOGRAPHY OF CHROMATIN CIRCLES 130
5.2.16 PROTEIN-BIOCHEMICAL METHODS 130
5.2.17 ANALYSIS OF HISTONE MODIFICATIONS BY MALDI TOF/TOF MASS
SPECTROMETRY
134
5.2.18 COMPARATIVE ITRAQ MALDI TOF/TOF MASS SPECTROMETRY 135
6 REFERENCES 137
7 ABBREVIATIONS 158
8 PUBLICATIONS 160
9 ACKNOWLEDGEMENTS 161
|
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| spelling | Hamperl, Stephan Verfasser aut Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae vorgelegt von Stephan Hamperl 2012 161 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Regensburg, Univ., Diss., 2012 Saccharomyces cerevisiae (DE-588)4178812-6 gnd rswk-swf Chromatin (DE-588)4010152-6 gnd rswk-swf Histone (DE-588)4159992-5 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Saccharomyces cerevisiae (DE-588)4178812-6 s Chromatin (DE-588)4010152-6 s Histone (DE-588)4159992-5 s b DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:355-epub-265861 http://epub.uni-regensburg.de/26586/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-265861 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025367346&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Hamperl, Stephan Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae Saccharomyces cerevisiae (DE-588)4178812-6 gnd Chromatin (DE-588)4010152-6 gnd Histone (DE-588)4159992-5 gnd |
| subject_GND | (DE-588)4178812-6 (DE-588)4010152-6 (DE-588)4159992-5 (DE-588)4113937-9 |
| title | Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae |
| title_auth | Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae |
| title_exact_search | Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae |
| title_full | Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae vorgelegt von Stephan Hamperl |
| title_fullStr | Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae vorgelegt von Stephan Hamperl |
| title_full_unstemmed | Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae vorgelegt von Stephan Hamperl |
| title_short | Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae |
| title_sort | compositional and structural analysis of selected chromosomal domains from saccharomyces cerevisiae |
| topic | Saccharomyces cerevisiae (DE-588)4178812-6 gnd Chromatin (DE-588)4010152-6 gnd Histone (DE-588)4159992-5 gnd |
| topic_facet | Saccharomyces cerevisiae Chromatin Histone Hochschulschrift |
| url | http://epub.uni-regensburg.de/26586/ https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-265861 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025367346&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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