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Language(s):
eng - English
Dates:
2004-10-20
Publication Status:
Accepted / In Press
Pages:
87 pp
Publishing info:
Berlin : Humboldt-Universität
Table of Contents:
1. Introduction 6
1.1. Organisation of chromatin 6
1.1.1. Chromatin assembly 7
1.1.2. Post-translational modification of histones 10
1.1.3. Silencing in S. cerevisiae 14
1.2. The centromere-kinetochore complex 18
1.2.1. The centromere in S. cerevisiae 20
1.2.2. The histone H3 variant Cse4 21
2. Materials and Methods 25
2.1. Material 25
2.1.1. Bacterial strains 25
2.1.2. Yeast strains 25
2.1.3. Plasmids 26
2.1.4. Media 27
2.1.5. Buffers and Solutions 28
2.1.6. Antibodies 29
2.1.7. Peptides 30
2.1.8. Primer 30
2.2. Methods 30
2.2.1. Molecular methods 30
2.2.1.1. Cell cultivation 30
2.2.1.2. Transformation of E. coli and S. cerevisiae 31
2.2.1.3. DNA isolation 31
2.2.1.4. Plasmid constructions 32
2.2.1.5. S. cerevisiae strain construction 32
2.2.1.6. Polymerase chain reaction 33
2.2.1.7. DNA sequencing 33
2.2.1.8. Two-hybrid system 34
2.2.1.8.1. β-galactosidase filter assay 35
2.2.1.8.2. HIS3 reporter assay 35
2.2.1.9. FACS – fluorescent activating cell sorting 35
2.2.2. Biochemical methods 36
2.2.2.1. Protein extract preparation 36
2.2.2.2. SDS-PAGE and immunoblotting 37
2.2.2.3. Detection methods for proteins 37
2.2.2.4. Concentration of protein solutions 37
2.2.2.5. Solo- and Co-immunoprecipitation 38
2.2.2.6. Bacterial expression of Cse4 38
2.2.2.7. Acetylation assay 39
3. Results 40
3.1. Interactions between Cse4, SAS-I and chromatin assembly
factors 40
3.2. Effect of mutations in SAS-I, CAF-I and Asf1 on centrome refunction 47
3.3. A SAS2-deletion abrogated the interaction between Cse4 and Ctf19 52
3.4. Does Sas2 acetylate the histone H3 variant Cse4 ? 53
4. Discussion 57
4.1. Cse4 interacts with the SAS-I complex and the chromatin
assembly factors Cac1 and Asf1 57
4.2. The histone acetyltransferase Sas2 has a function at the centromere 61
4.3. Cse4 exists in an acetylated state in the cell 63
4.4. A model for chromatin-assembly at the centromere 64
5. Literature 67
6. Figure index 86
Rev. Type:
-
Identifiers:
eDoc: 226389
Degree:
PhD
