Dynamics of histone modifications [Elektronische Ressource] / Annette N. D. Scharf

Annette Scharf

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 25
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Naturwissenschaften an der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
Dynamics of histone modifications
Annette N. D. Scharf
aus
OffenburgEingereicht am 23. Juli 2009
Mündliche Prüfung am 24. November 2009
1. Gutachter: Prof. Dr. Peter Becker
2. Gutachter: Prof. Dr. Dirk Eick
3. Gutachter: Prof. Dr. Thomas Cremer
4. Gutachter: Prof. Dr. Michael Boshart
5. Gutachter: PD Dr. Stefan Müller
6. Gutachter: Prof. Dr. Charles David
7. SV: Prof. Dr. Axel ImhofEhrenwörtliche Versicherung
Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation von mir selbständig und
ohne unerlaubte Hilfe angefertigt ist.
München, den .......................... ..................................................................................
(Unterschift)Dedicated to my fatherTable of contents
Contents
A c kn o wledgm en ts ........................................................... 13
S umm a ry ................................................................... 14
Zusammenfassung ........................................................... 15
1. Introduction ............................................................. 17
1.1 Epigenetics .............................................................. 18
1.2 Chromatin 18
1.3 Histone modifications..................................................... 21
1.3.1 Acetylation 23
1.3.2 Methylation .......................................................... 24
1.3.2.1 H3K36 ........................................................... 25
1.3.2.2 H3K9 ............................................................ 26
1.3.2.3 H3K27 26
1.3.2.4 H4K20 27
1.3.3 Demethylation ........................................................ 28
1.4 Histone code 28
1.5 Replicating chromatin .................................................... 30
1.5.1 Disassembly of parental nucleosomes .................................... 30
1.5.2 Relocation of parental histones .......................................... 31
1.5.3 Deposition of newly synthesized histones ................................. 32
1.5.4 Chromatin maturation ................................................. 33
1.6 Objective ................................................................ 35
2. M a t e rials a nd M ethods .................................................... 37
2.1 Materials 38
2.1.1 Technical devices ...................................................... 38
2.1.2 Chemicals and consumables ............................................ 39
2.1.3 Kits and enzymes ..................................................... 41
2.1.4 Plasmids ............................................................. 41
2.1.5 Media ............................................................... 42
2.1.5.1 Media for E. coli ................................................... 42
2.1.5.2 Media for HeLa cells ................................................ 42
9Table of contents
2.1.6 Antibiotics ........................................................... 43
2.1.7 Antibodies 43
2.1.7.1 Primary antibodies ................................................. 43
2.1.7.2 Secondary antibodies ............................................... 43
2.1.8 E. coli strains ......................................................... 43
2.1.9 DNA and protein markers .............................................. 44
2.1.10 Protease inhibitors ................................................... 44
2.1.11 Mass spectrometry material ........................................... 44
2.1.12 Bioinformatic tool .................................................... 45
2.2 Methods ................................................................ 45
2.2.1 Microbiology methods ................................................. 45
2.2.1.1 Preparation of competent cells ....................................... 45
2.2.1.2 Plasmid transformation ............................................. 46
2.2.1.3 Isolation of Plasmid DNA from E. coli ................................ 46
2.2.2 Nucleic acid methods .................................................. 47
2.2.2.1 Storage of DNA .................................................... 47
2.2.2.2 DNA quantification ................................................ 47
2.2.2.3 Agarose gel electrophoresis .......................................... 48
2.2.2.4 Restriction digest 48
2.2.2.5 Polymerase Chain Reaction ......................................... 48
2.2.2.6 RT PCR .......................................................... 49
2.2.3 Tissue culture methods 49
2.2.3.1 Cultivation of HeLa cells ............................................ 49
2.2.3.2 Harvesting of HeLa cells 50
2.2.3.3 Storage of HeLa cells ............................................... 50
2.2.3.4 Synchronization of HeLa cells ........................................ 50
2.2.3.5 SILAC labeling .................................................... 51
2.2.3.6 Flow cytometric analysis of the cell cycle .............................. 51
2.2.4 Protein methods ...................................................... 52
2.2.4.1 Protein quantification .............................................. 52
2.2.4.2 SDS-Polyacrylamid-Gelelectrophoresis ............................... 52
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