Analysis of developmental epistasis by chromatin immunoprecipitation in Xenopus laevis [Elektronische Ressource] / vorgelegt von Katrin Mansperger

Katrin Mansperger

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Analysis of Developmental Epistasis
by Chromatin Immunoprecipitation in
Xenopus laevis










Dissertation zur Erlangung der Doktorwürde des Dr. rer. nat.
an der Fakultät für Biologie der
Ludwig-Maximilians-Universität München










vorgelegt von

Katrin Mansperger

München 2007










Erklärung und ehrenwörtliche Versicherung:

Ich versichere, dass ich die vorliegende Arbeit selbstständig durchgeführt habe
und keine anderen als die aufgeführten Hilfsmittel und Quellen benutzt habe.

Hiermit erkläre ich, dass ich mich einer Doktorprüfung anderweitig ohne Erfolg
nicht unterzogen habe.

























































Mündliche Prüfung ablegt am 25. Juli 2007

1. Gutachter: Prof. Dr. Peter Becker

2. Gutachter: Prof. Dr. Heinrich Leonhardt





für meine Eltern

































„The final aim of all love intrigues, be they comic or tragic, is really of more
importance than all other ends in human life. What it turns upon is nothing less
than the composition of the next generation.“
Arthur Schopenhauer, quoted by Charles Darwin

































This work gave rise to the following publication:


Linder, B., Mentele, E., Mansperger, K., Straub, T., Kremmer, E. and Rupp, R.A.
(2007) CHD4/Mi-2beta activity is required for the positioning of the
mesoderm/neuroectoderm boundary in Xenopus. Genes Dev, 21, 973-983. Table of Contents I
Table of Contents
1 Summary..........................................................................1
2 Introduction .....................................................................3
2.1 The live cycle of the African clawed frog Xenopus laevis................... 3
2.2 Determination signals and induction events in Xenopus laevis......... 4
2.2.1 Transcriptional regulation of the muscle determination factor MyoD....................6
2.2.2 Canonical Wnt/β-catenin signaling during embryonic development.....................8
2.2.3 Distinct regulatory input of the SNF2-like chromatin remodeling
ATPase CHD4 ........................................................................................................12
2.3 Epigenetics - from genotype to phenotype ........................................ 13
2.4 Chromatin ............................................................................................. 15
2.4.1 Structural features of chromatin ............................................................................15
2.4.1.1 The nucleosome............................................................................................16
2.4.1.2 The 30nm fiber ..............................................................................................16
2.4.1.3 Higher order chromatin structure .................................................................17
2.4.2 ATP-dependent chromatin remodeling .................................................................18
2.4.2.1 SWI/SNF-containing chromatin remodeling complexes.............................19
2.4.2.2 CHD class of remodelers..............................................................................20
2.4.3 Covalent, post-translational histone modifications...............................................21
2.4.3.1 Acetylation .....................................................................................................23
2.4.3.2 Methylation.....................................................................................................24
2.4.3.3 Other modifications .......................................................................................27
2.5 Chromatin immunoprecipitation (ChIP).............................................. 27
2.6 Objectives............................................................................................. 30
3 Materials and Methods .................................................31
3.1 Reagents............................................................................................... 31
3.1.1 Fine chemicals........................................................................................................31
3.1.2 Enzymes and proteins............................................................................................31
3.2 Laboratory Equipment ......................................................................... 31
3.3 Antibodies ............................................................................................ 32
3.3.1 Primary Antibodies .................................................................................................32
3.3.1.1 Primary Antibodies, commercially available or published ..........................32
3.3.1.2 Rat monoclonal antibodies ...........................................................................32
3.3.2 Secondary Antibodies ............................................................................................34 Table of Contents II
3.3.2.1 Immunocytochemistry ...................................................................................34
3.3.2.2 Immunofluorescence.....................................................................................34
3.3.2.3 In Situ Hybridization ......................................................................................34
3.3.2.4 Western Blot analysis ...................................................................................34
3.4 Nucleic acids ........................................................................................ 34
3.4.1 Solutions..................................................................................................................34
3.4.2 Size standard..........................................................................................................34
3.4.3 Oligonucleotides .....................................................................................................35
3.4.3.1 Oligonucleotides for RT-PCR .......................................................................35
3.4.3.2 Oligonucleotides for cloning .........................................................................35
3.4.3.3 Oligonucleotides for real-time PCR..............................................................36
3.4.4 Plasmids..................................................................................................................38
3.4.4.1 Vectors for cloning.........................................................................................38
3.4.4.2 Plasmids for in vitro transcription .................................................................39
3.4.4.3 Plasmids for dig-labeled RNA in situ hybridization probes.........................40
3.4.4.4 Plasmids for recombinant GST-Fusion-Proteins.........................................40
3.4.4.5 Plasmids for real-time PCR tests .................................................................40
3.4.5 Handling of bacteria ...............................................................................................40
3.4.6 Bacteria strains .......................................................................................................40
3.5 Molecular biological methods ............................................................. 40
3.5.1 Solutions..................................................................................................................40
3.5.2 Isolation of nucleic acids ........................................................................................41
3.5.2.1 Mini-preparation with Qiagen kit...................................................................41
3.5.2.2 Isolation of RNA.............................................................................................42
3.5.3 Analysis and manipulation of nucleic acids ..........................................................42
3.5.3.1 Cloning methods ...........................................................................................42
3.5.3.2 Gel electrophoresis of nucleic acids ............................................................42
3.5.3.3 Isolation of DNA fragments from agarose gel .............................................42
3.5.4 Polymerase chain reaction (PCR).........................................................................42
3.5.4.1 PCR amplification of DNA fragments for cloning ..............................