hp66 protein paralogs [Elektronische Ressource] : SUMO modification and complex formation / vorgelegt von Zihua Gong

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2006
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

hp66 Protein Paralogs:
SUMO Modification and Complex Formation



Inaugural-Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenshaftlichen Fachbereiche
der Justus-Liebig-Universität Gießen
-Dr.rer.nat.-


vorgelegt von
Zihua Gong
aus China



Gießen, September 2006


Die vorliegende Arbeit wurde am Institut für Genetik des Fachbereiches 08 der
Justus-Liebig-Universität Gießen, in der Zeit von March 2003 bis September
2006 unter der Leitung von Prof. Dr. Rainer Renkawitz angefertigt.


PUBLICATIONS

Gong Z, Brackertz M, Renkawitz R.
SUMO modification enhances p66 mediated transcriptional repression of the Mi-
2/NuRD complex. Molecular and Cellular Biology, 2006, 26: 4519-28.

Brackertz M, Gong Z, Leers J, Renkawitz R.
p66 alpha and p66 beta of the Mi-2/NuRD complex mediated MBD2 and histone
interaction. Nucleic Acids Research, 2006, 34: 397-406.






1. Gutachter: Prof.Dr.Rainer Renkawitz
Institut für Genetik
Justus-Liebig-Universität Gießen

2. Gutachter: Prof.Dr. Ewald Beck
Institut für Biochemie
Universitätsklinikum Giessen und Marburg GmBH

3. Prüfer: Prof.Dr. Wolfgang Clauss
Institut für Tierphysiologie
Justus-Liebig-Universität Gießen

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Contents
Zusammenfassung................................................................................. vi
Summary ................................................................................................ vii
1 INTRODUCTION.................................................................................. 1
1.1 Molecular mechanisms of gene silencing………………………………………………. 1
1.1.1 Basic principles of gene expression ............................................................................ 1
1.1.2 DNA methylation.......................................................................................................... 1
1.1.3 DNA methyltransferases.............................................................................................. 1
1.1.4 DNA methylation and gene silencing ........................................................................... 2
1.1.5 Methyl-CpG binding proteins (MBPs) 3
1.1.5.1 MeCP2 .......................................................................................................................................4
1.1.5.2 MBD1.........................................................................................................................................7
1.1.5.3 MBD27
1.1.5.4 MBD3..............8
1.1.5.5 MBD49
1.1.5.6 Kaiso..............10
1.1.6 NuRD complex and MeCP1 complex......................................................................... 11
1.1.7 hp66 protein paralogs: hp66α and hp66β .................................................................. 13
1.2 SUMO: a history of protein modification………………………………………………15
1.2.1 The family of SUMO proteins..................................................................................... 15
1.2.2 The SUMOylation machinery 17
1.2.3 Functions of SUMO modification ................................................................................ 19
1.2.3.1 SUMO modification and nuclear localization ......................................................................20
1.2.3.2 SUon and ubiquitination...............................................................................21
1.2.3.3 SUMO modification and transcriptional regulation ...........................................................22
1.3 Aim of the project……………………………………………………………………….25
2 MATERIALS AND METHODS........................................................... 27
2.1 Materials………………………………………………………………………………...27
2.1.1 Equipment.................................................................................................................. 27
2.1.2 Consumables............................................................................................................. 28
ii
2.1.3 Chemicals and reagents ............................................................................................ 29
2.1.4 Enzymes, reaction buffers and enzyme inhibitors...................................................... 31
2.1.5 Molecular weight markers.......................................................................................... 32
2.1.6 Antibodies.................................................................................................................. 32
2.1.7 Kits............................................................................................................................. 35
2.1.8 E.coli strains .............................................................................................................. 35
2.1.9 Eukaryotic Cell lines................................................................................................... 35
2.1.10 Plasmids .................................................................................................................... 36
2.2 Methods………………………………………………………………………………….39
2.2.1 Working with Escherichia coli (E.coli) ........................................................................ 39
2.2.1.1 Preparation of competent bacterial cells: classical CaCl method .....................................39 2
2.2.1.2 Transformation of competent cells........................................................................................40
2.2.2 Working with DNA...................................................................................................... 41
2.2.2.1 Storage of DNA .......................................................................................................................41
2.2.2.2 Small-scale preparation of plasmid DNA (Mini-prep) ........................................................41
2.2.2.3 Large-scale preparation of plasmid DNA (Maxi-prep).......................................................42
2.2.2.4 Measurement of DNA concentration ....................................................................................43
2.2.2.5 Molecular cloning ...................................................................................................................44
2.2.2.5.1 Restriction endonuclease digestion.......................................................................................44
2.2.2.5.2 Filling-in of recessed 3’-termini of DNA with Klenow Fragment........................................44
2.2.2.5.3 Removing of 3’ and 5’ protruding ends with Mung Bean Nuclease .....................................44
2.2.2.5.4 Dephosphorylation of DNA ends..........................................................................................44
2.2.2.5.5 Phenol/chloroform extraction and ethanol precipitation of DNA .........................................45
2.2.2.5.6 PCR cloning..........................................................................................................................45
2.2.2.5.7 Agarose gel electrophoresis........46
2.2.2.5.8 Extraction of DNA fragments from agarose gel ...................................................................46
2.2.2.5.9 Ligation.................................................................................................................................47
2.2.2.6 Site-directed mutagenesis.......................................................................................................47
2.2.3 Working with RNA 48
2.2.3.1 Isolation of total RNA from mammalian cells......................................................................48
2.2.3.2 Measurement of RNA concentration ....................................................................................48
2.2.3.3 cDNA synthesis from total RNA by reverse transcription..................................................48
2.2.3.4 PCR amplification ..................................................................................................................49
2.2.4 Working with eukaryotic cells..................................................................................... 50
2.2.4.1 Cell culture..............................................................................................................................50
2.2.4.2. Freezing, thawing and storage of eukaryotic cells ...............................................................51
2.2.4.3. Transfection of DNA into mammalian cells .........................................................................51
2.2.4.3.1 Calcium phosphate transfection ............................................................................................51
2.2.4.3.2 jetPEI transfection................................................................................................................52
iii
2.2.4.3.3 Luciferase reporter assay.......................................................................................................52
2.2.4.4. Establishment of stably transfected HEK293 cell lines .......................................................53
2.2.4.5. Cellular fractionations ...........................................................................................................54
2.2.4.