LINC, a novel protein complex involved in the regulation of G2-M genes [Elektronische Ressource] / vorgelegt von Fabienne Schmit

Fabienne Broders

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

LINC, a novel protein complex involved in the
regulation of G2/M genes






Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg






vorgelegt von
Fabienne Schmit

aus Mamer (Luxemburg)




Würzburg, 2008




































Eingereicht am:
…………………………………………………………………………………………


Mitglieder der Promotionskommission:

Vorsitzender:

1. Gutachter: Prof. Dr. Stefan Gaubatz

2. Gutachter: Prof. Dr. Georg Krohne


Tag des Promotionskolloquiums:
.............................................................................................



Doktorurkunde ausgehändigt am:
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CONTENT
1 Introduction .........................................................................................1
1.1 Pocket proteins and E2F transcription factors in cell cycle progression.. 1
1.1.1 Retinoblastoma protein pRB and the pocket protein family ........................ 1
1.1.2 E2F transcription factors............................................................................. 2
1.1.3 Mechanism of gene repression by pocket proteins..................................... 3
1.1.4 Regulation of the G2/M transition by E2F................................................... 5
1.2 Rb/E2F complexes in model organisms ....................................................... 5
1.2.1 The DRM complex in C. elegans is composed of synMuv class B proteins 6
1.2.2 dREAM/MybMuvB complexes in Drosophila melanogaster........................ 7
1.3 A human synMuv-like complex?.................................................................... 9
1.3.1 B-MYB ...................................................................................................... 10
1.3.2 The human LIN-9...................................................................................... 10
1.3.2.1 LIN-9 in transformation ...................................................................... 10
1.3.2.2 LIN-9 and its role in cell cycle progression......................................... 11
1.3.2.3 LIN-9 target genes ............................................................................. 11
1.3.3 Other human homologues of the dREAM complex................................... 13
1.4 Aim of this project......................................................................................... 14
2 Materials and methods .....................................................................15
2.1 Materials ........................................................................................................ 15
2.1.1 Chemical Stocks....................................................................................... 15
2.1.2 Buffers...................................................................................................... 16
2.1.2.1 General Buffers.................................................................................. 16
2.1.2.2 Buffers for lysates and nuclear extracts ............................................. 18
2.1.2.3 Buffers for immunoprecipitation and immunoblot............................... 19
2.1.2.4 Buffers for chromatin immunoprecipitation......................................... 20
2.1.2.5 Buffers for gelshift.............................................................................. 21
2.1.2.6 Buffers for GST pulldown................................................................... 21
2.1.3 Antibodies................................................................................................. 22
2.1.3.1 Primary antibodies ............................................................................. 22
I CONTENT
2.1.3.2 Secondary antibodies ........................................................................ 23
2.1.4 Plasmids................................................................................................... 24
2.1.4.1 Plasmids for overexpression.............................................................. 24
2.1.4.2 Plasmids for recombinant proteins..................................................... 25
2.1.4.3 Plasmids for knock-down ................................................................... 26
2.1.4.4 Plasmids for gelshift........................................................................... 26
2.1.5 Primers..................................................................................................... 27
2.1.5.1 Primers for cloning ............................................................................. 27
2.1.5.2 Primers for point mutagenesis ........................................................... 28
2.1.5.3 Primers for quantitative RT-PCR........................................................ 28
2.1.5.4 Primers for chromatin immunoprecipitation........................................ 29
2.1.5.5 Primers for gelshift competitions 29
2.1.6 shRNA sequences.................................................................................... 30
2.1.7 Cell lines and cell culture media ............................................................... 30
2.1.8 Enzymes................................................................................................... 30
2.1.9 Markers .................................................................................................... 31
2.1.10 Kits ......................................................................................................... 31
2.1.11 Beads ..................................................................................................... 31
2.2 Methods 31
2.2.1 Cell culture ............................................................................................... 31
2.2.1.1 Passageing of cells ............................................................................ 31
2.2.1.2 Transient transfection ........................................................................ 31
2.2.1.3 Infection of BJ-ET cells ...................................................................... 32
2.2.1.4 Growth curve...................................................................................... 32
2.2.1.5 Synchronization of T98G cells by serum starvation ........................... 32
2.2.1.6 Determination of cell cycle phases: flow cytometry............................ 32
2.2.1.7 Immunofluorescence.......................................................................... 33
2.2.2 Expression analysis.................................................................................. 34
2.2.2.1 RNA ................................................................................................... 34
2.2.2.2 Reverse transcription ......................................................................... 34
2.2.2.3 Quantitative PCR ............................................................................... 34
II CONTENT
2.2.3 Biochemical methods ............................................................................... 35
2.2.3.1 Whole cell lysates .............................................................................. 35
2.2.3.2 Nuclear extracts................................................................................. 35
2.2.3.3 Determination of protein concentration (Bradford) ............................. 36
2.2.3.4 Immunoprecipitation........................................................................... 36
2.2.3.5 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)...................... 36
2.2.3.6 Immunoblotting .................................................................................. 37
2.2.3.7 Affinity purification of polyclonal antisera ........................................... 37
2.2.3.8 Chromatin immunoprecipitation ......................................................... 38
2.2.3.9 Purification of GST-proteins from recombinant bacteria for antibody
production and GST-pulldowns...................................................................... 39
2.2.3.10 In-vitro translation ............................................................................ 39
2.2.3.11 GST-pulldown 40
2.2.4 Molecular biology...................................................................................... 40
2.2.4.1 Isolation of plasmid DNA from bacteria.............................................. 40
2.2.4.2 Isolation of plasmid DNA fragments from agarose gels ..................... 40
2.2.4.3 Site-directed mutagenesis ................................................................. 41
2.2.5 Gelshift ..................................................................................................... 42
2.2.5.1 Labeling of DNA fragments................................................................ 42
2.2.5.2 Purification of GST-proteins for gelshift analysis................................ 42
2.2.5.3 Preparation of DNA fragments for competition................................... 43
2.2.5.4