Molecular functional characterization of the human biglycan gene 5'-flanking region [Elektronische Ressource] = Molekular funktionelle Charakterisierung der 5'-flankierenden Region des humanen Biglykan Gen-Promotors / vorgelegt von Boris Schmitz

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• Biologie

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• Publié par: westfalische_wilhelms-universitat_munster
• Publié le: 01 janvier 2010
• Nombre de lectures: 28
• Poids de l'ouvrage: 7 Mo

Extrait

Biologie







Molecular functional characterization of the
human biglycan gene 5'-flanking region










Dipl. Biol. Boris Schmitz
-2010-




Molecular functional characterization of the
human biglycan gene 5'-flanking region


Molekular funktionelle Charakterisierung der 5'-flankierenden Region des
humanen Biglykan Gen-Promotors







Inaugural-Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften im Fachbereich Biologie
der Mathematisch-Naturwissenschaftlichen Fakultät
der Westfälischen Wilhelms-Universität Münster



vorgelegt von
Dip. Biol. Boris Schmitz
aus Remscheid
-2010-




































Dekan: Univ.-Prof. Dr. Christian Klämbt
Erster Gutachter: Univ.-Prof. Dr. Dirk Prüfer
Zweiter Gutachter: Univ.-Prof. Dr. Christian Klämbt
Datum der mündlichen Prüfung: 13.12.2010
Datum der Promotion: 17.12.2010

























The path is hard,
but will be amply rewarding.


Table of contents

TABLE OF CONTENTS
TABLE OF CONTENTS ............................................................................................................ I
LIST OF FIGURES ................................................................................................................... V
LIST OF TABLES .................................................................................................................. VII
ABREVIATIONS ................................................................................................................. VIII
1 INTRODUCTION ....................................................................................................... 1
1.1 Cardiovascular Disease (CVD) .......................................................................... 1
1.2 Arteriosclerosis ................................................................................................... 1
1.2.1 Pathophysiology of atherosclerosis ........................................................... 3
1.3 BGN as a candidate for CVD pathophysiology .................................................. 5
1.4 Polymorphic structure of the BGN gene ............................................................. 7
1.5 TGF-β1 cytokine signalling pathway ................................................................. 9
1.6 Gene expression control ................................................................................... 10
1.6.1 Transcriptional control ............................................................................ 11
1.6.2 The general transcription machinery ....................................................... 12
1.6.2.1 Different pathways for PIC assembly ....................................... 14
1.6.2.2 The sequential assembly pathway ............................................ 14
1.6.2.3 The Pol II holoenzyme pathway ............................................... 14
1.6.2.4 The TFIID complex .................................................................. 15
1.6.3 CpG islands promoters ............................................................................ 17
1.7 Linkage and association studies ....................................................................... 18
1.7.1 Family-based linkage analyses ................................................................ 18
1.7.2 Population-based association analyses .................................................... 18
1.8 Aim and design of the study ............................................................................. 19
2 MATERIAL ............................................................................................................... 21
2.1 Chemicals ......................................................................................................... 21
2.2 Other solutions and reagents ............................................................................. 22
I Table of contents

2.2.1 Sera and media ........................................................................................ 22
2.2.2 DNA ladder and protein marker .............................................................. 22
2.2.3 Enzymes and antibiotics .......................................................................... 22
2.2.4 Consumables and kits .............................................................................. 22
2.2.5 DNA-modifying enzymes ....................................................................... 23
2.2.6 Antibodies ............................................................................................... 25
2.2.7 Plasmids and vectors ............................................................................... 25
2.2.8 Bacteria (E. coli) ..................................................................................... 26
2.2.9 Eukaryotic cells ....................................................................................... 26
2.2.10 Laboratory equipment ............................................................................ 27
3 METHODS ................................................................................................................ 29
3.1 Molecular biological methods .......................................................................... 29
3.1.1 Preparation of genomic DNA .................................................................. 29
3.1.2 Preparation of total RNA ........................................................................ 29
3.1.3 Preparation of plasmid DNA ................................................................... 29
3.1.3.1 Quality and quantity control of nucleic acids ........................... 30
3.1.4 Polymerase Chain Reaction (PCR) ......................................................... 30
3.1.5 cDNA synthesis ....................................................................................... 32
3.1.6 5'RACE ................................................................................................... 32
3.1.7 DNA/RNA-modifying reactions ............................................................. 34
3.1.7.1 Hydrolysation with bacterial endonucleases ............................ 34
3.1.7.2 Dephosphorylation of DNA ..................................................... 34
3.1.7.3 Biotinylation of oligonucleotides for EMSA experiments ....... 34
3.1.8 Agarose gel electrophoresis .................................................................... 35
3.1.9 Site-directed mutagenesis ........................................................................ 35
3.1.10 Construction of reporter gene plasmids ......................................... 36
3.1.11 Purification of PCR products ......................................................... 39
3.1.12 Sequencing ..................................................................................... 40
3.1.13 EMSA ............................................................................................ 41
II Table of contents

3.1.14 ChIP ............................................................................................... 43
3.2 Protein biochemical methods ............................................................................ 45
3.2.1 Preparation of proteins ............................................................................ 45
3.2.2 Isolation of nuclear proteins .................................................................... 45
3.2.3 Protein quantification .............................................................................. 47
3.2.4 SDS-Polyacrylamide Gel Electrophoresis (PAGE) ................................ 47
3.2.5 Coomassie blue staining .......................................................................... 48
3.2.6 Western Blot (tank blot) .......................................................................... 48
3.3 Cell biological and microbiological methods ................................................... 49
3.3.1 Prokaryotic cells ...................................................................................... 49
3.3.1.1 Generation of chemically competent cells ............................... 50
3.3.1.2 Transformation of competent cells ........................................... 50
3.3.2 Eukaryotic cells ....................................................................................... 51
3.3.2.1 Eukaryotic cell culture .............................................................. 51
3.3.2.2 Storage ...................................................................................... 51
3.3.2.3 Transient transfection ............................................................... 52
3.3.2.4 Cotransfection ................................................