Functional characterization of promoter polymorphisms in the human cytochrome P450 2B6 gene (CYP2B6) [Elektronische Ressource] = Funktionelle Charakterisierung von Promotorpolymorphismen im humanen Cytochrom-P450-2B6-Gen (CYP2B6) / vorgelegt von Jörg Zukunft

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2005
• Nombre de lectures: 36
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Functional characterization of promoter
polymorphisms in the human
Cytochrome P450 2B6 gene (CYP2B6)

Funktionelle Charakterisierung von
Promotorpolymorphismen im humanen
Cytochrom P450 2B6-Gen (CYP2B6)




DISSERTATION


der Fakultät für Chemie und Pharmazie
der Eberhard-Karls-Universität Tübingen
zur Erlangung des Grades eines Doktors
der Naturwissenschaften

2005
vorgelegt von

Jörg Zukunft















Teile der Arbeit wurden bereits veröffentlicht in:

Zukunft J et al. (2005) A natural CYP2B6 TATA box polymorphism (-82T->C)
leading to enhanced transcription and relocation of the transcriptional start site.
Mol Pharmacol 67: 1772-1782.














Tag der mündlichen Prüfung: 3. Mai 2005

Dekan: Prof. Dr. S. Laufer
1. Berichterstatter: PD Dr. U. M. Zanger
2. Berichterstatter: Prof. Dr. K. W. Bock




“Musik ist Trumpf!
Musik ist Trumpf im Leben.”
(Hazy Osterwald)







Für Tatjana und Richard

Table of contents
TABLE OF CONTENTS
Table of contents....................................................................................................................... I
Abstract ....................................................................................................................................V
Zusammenfassung ................................................................................................................VII
Abbreviations......................................................................................................................... IX
I Introduction ......................................................................................................................1
1. The Cytochrome P450 enzymes.........................................................................................1
2. CYP2B6..............................................................................................................................3
2.1. Chromosomal localization and gene structure.............................................................3
2.2. Substrates.....................................................................................................................5
2.3. Expression ...................................................................................................................5
2.4. Regulation....................................................................................................................6
2.5. Pharmacogenetics of CYP2B6.....................................................................................8
3. Aims of this study...............................................................................................................9
II Materials and Methods ..................................................................................................10
1. DNA and liver samples ....................................................................................................10
2. PCR conditions.................................................................................................................10
3. Sequencing of double-stranded DNA...............................................................................10
4. Genotyping assays ............................................................................................................11
4.1. SNP -2320T>C ..........................................................................................................11
4.2. SNP c.1459C>T.........................................................................................................12
4.3. SNP g.24322C>T ......................................................................................................13
4.4. SNP -82T>C (DHPLC assay)....................................................................................13
4.4.1. Principle of DHPLC ...........................................................................................13
4.4.2. Genotyping assay................................................................................................14
5. Testing for deviation from Hardy-Weinberg Equilibrium ...............................................16
6. Reconstruction of haplotypes16
7. Construction of plasmids..................................................................................................16
7.1. Reporter gene vectors pGL3-2B6(-1641) and pGL3-2B6(-2033).............................16
7.2. Reporter gene vectors pGL3-2B6(-244)WT and -82T>C.........................................19
ITable of contents
7.3. Reporter gene vectors pGL3-2B6(-160)WT and -82T>C ........................................ 19
7.4. Expression plasmids pBJ5HNF1 α and pBJ5DCoH.................................................. 20
7.5. Expression plasmids pcDNA3-C/EBP β and pcDNAmHNF1 α ................................ 20
7.6. Expression plasmids pAC-LAP/LIP ......................................................................... 20
8. Cell culture and transient transfection of different cell types .......................................... 20
8.1. HepG2 cells............................................................................................................... 20
8.2. Primary human hepatocytes...................................................................................... 21
8.3. Primary rat hepatocytes............................................................................................. 22
9. RNA ligase-mediated rapid amplification of 5’-cDNA ends........................................... 22
10. Primer extension analysis............................................................................................... 25
10.1. Primer labeling........................................................................................................ 25
10.2. Primer hybridization and reverse transcription....................................................... 25
10.3. Electrophoresis and detection ................................................................................. 26
11. Electrophoretic mobility shift assay (EMSA)................................................................ 26
11.1. Annealing and labeling of oligonucleotides............................................................ 26
11.2. Preparation of nuclear cell extracts......................................................................... 27
11.3. In vitro translation................................................................................................... 28
11.4. Incubation and electrophoresis conditions.............................................................. 28
12. Quantitative real-time PCR............................................................................................ 29
12.1. Synthesis of cDNA ................................................................................................. 29
12.2. Conditions for RT-PCR .......................................................................................... 29
12.2.1. β-Actin ............................................................................................................. 30
12.2.2. CYP2B6 ........................................................................................................... 30
13. Gene copy number determination of CYP2B6............................................................... 30
13.1. TaqMan real-time PCR conditions ......................................................................... 30
13.2. Evaluation of specificity ......................................................................................... 31
13.3. Normalization and Quantification........................................................................... 32
III Results ......................................................................................................................... 33
1. Sequencing of the CYP2B6 promoter region ................................................................... 33
2. Genotyping SNP -2320T>C by RFLP ............................................................................. 34
3. Genotyping SNP c.1459C>T by RFLP............................................................................ 34
4. Haplotype structure of CYP2B6 ...................................................................................... 38
5. Promoter activity in different cell lines 39
II Table of contents
6. In silico analysis of the promoter .....................................................................................42
7. Electrophoretic mobility shift assays................................................................................43
7.1. C/EBP ........................................................................................................................43
7.2. TATA-box binding protein (TBP).............................................................................45
7.3. HNF1 .........................................................................................................................46
8. Transactivation of CY