Untersuchung und Charakterisierung des Hic2-Gens der Maus [Elektronische Ressource] / von Aleksandra Terzic

Terzic, Wasowicz Aleksandra

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Aus dem Institut für Entwicklungsgenetik
des GSF-Forschungszentrums für Umwelt und Gesundheit, GmbH
Direktor: Prof. Dr. Wolfgang Wurst


Anfertigung unter der Leitung von Prof. Dr. Jochen Graw


Vorgelegt über den Lehrstuhl für Molekulare Tierzucht und Biotechnologie
Der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München
Vorstand: Prof. Dr. Eckhard Wolf



Untersuchung und Charakterisierung des Hic2-Gens der Maus



Inaugural-Dissertation
Zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München

von

Aleksandra Terzic

aus Sarajevo/Bosnia und Herzegowina


München 2004 II




Aus dem Institut für Entwicklungsgenetik
des GSF-Forschungszentrums für Umwelt und Gesundheit, GmbH
Direktor: Prof. Dr. Wolfgang Wurst


Anfertigung unter der Leitung von Prof. Dr. Jochen Graw


Vorgelegt über den Lehrstuhl für Molekulare Tierzucht und Biotechnologie
Der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München
Vorstand: Prof. Dr. Eckhard Wolf




Analysis and characterisation of the mouse Hic2 gene

Inaugural-Dissertation
Zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München

von

Aleksandra Terzic

aus Sarajevo/Bosnia und Herzegowina


München 2004 III


Gedruckt mit Genehmigung der Tierärztlichen Fakultät der
Ludwig-Maximilians-Universität München












Dekan: Univ.-Prof. Dr. A.Stolle

Referent: Univ.-Prof. Dr. E. Wolf

Korreferent: Univ.-Prof. Dr. K. Heinritzi








Tag der Promotion: 13. Februar 2004

















IV
List of contents


1 INTRODUCTION.............................................................................................................1
1.1 Promoter CpG islands hypermethylation and gene expression…………………………1
1.2 POZ-Zincfinger Proteins……………………………………………………………….. 2
1.3 HIC1 gene, human HIC2 homologue……………………………………………………3
1.4 Hic1 a mouse gene……………………………………………………………………….4
1.5 HIC2 a human homologue of the HIC1 gene……………………………………………5
1.6 γFBP the avian homologue of the HIC1 and HIC2 gene…………………………………6
1.7 HIC1 and the Miller-Dieker syndrome…………………………………………………...7
1.8 Gene map locus 22q11.21………………………………………………………………...7
1.9 Tumor suppressor gene…………………………………………………………………...8
1.10 Aim of the work…………………………………………………………………………11

2 MATHERIALS AND METHODS……………………………………………………….12
2.1 Matherials………………………………………………………………………………..12
2.1.1Tools……………………………………………………………………………………..12
2.2 Cell culture material………………………………………………………………………13
2.3 Bacterial strains…………………………………………………………………………...14
2.4 Chemicals for cell culture………………………………………………………………...14
2.5 Plasmids…………………………………………………………………………………..15
2.6 Enzymes…………………………………………………………………………………..15
2.7 Solutions for cultivating of E.coli………………………………………………………...16
2.8 Solutions for agar electrophoresis………………………………………………………...16
2.9 Solutions for Acrylamide electrophoresis………………………………………………...17
2.10 Compositions of FA gel buffers…………………………………………………………17
2.11 Solutions for transfection………………………………………………………………..18
2.12 Other buffers…………………………………………………………………………….18
2.13 Solutions for Nortehrn Blot……………………………………………………………...19
2.14 Buffers for Midi DNA isolation…………………………………………………………20
2.15 Solutions for genomic DNA isolation…………………………………………………...20
2.16 Chemicals for RNA probes……………………………………………………………...21
2.17 Solutions for in situ hybridisation……………………………………………………….21
2.18 Primers list ……………………………………………………………………………...24 V

2.2 Methods…………………………………………………………………………………..26
2.2.1 PCR……………………………………………………………………………………..26
2.2.2 Gel Electrophoresis……………………………………………………………………..28
2.2.3 Computer Sequence analysis…………………………………………………………...29
2.2.4 Nucleic acids isolation………………………………………………………………….31
2.2.5 RNA isolation…………………………………………………………………………..32
2.2.6 In situ hybridisation..…………………………………………………………………...35
2.2.6.1 In situ hybridisation on paraffin sections……………………………………………..35
2.2.6.2 Whole mount In situ hybridisation……………………………………………………40
2.2.6.3 Staging of embryos…………………………………………………………………...41
2.2.7 Northern Blot…………………………………………………………………………...42
2.2.8 Promoter analysis……………………………………………………………………….43

3 RESULTS………………………………………………………………………………….49
3.1 Isolation of the mouse Hic2 gene…………………………………………………………49
3.2 Genomic organisation of the Hic2 locus………………………………………………….51
3.3 Deduced amino acid sequence and domain structure…………………………………….58
3.4 Characterisation of the Hic2 promoter……………………………………………………63
3.4.1 Binding sites and deletions constructs………………………………………………….63
3.4.2 Hic2 promoter activity………………………………………………………………….66
3.5 Expression of the mouse Hic2 mRNA……………………………………………………69
3.5.1 WMISH Whole mount in situ hybridisation……………………………………………69
3.5.2 Hic2 is expressed in central and peripheral nervous system……………………………70
3.5.3 Hic2 is expressed in the limbic region of the brain……………………………………..72
3.5.4 Hic2 is expressed in the embryonic
ectoderm…………………………………………..74
3.5.5 Hic2 is expressed in small intestine…………………………………………………….76
3.5.6 Hic2 is not expressed in eye……………………………………………………………77

4 DISCUSSION……………………………………………………………………………..81
4.1 Hic2 promoter and its binding sites………………………………………………………81
4.2 Conserved domains……………………………………………………………………….84
4.2.1 Hic2 BTB/POZ domain………………………………………………………………...84 VI
4.2.2 GLDLSKK/R polypeptide……………………………………………………………...85
4.2.3 Hic2 zinc finger domain………………………………………………………………85
4.3 Comparasion of the Hic2 and Hic1 expression patterns………………………………..87
4.4 Hic2 and its avian homolog γFBP………………………………………………………88
4.5 Hic2 exonic CpG islands………………………………………………………………..88
4.6 Hic2 a candidate for tumor suppressor gene...................................................................89

5 SUMMARY………………………………………………………………………………92
5 ZUSAMMENFASSUNG…………………………………………………………………93
6 REFERENCES……………………………………………………………………………95
7 ACKNOWLEDGEMENTS……………………………………………………………...104
8 CURRICULUM VITAE…………………………………………………………………105
9 APENDIX………………………………………………………………………………...106


























VII

























I dedicate this work to my parents






















1
1. Introduction

1.1 Promoter CpG island hypermethylation and gene expression

Approximately 50% of mammalian gene promoters are associated with one or more CpG
islands (Ioshikhes and Zhang, 2000).
Clusters of CpG dinucleotides or “CpG islands” (Gardiner-Garden M. and Frommer M.,
1987) are present in the promoter and exonic regions of approximately to 50% of mammalian
genes.

By contrast, other regions of the mammalian genome contain few CpG dinucleotides and
these are largely methylated (Larsen et al., 1992). The decreased occurrence of CpG is best
explained by the fact that methylated cytosines are mutational hotspots leading to CpG
depletion (Coulondre et al., 1978). A large number of experiments have shown that
methylation of promoter CpG islands plays an important role in gene silencing (Bird, 2002),
genomic imprinting, X-chromosome inactivation, the silencing of intragenomic parasites, and
carcinogenesis (Baylin et al., 1998).

The extent of aberrant promoter hypermethylation and its association with loss of gene
function in cancer suggests that CpG island methylation is an important mechanism in
inactivating tumor suppressor genes (TSGs) (Narayan et al., 2003).

For tumor suppressor genes, promoter hypermethylation is very important and can change
alone ever designate true tumor suppressor gene function (Baylin at al., 2001). Aberrant
promoter methylation changes that occur in cancer are associated with transcriptional
repression and loss of function of the gene by interrupting the binding of proteins involved in
transcription activator complex (Jones and Baylin, 2002). There are a lot of the genes which
are affected by promoter CpG island methylation in aging and / or cancer such as BRCA1,
HIC1, MYOD1, p57, PAX6, WT1.



2
1.2 POZ-Zincfinger Proteins

The C H zinc finger domain defines a family of genes that has bee