TABLE OF CONTENT TABLE OF CONTENT INTRODUCTION . 1
1.1 Homeostasis . 11.2 The family of MYC genes. 1 1.3 The structure of the c-MYC protein and c-MYC isoforms . 2
1.3.1 The amino-terminal domain (NTD) of c-MYC. 4 1.3.2 The central region of c-MYC . 5 1.3.3 The carboxy-terminal domain (CTD) of c-MYC . 5
1.4 The function of c-MYC as a transcription factor . 6
1.4.1 Activation of transcription . 8 1.4.2 Repression
1.5.1 Proteins interacting with the NTD of c-MYC . 13 1.5.2 Central domain-interacting proteins. 16 1.5.3 Proteins associated with the CTD of c-MYC. 16 1.5.4 Proteins binding to multiple regions of c-MYC. 17
1.6 The biological activity of c-MYC . 18
1.6.1 Cell cycle progression . 20 1.6.2 Cell
1.6.3 Block of differentiation . 22 1.6.4 Apoptosis. 23
1.7 c-MYC and cancer . 23 1.8 Aim of the study. 27
MATERIALS. 28
2.1.1 Chemicals and reagents. 28 2.1.2 Enzymes . 30 2.1.3 Antibodies . 30
2.2.1 Oligonucleotides. 33 2.2.2 Expression
2.2.2.1 Standard expression plasmids . 34 2.2.2.2 Constructed
2.4 Stock solutions, buffers, and media . 38
METHODS . 42
3.1.2 Preparation of competent bacteria. 42 3.1.3 Transformation of competent bacteria . 42
TABLE OF CONTENT
3.2.1 Isolation of plasmid DNA from E. coli . 43 3.2.2 Enzymatic digestion of DNA . 43 3.2.3 Generation of DNA fragments for ligation . 43 3.2.4 Amplification of DNA by polymerase chain reaction . 44 3.2.5 Agarose gel electrophoresis and purification of DNA fragments . 44 3.2.6 DNA ligation and sequencing . 45
3.3.1 Cultivation of eukaryotic cell lines . 45 3.3.2 Cryopreservation
3.3.3 Transient and stable transfection of eukaryotic cells . 46 3.3.4 Expression of tetracycline regulated proteins . 47
3.4 Methods for protein purification and analyses. 47
3.4.1 Protein lysate preparation from eukaryotic cells. 47 3.4.2 Protein gel electrophoresis and Western blot analysis . 47 3.4.3 Tandem affinity purification . 48 3.4.4 Multidimensional protein identification technology . 50 3.4.5 Generation of antigen for anti-DBC-1 antibody production . 51 3.4.6 Antibody
3.4.7 Co-immunoprecipitation . 52 3.4.8 Indirect
3.5 Microscopy. 543.6 Bimolecular fluorescence complementation assay . 54 3.7 Cell-based reporter gene assay. 54 3.8 Yeast two-hybrid assay . 55
RESULTS. 56
4.1 Generation of the c-MYC-HA-TAP fusion. 56 4.2 Activity of the c-MYC-HA-TAP fusion protein . 57
4.2.1 Transactivation potential of c-MYC-HA-TAP . 57 4.2.2 Binding properties of c-MYC-HA-TAP . 58
4.3 Purification and identification of c-MYC-binding proteins. 59
4.3.1 Characterization of cell lines for tandem affinity purification . 59 4.3.2 Tandem affinity purification of proteins associated with c-MYC . 61 4.3.3 MudPIT identification of c-MYC-associated proteins. 62
4.4 Validation of identified proteins and their interaction with c-MYC . 68
4.4.1 Confirmation of c-MYC-associated proteins by Western blot analysis. 68 4.4.2 Co-localization of identified proteins with c-MYC . 69 4.4.3 Confirmation of c-MYC-interacting proteins by co-immunoprecipitation. 70 4.4.4 Validation of selected interactions by yeast two-hybrid assay . 72 4.4.5 Identification of binding regions for interaction partners within c-MYC . 72 4.4.6 Competition of DBC-1 and MCM7 with MAX for binding to c-MYC. 74
4.5.1 Generation of anti-DBC-1 antibodies . 76 4.5.2 Characterization of a NLS in the DBC-1 protein . 77 4.5.3 Mapping of the binding domain for c-MYC within DBC-1 . 79 4.5.4 Protein level of DBC-1 in senescent fibroblasts . 81
TABLE OF CONTENT DISCUSSION . 83
5.1 The tandem affinity purification method . 83 5.2 Identification of proteins associated with the c-MYC oncoprotein . 84
5.2.6 Metabolism. 94 5.2.7 Protein modification and degradation . 94
5.3 Identification of DBC-1 as a potential co-factor for c-MYC . 95 5.4 Conclusions and perspectives. 97
SUMMARY. 98 ABBREVIATIONS . 99 APPENDIX . 103 REFERENCES . 113 10 PUBLICATION LIST. 128 11 ACKNOWLEDGEMENT . 129 12 CURRICULUM VITAE . 130
Simon Thorley QC Practice Area Simon Thorley has an extensive intellectual property practice, particularly in the field of chemical and biotechnology patents but extending to cover all other patent matters, passing off, infringement of trade marks, copyright and designs and breach of confidence. He also advises on EEC law relating to intellectual property and in technical commercial di
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