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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

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