TITLE PAGE
ACKNOWLEDGEMENT
CONTENTS
ABBREVIATION 9
ABSTRACT 11
Ⅰ. Introduction 12
Ⅱ. Materials and methods 14
Ⅱ. 1. Cell culture 14
Ⅱ. 2. Database screening 14
Ⅱ. 3. Isolation of full-length cDNA for hUBH1 14
Ⅱ. 4. Cloning of mUBH1 and hUBH1 15
Ⅱ. 5. Northern blot analysis 15
Ⅱ. 6. Site-directed mutagenesis 15
Ⅱ. 7. In vivo deubiquitinating enzyme activity assay 16
Ⅱ. 8. In vivo deneddylation, desumoylation, and de-ISG15 enzyme activity assays 16
Ⅱ. 9. Localizationan alysis and DAPI staining 17
Ⅲ. Results 18
Ⅲ. 1. Expression pattern analysis of mUBH1 and hUBH1 18
Ⅲ. 2. In vivo deubiquitinating enzyme activity 21
Ⅲ. 3. In vivo deneddylation, desumoylation and de-ISG15 enzyme activity 23
Ⅲ. 4. Localization of mUBH1 and hUBH1 at the nuclear envelope (NE)/endoplasmic reticulum (ER) 26
Ⅳ. Discussion 29
Ⅴ. Conclusions 31
References 32
Summary in Korean 37
Figure 1. Expression pattern analysis of mUBH1and hUBH1 by Northern blotting. (A) Expression pattern of mUBH1 in murine embryo stages (a) The 770 bp mUBH1 cDNA probe detects an approximately 4.5 kb transcript in each stage of embryonic development. (b) The 500 bp murine GAPDH cDNA probe was used as a control 19
Figure 2. In vivo deubiquitinating enzyme activity assay. Both mUBH1 and hUBH1 have deubiquitinating enzyme activity in vivo. HA-tagged ubiquitin was transiently expressed in NIH3T3 cells with or without myc-tagged mUBH1,ormyc-tagged hUBH1, and its mutant form myc-tagged mUBH (C33S), ormyc-tagged hUBH1(C33S), followed by immunoblot analysis of the cell extracts using an anti-HA antibody 22
Figure 3. In vivo deneddylating enzyme activity assay. Both mUBH1 and hUBH1 have deneddylating enzyme activity in vivo in a dose-dependent manner. (A) Myc-tagged NEDD8 was transiently expressed in NIH3T3 cels with or without myc-tagged mUBH1 and its mutant form myc-tagged mUBH1 (C33S) 24
Figure 4. Localization of mUBH1 in NIH3T3 cells and hUBH1 in HeLa cells and DAPI staining. (A) pEGFP-C1-mUBH1 was expressed at the nuclear envelope (NE)/endopalsmic reticiulum (ER) 27