표제지
ACKNOWLEDGEMENT
목차
ABBREVIATION 10
ABSTRACT 11
I. Introduction 12
Ⅱ. Materials and methods 16
Ⅱ. 1. Cell culture and tissue specimens 16
Ⅱ. 2. Total RNA preparation and cDNA synthesis 16
Ⅱ. 3. Degenerate PCR 17
Ⅱ. 4. Cloning of vDUB1, vDUB2, vDUB3 and vDUB4 20
Ⅱ. 5. Northern blot analysis 20
Ⅱ. 6. Site-directed mutagenesis 21
Ⅱ. 7. In vitro deubiquitination assay 21
Ⅱ. 8. Mammalian transfection 22
Ⅱ. 9. In vivo deubiquitination assay 22
Ⅱ. 10. In vitro isopeptidase enzyme assay 23
Ⅲ. Results 25
Ⅲ. 1. Degenerate PCR 25
Ⅲ. 2. Cloning of vDUBs in human chorionic villi and various human cell lines 25
Ⅲ. 3. Expression analysis of vDUBs 30
1) Northern blot analysis of vDUBs 30
2) Expression of vDUB genes in various human cell lines detected by RT-PCR 32
Ⅲ. 4. In vivo and in vitro functional assays of vDUBs 34
1) In vitro deubiquitinating enzyme activity of vDUB1, vDUB2, vDUB3, and vDUB4 34
2) In vivo deubiquitinating enzyme assay of vDUB2 36
Ⅲ. 5. Isopeptidase enzyme assay of vDUB2 38
Ⅳ. Discussion 40
Ⅴ. Conclusions 43
References 44
Summary in Korean 49
Table 1. Primers used for degenerate PCR for the isolation of putative deubiquitinating enzymes 19
Table 2. The amino acids similarity between vDUBs and other murine DUB family 28
Figure 1. Several functions of deubiquitinating enzymes 13
Figure 2. Design for degenerate PCR primers 18
Figure 3. Alignment of amino acid sequences for vDUB1, vDUB2, vDUB3, vDUB4 and USP17 27
Figure 4. Conservation domains of vDUBs with some of the deubiquitinating enzyme family members 29
Figure 5. Northern analysis of vDUBs in human tissues 31
Figure 6. Expression of vDUBs in various human cell lines detected by RT-PCR 33
Figure 7. In vitro deubiquitinating enzyme activity of vDUBs 35
Figure 8. In vivo deubiquitination enzyme activity of vDUB2 37
Figure 9. In vitro isopeptidase assay of vDUB2 39