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Ⅰ. 서론 11
1. 1-Nitropyrene 11
2. Metabolism of 1-nitropyrene 11
3. Mutagenicity of 1-nitropyrene 12
4. 1-Nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide 13
Ⅱ. 실험재료 20
1. Chemicals and reagents 20
2. Equipments 20
3. Cell lines and cultures 21
4. Bacterial strains and plasmids 21
Ⅲ. 실험방법 22
1. Preparation of pSP189 shuttle vector 22
2. Treatment of pSP189 shuttle vector with 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in vitro 22
3. Transfection of bacterial pSP189 shuttle vector into NER-deficient XP-A (XP12BE) 23
4. Treatments of DpnⅠand RNase A to remove bacterial pSP189 shuttle vector and RNA 24
5. Preparation of electrocompetent cell 24
6. Electroporation of progeny pSP189 shuttle vector into electrocompetent MBM7070 25
7. Identification of mutant 26
8. Mutation spectrum 26
Ⅳ. 실험결과 27
1. Isolation of bacterial and progeny pSP189 shuttle vector from XP-A fibroblasts 27
2. Mutant frequencies induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide. 27
3. Mutation spectrum induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene. 29
Ⅴ. 토의 42
요약 51
Abstract 53
참고문헌 55
Table 1. Mutant frequencies induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide. 32
Table 2. Types and locations of mutations induced by 1% DMSO (control) in the supF gene of pSP189 shuttle vectors. 34
Table 3. Types and locations of mutations induced by 1-nitropyrene 4,5-oxide in the supF gene of pSP189 shuttle vectors. 35
Table 4. Types and locations of mutations induced by 1-nitropyrene 9,10-oxide in the supF gene of pSP189 shuttle vectors. 38
Table 5. Summary of types of mutations induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene of pSP189 shuttle vectors. 48
Figure 1. Metabolic pathways of 1-nitropyrene. 16
Figure 2. Structures of DNA adducts ( trans and cis) obtained from 1-nitropyrene 4,5-oxide with calf thymus DNA in vitro (Roy et al., 1991). 17
Figure 3. Suppressions of a nonsense mutation by an amber suppressor tRNA ( supF gene). 18
Figure 4. supF mutagenesis assay. 19
Figure 5. Comparisons of pSP189 shuttle vector before and after treatments with DpnⅠ and RNase A. The pSP189 shuttle vector was reacted with Dpn Ⅰ and RNase A for 30 min at 37℃ in the dark. Lane M: lamda/HindⅢ marker. Lane 1: bacterial pSP189 shuttle vector. Lane 2: pSP189 shuttle vector from XP-A. Lane 3-5: pSP189 shuttle vector for 1-nitropyrene 4,5-oxide from XP-A (2, 20, 200 μM, respectively). Lane 6-8: pSP189 shuttle vector for 1-nitropyrene 9,10-oxide from XP-A (2, 20, 200 μM, respectively). Lane 9: 0
Figure 6. Mutant frequencies induced by 1-nitropyrene 4,5-oxide (○) and 1-nitropyrene 9,10-oxide (●). 33
Figure 7. Comparisons of types of mutations induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene. The percentage of each type of mutations is indicated for 1-nitropyrene 4,5-oxide (open bars) and 1-nitropyrene 9,10-oxide (black bars). 49
Figure 8. Mutation spectrum of base substitutions induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene. The sequence shown represents the transcribed strand of supF tRAN gene sequence. The mutations for 1-nitropyrene 4,5-oxide are shown above, the mutation for 1-nitropyrene 9,10-oxide are shown below. Mutations were induced with treatment of 200 μM of each K-region oxides. Tandem mutations are underlined. 50
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