| Project/Area Number |
10470052
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Experimental pathology
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
MORIUCHI Tetsuya Hokkaido University, School of Medicine, Professor, 医学部, 教授 (20174394)
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| Co-Investigator(Kenkyū-buntansha) |
HAMADA Jun-ichi Hokkaido University, School of Medicine, Associate Professor, 医学部, 助教授 (50192703)
外木 秀文 北海道大学, 医学部, 助手 (50281803)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1998: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Keywords | LEC rat / p53 / slippage / hepatoma / polyadenine / 細胞障害 / slippage |
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| Research Abstract |
The LEC rat is a mutant strain in which hepatitis and hepatoma spontaneously develop at an extremely high incidence. Using a yeast-based assay which scores the ratio of mutants in p53 gene transcripts as red colonies, we detected frequent mutations in the liver of LEC rats. The majority of them (50-65%) were frameshift mutations caused by insertion of an extra adenine (A) and all the A insertions were found in the regions containing 6 consecutive adenines. Insertions of an extra adenine were found almost exclusively in the mRNA (cDNA), especlally in the (A)6 tract located in the most 5'-side (exon 4) among the three (A)6 tracts (exon 4, 7, and 8), and rarely in the clones of the corresponding genomic DNA part. Artifrcially synthesized p53 mRNA with the use of SP6 RNA polymerase gave insertions of an extra adenine in two clones in exon 7 and one in exon 8, but none in exon 4 out of 1 2 analyzed clones recovered from red colonies; overall accounting for 1.6% of the transcribed mRNA, indicating that the adenine insertion in exon4 (A)6 tract was an in vivo phenomenon rather than an artifact in reverse-transcription. The incidence of A insertion sharply increased up to about 20% in acute hepatitis stage, returned to control level in chronic hepatitis stage, and increased again slightly in neoplastic stage. The mutation rate correlated with serum AST level (r = 0.96, p<0.00 1) but not with contents of copper and 8-hydroxydeoxyguanosine in the liver of LEC rats. Ethanol treatment of cultured liver-derived cells also increased the rate of transcriptlonal slippage at the (A)6 tract, together suggesting that non-specific cellular damage is responsible to the increased ratios of the transcriptional mutations. These results suggest that cellular damage reduces transcriptional fidelity, thereby further enhancing the impairment of cellular functions.
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