[Establishment of mouse p53 yeast functional assay and evaluation of its detectability of p53 gene mutation].
Mice have widely been used as an experimental model for carcinogenesis induced by chemicals or irradiation. Recently, transgenic mice expressing oncogene proteins and knockout mice lacking for tumor suppressor genes are available, and used for the analysis of mechanisms underlying carcinogenesis. In such experimental carcinogenesis, a rapid and sensitive method for screening p53 mutations is desired. In human carcinogenesis, p53 yeast functional assay has been proved to be a very useful method for screening p53 mutations. However, the p53 yeast functional assay has been unsuccessful in mice because of the high background mutations in them. In the present study, the author developed a mouse p53 yeast functional assay by reducing the background mutations. Initially, 25.8 +/- 2.8% of background mutant red colonies were given by total RNA from normal mouse liver. The background level was lowered to 13.6 +/- 3.3% by improvement of RT-PCR conditions. Then the p53 cDNA-containing plasmids were rescued from the red colonies and the cDNA sequences were determined. The analysis revealed that many background mutations were caused by insertions of extra adenine (A) and thymine (T) at A and T homopolymeric runs, respectively. Majority of the insertion mutations occurred at 5' terminus of murine p53 cDNA. Based on these findings, we constructed a new vector and designed an optimal PCR primer set to exclude 5' terminal sequence in the yeast assay. Finally, the background mutation rate was reduced to 8.0 +/- 1.4%, which was comparable with the rate of 5.2 +/- 2.7% in human p53 yeast functional assay. Using the murine p53 yeast functional assay, we screened several cell lines for p53 mutations and determined those mutations by DNA sequencing. Furthermore, we investigated p53 mutations in UV-irradiated skins of XPC-gene-knockout mice. The yeast functional assay followed by DNA sequencing analysis revealed predominant mutations in dipyrimidines in the p53 coding sequence. These results indicate that mouse p53 yeast functional assay will be very useful for the analysis of p53 mutations in experimental carcinogenesis.
Tsunematsu
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I
Third Department of Internal Medicine, Hokkaido University School of Medicine, Sapporo, Japan.
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000032:analysis; D016158:Genes, p53 / Q000235:genetics*; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000166:cytology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000235:genetics; D014407:Tumor Cells, Cultured; D015003:Yeasts / Q000235:genetics
D018076:DNA, Complementary