酵母アッセイを用いたラットp53癌抑制遺伝子の転写スリップの解析 Analysis of Transcript Mutations Due to Transcriptional Slippage in Rat p53 Tumor Suppressor Gene with the Use of Yeast Functional Assay
Analysis of Transcript Mutations Due to Transcriptional Slippage in Rat p53 Tumor Suppressor Gene with the Use of Yeast Functional Assay
Transcriptional slippage was previously found in Escherichia coli during RNA elongation at runs of 10 or more As or Ts, resulting in the addition of untemplated A or U residues. To evaluate the incidence of transcriptional slippage in vivo, we employed a yeast functional assay, and analyzed the frequency and spectrum of mutations in mRNA of the tumor suppressor p53 in rat tissues. In this assay, yeast are transfected with p53 PCR products and a gapped p53 expression vector allowing homologous recombination in vivo and yielding a percentage of red colonies which refiects the proportion of mutant PCR products. Insertion mutations of single base of adenine (A) at stretches of 6As were frequently detected in the liver samples of LEC rats which develop spontaneous hepatitis and hepatocellular carcinoma. For excluding the possibility of artifacts, p53 cDNA was amplified by PCR from plasmids containing wild-type p53 and tested with the yeast functional assay, resulting in no A insertion after sequencing 23 mutant clones. Furthermore, in vitro transcript of wild-type p53 was synthesized by SP6 RNA polymerase, and then, reverse-transcribed, PCR-amplified, and tested with the yeast functional assay. The overall rate of A insertion was much lower than that in the LEC rat liver. Since A insertions were found predominantly at nucleotides 293-298 in exon 4, an exon 4-specific yeast functional assay was developed. A insertion was detected in 4.8% of the PCR product of mRNA but 0-0.1% from genomic DNA, which suggested that such A insertion was caused by transcriptional slippage in vivo. The A insertion rate was increased in acute hepatitis stage in the LEC rat liver, while the rate slowly increased by aging in control WKAH rat liver. It was suggested that cell damage and aging were primarily responsible for increased rate of transcriptional slippage.
Hokkaido University（北海道大学）. 博士(医学)