[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, which allow homologous recombination in vivo and yield a percentage of red colonies which reflects the proportion of mutant PCR products. Insertion mutations of single base of adenine (A) at stretches of 6 As were frequently detected in the liver samples of LEC rats which develop spontaneous hepatitis and hepatocellular carcinoma. For excluding the possibility of artifacts involvement, p53 cDNA was amplified by PCR from plasmids containing wild-type p53 and tested with the yeast functional assay, which resulted 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 abruptly 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 the increased rate of transcriptional slippage.
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Division of Cell Biology, Hokkaido University School of Medicine, Sapporo, Japan.
D000225:Adenine; D000375:Aging / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D004247:DNA; D004252:DNA Mutational Analysis / Q000379:methods*; D016158:Genes, p53 / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D012313:RNA; D051381:Rats; D020332:Rats, Inbred LEC; D017208:Rats, Wistar; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*
D012313:RNA; D004247:DNA; D000225:Adenine