Mutation analysis in two newly identified rat p53 pseudogenes.

Abstract

We have analysed the genomic organization of the rat p53 gene in normal intestinal rat cells. Exons 5-8 of the p53 gene were amplified by PCR from rat genomic DNA using primers complementary to stretches of nucleotides identical in rat and human cDNAs. Two amplification products of 727 and 339 bp were obtained from the rat DNA. The PCR products were subcloned into M13mp18 and sequenced. By comparison with the rat cDNA sequence the 727 bp band was identified as the functional p53 gene containing exons 5, 6 7 and 8 and introns 5 and 7. The sequences corresponding to human intron 6 are absent from the rat p53. The second amplification product was a mixture of two different 'processed' pseudogenes, in which 42 mutations accumulated in a sequence corresponding to the cDNA between exons 5 and 8. Analysis of these mutations shows that in both pseudogenes the vast majority is constituted by base substitutions, with transitions being more frequent than transversions. The most prominent mutational class is formed by G-->A transitions which are predominantly located at CpG sites. Since a high level of homology is present between the rat and the human cDNA, the type and the positions where mutations occur in the two rat pseudogenes is discussed in relation to the possible origin of these mutations in human tumors.