Abstract

We have used a polymerase chain reaction (PCR)-based exon screening assay to determine the spectrum of spontaneous hypoxanthine phosphoribosyltransferase (hprt) gene mutations occurring in an aphidicolin-resistant V79 Chinese hamster cell line (designated Aphr-4-2) that contains a mutant DNA polymerase-alpha and displays a spontaneous mutator phenotype. PCR analyses of 71 independent, 6-thioguanine (TG)-resistant sublines isolated from Aphr-4-2 or parental V79-743X cells using hprt exon 3- and exon 9-specific oligonucleotide primer pairs revealed the loss of exon 3 or 9 from 6 of 60 Aphr-4-2 derived-, and from 1 of 11 parental V79-derived, TG-resistant mutants. Exons 3 and 9 were both lost from 5 of 60 Aphr-4-2-derived mutants, while none of the 11 V79-derived mutants had lost both exons. The results of these PCR-screening assays were further corroborated by Southern and Northern blot hybridization analyses of 28 mutants: 22 of 28 mutants contained an intact hprt gene by Southern analysis; of these 22 mutants 6 of 11 Aphr-4-2-derived mutants contained either reduced or undetectable steady state mRNA levels in contrast to all 11 V79-derived mutants that contained normal amounts of a normal-sized hprt mRNA. The results of our PCR and blot hybridization analyses indicate that the rates of base substitution and deletion mutagenesis are elevated in Aphr-4-2 cells, and suggest that DNA polymerase-alpha may play a role in determining the rate of different molecular types of spontaneous mutations in vivo.