Pk. Liu et al., SPECTRUM OF SPONTANEOUS MUTATION IN ANIMAL-CELLS CONTAINING AN APHIDICOLIN-RESISTANT DNA POLYMERASE-ALPHA, MUTATION RESEARCH, 288(2), 1993, pp. 229-236
We have used a polymerase chain reaction (PCR)-based exon screening as
say to determine the spectrum of spontaneous hypoxanthine phosphoribos
yltransferase (hprt) gene mutations occurring in an aphidicolin-resist
ant V79 Chinese hamster cell line (designated Aph(r)-4-2) that contain
s a mutant DNA polymerase-alpha and displays a spontaneous mutator phe
notype. PCR analyses of 71 independent, 6-thioguanine ( TG)-resistant
sublines isolated from Aph(r)-4-2 or parental V79-743X cells using hpr
t exon 3- and exon 9-specific oligonucleotide primer pairs revealed th
e loss of exon 3 or 9 from 6 of 60 Aph(r)-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 Aph(r)-4-2-derived mutants, while none of the 11 V7
9-derived mutants had lost both exons. The results of these PCR-screen
ing assays were further corroborated by Southern and Northern blot hyb
ridization analyses of 28 mutants: 22 of 28 mutants contained an intac
t hprt gene by Southern analysis; of these 22 mutants 6 of 11 Aph(r)-4
-2-derived mutants contained either reduced or undetectable steady sta
te mRNA levels in contrast to all 11 V79-derived mutants that containe
d normal amounts of a normal-sized hprt mRNA. The results of our PCR a
nd blot hybridization analyses indicate that the rates of base substit
ution and deletion mutagenesis are elevated in Aph(r)-4-2 cells, and s
uggest that DNA polymerase-alpha may play a role in determining the ra
te of different molecular types of spontaneous mutations in vivo.