Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells.
AS52 cells are Chinese hamster ovary (CHO) cells that carry a single functional copy of the bacterial gpt gene and allow the isolation of 6-thioguanine-resistant (6TGr)mutants arising from mutation at the chromosally integrated gpt locus. The gpt locus in AS52 cells is extremely stable, giving rise to 6TGr mutants at frequencies comparable to the endogenous CHO hprt locus. In this study, we describe the spectrum of spontaneous mutations observed in AS52 cells by Southern blot and DNA sequence analyses. Using the polymerase chain reaction (PCR) and the Thermus aquaticus (Taq) polymerase, we have enzymatically amplified 6TGr mutant gpt sequences in vitro. The PCR product was then sequenced without further cloning manipulations to directly identify gpt structural gene mutations. Deletions predominant among the 62 spontaneous 6TGr-AS52 mutant clones analyzed in this study. Of these, 79% (49/62) of the mutations were identified as deletions either by Southern blotting, PCR amplification or DNA sequence analysis. Among these deletions is a predominant 3-base deletion that was observed in 31% (19/62) of the mutants. These data provide a basis for future comparisons of induced point mutational spectra derived in the AS52 cell line, and demonstrate the utility of PCR in the generation of DNA sequence spectra derived from chromosomally integrated mammalian loci.
Tindall
K R
KR
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Stankowski
L F
LF
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005784:Gene Amplification; D007041:Hypoxanthine Phosphoribosyltransferase; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation; D010430:Pentosyltransferases / Q000235:genetics*; D013866:Thioguanine / Q000633:toxicity
D010430:Pentosyltransferases; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine