We have continued our studies of point mutational changes in mammalian cells using the AS52 cell line. AS52 cells carry and express (using the SV4O early promotor) a single copy of the bacterial gpt gene stably integrated into the genome of hprt- deficient Chinese hamster ovary (CHO) cells. The gpt gene is analogous to hprt and one can readily isolate 6-thioguanine resistant (6TGr) mutant AS52 cells. We have begun a detailed characterization of spontaneous mutations in AS52 cells. In two separate experiments, independent spontaneous 6TGr AS52 mutants were characterized by Southern blot analysis. Both surveys indicate that approximately 60% of the spontaneous mutants are deletions or rearrangements of the gpt sequences. The remaining 40% show no alteration from the Southern blot pattern of the nonmutant parental AS52 cell line and have been designated putative gpt point mutations. We have simplified the process of AS52 mutant sequence analysis by using the polymerase chain reaction (PCR) technique to amplify spontaneous gpt mutant sequences in vitro using the Taq DNA polymerase. We have measured the replication fidelity of the Taq polymerase. This relatively error-prone enzyme yields an average of 1 base substitution per 9000 nucleotides synthesized and an average of 1 frameshift mutation per 40,000 nucleotides synthesized. In addition, we have developed techniques to allow for the direct DNA sequence analysis of the amplified PCR product, eliminating the effect of any mutations that occurred during PCR. Thus, any mutation observed must have been present in the initial mutant colony. Results thus for indicate that most spontaneous mutations are small deletions, although a limited number of base pair substitutions have been observed. Notably, there is a 3-base deletion hot spot in the gpt gene that has been observed in approximately 50% of the gpt mutants sequenced. We intend to continue to use PCR and direct DNA sequence analysis to generate mammalian point mutational spectra derived as a result of induced mutations in AS52 cells. The first induced spectrum to be analyzed will be point mutations occurring at lower treatment doses of the anti-tumor agent and mutagen, mitomycin C.
