MOLECULAR ANALYSIS OF POINT MUTATIONS IN CHINESE HAMSTER OVARY CELLS

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We use the Chinese hamster ovary (CHO) cell line (AS52) with a single copy of the bacterial gpt gene stably integrated into the genome to study point mutational changes in mammalian cells. Mutants arise as 6-thioguanine resistant (6TGr) colonies and mutant sequences are recovered using the polymerase chain reaction (PCR) followed by DNA sequence analysis to generate mutational spectra. We have identified a specific 3-base deletion represented in approximately 30% of the spontaneous AS52 mutants analyzed. However, in parallel studies at the gpt locus in E. coli, this 3-base deletion has not been observed. Differences between eukaryotic and prokaryotic DNA metabolism and/or chromosome structure may account for this striking difference in spectrum. Based on the specificity of this 3-base deletion in mammalian cells, the DNA sequences affected and the high frequency of this event, we suspect the involvement of eukaryotic DNA topoisomerase I. Experiments are underway using the topoisomerase I inhibitor, Camptothecin, as well as reversion analyses and targeted gene conversion in AS52 cells to evaluate the mechanistic basis of this deletion. In addition, we are generating comparative induced mutational spectra using the E. coli and AS52 cell systems. Evaluation of mutations induced by both UV-irradiation and H202 are currently in progress. Finally, we are in the process of modifying the AS52 cell system to yield an accelerated phenotypic expression time and, therefore, more rapid generation of independent mutants for DNA sequence analysis.