Quantitative mutagenesis at the adenosine kinase locus in Chinese hamster ovary cells Development and characteristics of the selection system

Stable mutants exhibiting high degree of resistance (100-1000-fold) to various nucleoside analogs viz, toyocamycin, tubercidin, 6-methyl mercaptopurine riboside (6-MeMPR) and pyrazofurin, are obtained at similar frequency (congruent to 1 X 10(-4] in CHO cells. The mutants resistant to any of the above analogs exhibit similar degree of cross-resistance to the other three nucleoside analogs, and all of the mutants examined contained no measurable activity of the purine salvage pathway enzyme adenosine kinase (AK) which converts these analogs to their phosphorylated derivatives. These results indicate that very similar mutants are selected using any of these analogs. The recovery of AK- mutants in CHO cells is not affected by cell density (up to at least 5 X 10(5) cells per 100-mm diameter dish) and after treatment with mutagen(s) maximum mutagenic effect is observed after 7-8 days, which then remains unchanged for the next several days. Treatment of CHO cells with a number of mutagenic agents e.g. ethyl methanesulfonate, ICR170, ultraviolet light, and benzo[a]pyrene, led to a nearly linear concentration-dependent increase in the frequency of the AK- mutants in cultures. The mutagenic response of the AK locus to these agents compared favorably with that of the HGPRT locus (6-thioguanine resistance) within the same experiments. These results show that the selection system for AK- mutants provides an additional valuable genetic marker for quantitative mutagenesis studies in CHO cells.