Selection of mammalian thymidine auxotrophic cell mutants defective in thymidylate synthase by their reduced sensitivity to methotrexate.
Thymidine auxotrophic mutants were selectively isolated from mutagenized mouse FM3A cells by resistance to methotrexate in the presence of thymidine and 5-methyl-tetrahydrofolate with a frequency of 10(-5)-10(-6). In most of the thymidine auxotrophs the activity of thymidylate synthase was very low or undetectable, but dihydrofolate reductase activity was normal. Upon starvation of thymidine, the mutant cells immediately stopped growing and started to lyse within one day. In the presence of thymidine, the mutant cells grew quite normally. This phenotype behaved recessively in cell-cell hybrids, and the segregation profile of its marker indicated that the lesions in the mutants are not linked to the X chromosome. Prototrophic revertants could be isolated from these mutants, and they showed almost the normal level of thymidylate synthase activity. The selection method described here should be useful for isolating large numbers of thymidylate synthase-negative mutants from various mammalian cell lines.
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D005260:Female; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental; D008727:Methotrexate / Q000494:pharmacology*; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008780:Methyltransferases / Q000172:deficiency*; D051379:Mice; D008809:Mice, Inbred C3H; D009154:Mutation; D013936:Thymidine / Q000378:metabolism* / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000172:deficiency* / Q000235:genetics
D008769:Methylnitronitrosoguanidine; D008780:Methyltransferases; D013940:Thymidylate Synthase; D013936:Thymidine; D008727:Methotrexate