Single-step selection of mouse FM3A cell mutants defective in thymidylate synthetase

A tritium-suicide method for isolating thymidine auxotrophic mutants is described. Mutagenized mouse FM3A cells were cultured in medium containing [3H] deoxyuridine. Most of the surviving clones examined showed a phenotype of absolute thymidine auxotrophy. This phenotype is very stable and was found to be genetically recessive in cell-cell hybridization experiments. The growth of these variant clones was not supported by various pyrimidine nucleosides other than thymidine. The activity of thymidylate synthetase in crude extracts of these clones was less than 1% of that of FM3A cells. These results strongly indicate that the thymidine auxotrophic phenotype resulted from a genetic defect in thymidylate synthetase.