Single-step selection of mammalian cell mutants deficient in CTP synthetase

Abstract

A single-step selection of Chinese hamster V79 cells deficient in CTP synthetase (CTPS − ) is presented. The underlying principle of the direct selection is the differential and efficient killing of synchronized wild-type cells through incorporation of [ 3 H] uridine and [ 3 H]thymidine. The CTPS − mutant cells were recovered by virtue of their not engaging in DNA synthesis, because (1) CTPS − cells are deficient in CTP synthetase and thus are unable to convert [ 3 H]UTP into [ 3 H]CTP, which eventually is converted into [ 3 H]dCTP and incorporated into DNA; (2) the growth of CTPS − mutant cells was arrested as a result of cytidine deprivation, thus escaping the killing by the incorporation of [ 3 H]thymidine. The isolated mutant clones are auxotrophic for cytidine and are stable in phenotype with a reversion frequency of less than 1 × 10 −7 . The mutant cells have no or very low CTP synthetase activity when tested by in vitro CTP synthetase assay or by whole-cell [ 3 H]uridine labeling assay. This modified “tritium suicide” method combined with the S-phase cell synchronization could provide a powerful means for the recovery from the cell population of nondividing mutant cells that are auxotrophic for some special nutrient requirement .