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 [³H]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.