Single-step selection of mammalian cell mutants deficient in CTP synthetase.
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 [3H]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 [3H]UTP into [3H]CTP, which eventually is converted into [3H]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 [3H]thymidine. The isolated mutant clones are auxotrophic for cytidine and are stable in phenotype with a reversion frequency of less than 1 x 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 [3H]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.
Li
I C
IC
Department of Medicine, Medical College, National Cheng Kung University, Tainan, Taiwan.
Wu
C L
CL
Chu
E H
EH
D000818:Animals; D019731:Carbon-Nitrogen Ligases; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D006224:Cricetinae; D003562:Cytidine / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D003841:Deoxycytidine / Q000378:metabolism; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D013936:Thymidine / Q000378:metabolism; D014529:Uridine / Q000378:metabolism
D003841:Deoxycytidine; D003562:Cytidine; D004247:DNA; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D013936:Thymidine; D014529:Uridine