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

I. Chian Li, Chao Liang Wu, Ernest H.Y. Chu

Research output: Contribution to journalArticle

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 [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 × 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.

Original languageEnglish
Pages (from-to)85-91
Number of pages7
JournalSomatic Cell and Molecular Genetics
Volume15
Issue number1
DOIs
Publication statusPublished - 1989 Jan 1

All Science Journal Classification (ASJC) codes

  • Genetics
  • Cell Biology

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