Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity.

TLDR

The mutants were generated to aid metabolic studies of cancer chemotherapy and fine‑structure genetic mapping of the dihydrofolate reductase gene. Chinese hamster ovary cells were mutagenized and selected with high‑specific‑activity [3H]deoxyuridine, and a heterozygous intermediate was obtained by combining [3H]deoxyuridine with methotrexate to reduce DHFR activity. Fully deficient DHFR mutants could only be isolated from a heterozygous intermediate, are glycine‑, purine‑, and thymidine‑dependent, display a recessive phenotype in hybrids, and revertants including a heat‑labile DHFR variant have been recovered.

Abstract

Mutants of Chinese hamster ovary cells lacking dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) activity were isolated after mutagenesis and exposure to high-specific-activity [3H]deoxyuridine as a selective agent. Fully deficient mutants could not be isolated starting with wild-type cells, but could readily be selected from a putative heterozygote that contains half of the wild-type level of dihydrofolate reductase activity. The heterozygote itself was selected from wild-type cells by using [3H]deoxyuridine together with methotrexate to reduce intracellular dihydrofolate reductase activity. Fully deficient mutants require glycine, a purine, and thymidine for growth; this phenotype is recessive to wild type in cell hybrids. Revertants have been isolated, one of which produces a heat-labile dihydrofolate reductase activity. These mutants may be useful for metabolic studies relating to cancer chemotherapy and for fine-structure genetic mapping of mutations by using available molecular probes for this gene.