Chromosomal assignment of the gene for folylpolyglutamate synthetase to human chromosome 9

Abstract

An auxotrophic mutant, GAT-, derived from the Chinese hamster cell line CHO-K1 and exhibiting multiple growth requirements for glycine, adenine, and thymidine, has been shown to be deficient in one of the folate-dependent enzymes, folylpolyglutamate synthetase (FPGS). This mutant was fused with normal human lymphocytes and human lymphoblasts and 41 GAT+ primary hybrid clones were isolated in medium lacking glycine, adenine, and thymidine. In addition, 71 secondary clones were isolated after growth of four primary hybrid clones in enriched medium, which allows losing the complementing human chromosome without losing cell viability. Analysis of human isozyme markers in the 112 primary and secondary clones established a syntenic relationship between the GAT marker and AK1, an isozyme marker for human chromosome 9. Enzyme studies of the parental and hybrid cells not only showed restored enzyme activities of FPGS in the hybrids, but also demonstrated that several enzyme characteristics of FPGS in the hybrids are consistent with those of the human enzyme. Thus, it is concluded that the human gene coding for the enzyme FPGS which complements the auxotrophic mutant GAT- in the CHO-K1 cells can be assigned to human chromosome 9. This assignment provides an additional selective marker for mammalian cell genetic analysis in which human chromosome 9 can be selectively and stably retained in the cell hybrids.