Expression of a human cDNA encoding a protein containing GAR synthetase, AIR synthetase, and GAR transformylase corrects the defects in mutant Chinese hamster ovary cells lacking these activities

Abstract

The isolation of a human cDNA encoding the multifunctional protein containing GAR synthetase, AIR synthetase, and GAR transformylase by functional complementation of purine auxotrophy in yeast has been reported. Chinese hamster ovary (CHO) cell mutant purine auxotrophs deficient in GAR synthetase (Ade-C) or AIR synthetase plus GAR transformylase (Ade-G) activities were transfected with this human GART cDNA subcloned into a mammalian expression vector. This restored 49-140% of the activities of GAR synthetase, AIR synthetase, and GAR transformylase in transfected cells when compared to wild-type CHO K1 parental cells. Study of one stably expressing transfectant, AdeC2, revealed that the human GART cDNA was incorporated into the CHO genome. The enzyme activities appear to be associated with an expressed protein of 110 kDa, very similar to that of purified human GART trifunctional enzyme. The Ade-C mutant shows reduced amounts of GART mRNA compared to CHO K1 and a protein of apparently reduced size, results consistent with the purine requirement and enzyme deficiency observed in the mutant. These experiments provide definitive evidence that the human GART cDNA encodes and can direct the production of active human GART trifunctional protein in mammalian cells. They also provide important evidence that the Ade-C and Ade-G mutants of CHO cells are defective in this gene.

Mutations in the Chinese hamster ovary cell GART gene of de novo purine synthesis

“…Similar mobility shifts for glu to lys substitution have been observed previously (Yannoukakos et al, 1991). In general agreement with our previous studies (Oates et al, 1977; Irwin et al, 1979; Chang et al, 1991; Brodsky et al, 1997; Bleskan and Patterson, unpublished results) AdeC and AdeG (55-1) both contain at least as much GARS and GART protein as CHO-K1 lysates (Fig. S2).…”

“…Enzyme assays of GARS, AIRS, and GART were carried out using our previously published procedures (Chang et al, 1991). All assays were linear with time and amount of protein and were run in duplicate.…”

Intronic polyadenylation in the human glycinamide ribonucleotide formyltransferase gene

“…To our knowledge, all of the other cases analyzed to date involve a switch from one isotype of a protein to another in response to the needs of a tissue-specific or developmental program of gene expression. Although it is not clear whether there is a developmental role for the GART gene, others have implicated this locus in Down's syndrome (3). From the striking sequence similarities and differences in the mouse and human GART genes near the region involved in intronic polyadenylation, this locus seems to be an interesting and informative model system to dissect the mechanisms involved in the choice of intronic polyadenylation versus downstream splicing.…”