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.
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.
Chang
F H
FH
Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado 80206.
Barnes
T S
TS
Schild
D
D
Gnirke
A
A
Bleskan
J
J
Patterson
D
D
D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D019731:Carbon-Nitrogen Ligases; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000302:isolation & purification*; D005260:Female; D015870:Gene Expression; D005796:Genes; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases; D008025:Ligases / Q000096:biosynthesis / Q000235:genetics*; D009154:Mutation; D010053:Ovary / Q000201:enzymology*; D051026:Phosphoribosylglycinamide Formyltransferase; D014162:Transfection
D004247:DNA; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D000217:Acyltransferases; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C022387:phosphoribosylamine-glycine ligase