Isolation of a human cDNA encoding amidophosphoribosyltransferase and functional complementation of a CHO Ade-A mutant deficient in this activity.
We report here the isolation of a human cDNA encoding the first step in de novo purine biosynthesis, amidophosphoribosyltransferase (PRAT). The human PRAT cDNA was isolated by complementation of a Saccharomyces cerevisiae ade4 mutant deficient in PRAT enzymatic activity. The identity of the isolated cDNA, designated pAdeA-3, was confirmed by several independent methods. Genomic DNA sequences homologous to pAdeA-3 show coordinate segregation with the hypoxanthine nutritional requirement in Chinese hamster ovary (CHO) cell Ade-A-human hybrids, segregants of these hybrids, and irradiation reduction hybrids. The PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT enzyme deficiency in CHO Ade-A mutants. This correction was monitored by both cell-free PRAT assays and in vivo phosphoribosylformylglycinamide (FGAR) accumulation studies. FGAR accumulation is a classic method for assessment of the early steps of purine nucleotide biosynthesis. Two of the isolated transformants, designated PRAT-1 and PRAT-2, exhibited 22% and 53%, respectively, of wild-type CHO K1 PRAT enzymatic activity using a cell-free enzyme assay. These same two transformants plus an additional transformant, designated PRAT-13, showed FGAR accumulations of 150%, 260%, and 140%, respectively, compared to the levels of accumulation seen in CHO K1. Transformants PRAT-1 and PRAT-2 both contained a mRNA species recognized by the PRAT cDNA of identical size to a mRNA species in human fibroblasts homologous to the PRAT cDNA. This observation, along with the functionality of the cDNA in both yeast and CHO cells deficient in PRAT activity, suggests the isolated cDNA is full length.
Barton
J W
JW
Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado.
Bleskan
J
J
Patterson
D
D
D000582:Amidophosphoribosyltransferase / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics*; D000818:Animals; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000302:isolation & purification*; D005816:Genetic Complementation Test; D005819:Genetic Markers; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism; D008114:Liver Neoplasms, Experimental; D009154:Mutation; D012333:RNA, Messenger / Q000032:analysis; D012265:Ribonucleotides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014407:Tumor Cells, Cultured
D005819:Genetic Markers; D007042:Hypoxanthines; D012333:RNA, Messenger; D012265:Ribonucleotides; D019271:Hypoxanthine; C002824:phosphoribosyl-N-formylglycineamide; D004247:DNA; D000582:Amidophosphoribosyltransferase; D005998:Glycine