Isolation of a human cDNA encoding amidophosphoribosyltransferase and functional complementation of a CHO Ade?A mutant deficient in this activity

Abstract

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.

Isolating the Arabidopsis thaliana Genes for de Novo Purine Synthesis by Suppression of Escherichia coli Mutants (I. 5[prime]-Phosphoribosyl-5-Aminoimidazole Synthetase)

“…1). A number of chicken pur genes have been isolated by suppression of E. coli pur mutations (Chen et al, 1990), whereas human and Drosophila pur cDNAs have been cloned by suppression of yeast ade mutations (Henikoff et al, 1981;Schild et al, 1990;Barton et al, 1991). In plants, suppression of E. coli mutant phenotypes has been used successfully to isolate the phosphoenolpyruvate carboxylase cDNAs from a maize library (Izui et al, 1986) and a superoxide dismutase cDNAs from Arabidopsis and tobacco libraries (Van Camp et al, 1990).…”

Inhibitors of dihydro-orotase, amidophosphoribosyltransferase and IMP cyclohydrolase as potential drugs

“…The purine nucleotides IMP, AMP and GMP induce formation of an inactive tetramer (224 kDa) of amidophosphoribosyltransferase from an active dimer (112 kDa) found in the presence of P-Rib-PP [22,28], Using sucrose density centrifugation, we showed that piritrexim 7.2 s) from the active dimer (6.7 s) as shown in …”

Report of the committee on the genetic constitution of chromosome 4

“…A gene involved in de novo purine biosynthesis, PA1CS (phosphoribosylaminoimidazole carboxylase (EC 4.1.1.21)) has been newly cloned (Schild et al, 1990) and mapped to 4pl6.3-4q21 using somatic cell hybrids, (Barton et al, 1991a). It joins a previously mapped and more recently cloned (Barton et al, 1991b) member of this enzyme pathway (PPAT) on chromosome 4. The eucaryotic initiation factor 4E (EIF4EL1) has been mapped to4pl5-4qter using somatic cell hybrids (Pelletier et al, 1991).…”