MOLECULAR AND GENETIC ANALYSES OF DROSOPHILA-PRAT, WHICH ENCODES THE FIRST ENZYME OF DE-NOVO PURINE BIOSYNTHESIS

The Drosophila Prat gene encodes phosphoribosylamidotransferase (PRAT) , the enzyme that performs the first committed step of the de novo pur ine nucleotide biosynthesis pathway. Using information from amino acid sequence alignments of PRAT from other organisms, a polymerase chain reaction-based approach was employed to clone Prat, Amino acid sequenc e alignment of Drosophila PRAT with PRAT from bacteria, yeast, and ver tebrates indicates that it is most identical (at least 60%) to the ver tebrate PRATs. It shares putative amino-terminal propeptide and iron-b inding domains seen only in Bacillus subtilis and vertebrate PRATs. Pr at was localized to the right arm of chromosome 3 at polytene band 84E 1-2. Owing to the fact that this region had been well characterized pr eviously, Prat was localized to a 30-kilobase region between two defic iency breakpoints. By making the prediction that Prat would have a sim ilar ''purine syndrome'' phenotype as mutations in the genes ade2 and ade3, which encode enzymes downstream in the pathway, five alleles of Prat were isolated. Three of the alleles were identified as missense m utations. A comparison of PRAT enzyme activity with phenotype in three of the mutants indicates that a reduction to 40% of the wild-type all ele's activity is sufficient to cause the purine syndrome, suggesting that PRAT activity is limiting in Drosophila.