The objective of this research is to delineate biochemical and genetic mechanisms regulating purine nucleotide synthesis in man. Studies of inherited enzyme defects underlying excessive purine nucleotide and uric acid production in some patients with gout have contributed to concepts of the control of rates of purine synthesis de novo. Superactivity of phosphoribosylpyrophosphate (PRPP) synthetase (PRS), which catalyzes synthesis of the regulatory substrate PRPP, is one such defect. The proposed investigations focus on defining genetic and protein structural determinants of the activity of normal human PRS and on identifying genetic and protein structural aberrations underlying inherited PRS superactivity. Molecular cloning, protein structural, and enzyme kinetic methods will be employed to pursue this Specific Aim. Two cDNAs encoding proteins highly homologous to authentic PRSs have been cloned from human cDNA libraries. Despite striking sequence identity in protein coding regions, 3' noncoding regions of these hPRS cDNAs are divergent, suggesting separate hPRS genes coding for each. Full-length hPRS cDNAs complementary to hPRS mRNAs identified in human tissues will be isolated, characterized, and utilized as probes in several projects. 1) Kinetic characteristics and deduced amino acid sequences of normal and mutant hPRSs and PRSs characterized from bacterial and mammalian sources will be compared in order to define the structural basis of PRS activity. 2) Patterns of hPRS gene restriction and transcription and translation of hPRS genes will be examined to evaluate normal hPRS gene structure and expression and to identify provisional sites of derangement in genetic control of enzyme activity in cells with PRS superactivity. 3) Utilizing PCR and cDNA cloning methods, hPRS cDNAs will be cloned and characterized from cells of affected patients in order to define precise genetic defects underlying PRS superactivity. In these studies and in studies of the structure and function of normal hPRS gene products, expression of hPRS cDNAs will be attempted using an expression system in an E coli strain engineered to lack bacterial PRS. 4) Mapping of hPRS 1 and hPRS 2 genetic loci and identification of additional hPRS (pseudo)genes will be undertaken utilizing in situ chromosomal hybridization, pulsed field gel electrophoresis mapping and genomic DNA cloning. 5) Finally, genomic DNA libraries will be probed with hPRS cDNAs in order to clone and characterize normal and variant hPRS genes.
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