The long-term objective of this research is to define and characterize the biochemical and genetic mechanisms regulating human purine nucleotide synthesis. Studies of inherited enzyme defects underlying excessive purine nucleotide and uric acid production in some families with gout have contributed to concepts of the control of rates of purine synthesis de novo. Superactively of phosphoribosylpyrophosphate (PRPP) synthetase (PRS), which catalyzes abnormal expression of two X chromosome-encoded human PRS isoforms (PRS1 and PRS2) and their respective PRPS genes.
The specific aims are: 1) to delineate the structural and regulatory determinants of expression of normal human (h) PRPS1 and PRPS2 genes 2) to identify independent and/or interactive roles of the corresponding highly homologous hPRS isoforms in the expression of PRS activity; and 3) to define the precise genetic defects and resulting aberrant molecular mechanisms underlying inherited superactivity of hPRS. In pursuit of these specific aims, molecular cloning, protein chemical and enzyme kinetic methods will be employed. The structure of hPRPS1 and hPRPS2 gene promoter and adjacent 5' flanking sequences will be defined by sequencing of cloned PRPS genomic DNA. hPRPS promoter function and cis-acting regulatory elements in the 5' flanking DNAs will be studied in murine and human cell lines transfected with hPRPS promoter region reporter gene plasmid constructs. Pertinent DNA sequences will be tested for nuclear protein and specific transcription factor binding by gel mobility shift assays and, where appropriate, DNA footprinting and site-directed mutation will be utilized to define and delimit key protein-binding sequences in the DNA. PRPS gene promoter activities will be correlated with PRS transcript levels PRS isoform contents, and PRPS and purine nucleotide synthesis in differentiated human cell lines representative of tissues showing differential expression of PRS1 and/or PRS2 transcript abundance. The influences of growth promoting agents, viral transformation, and cell cycle traversal on hPRPS promoter activities and mouse PRS transcript levels will be tested in appropriate murine cell lines. These studies are aimed at providing models for cell-specific and gene-differential regulation of PRPS gene expression and at defining the levels of genetic information transfer at which control occurs. The presence of active hybrid PRS multimers, composed of hPRPS1 and hPRS2 subunit, will be sought in studies in which dissociated recombinant PRS1 and PRS2 are mixed and re-association is promoted. Evidence for a specific protein or proteins modulating PRS activity will also be sought. hPRS1 cDNA derived from patients with feedback-resistant PRS superactivity will be overexpressed in E. coli (or alternatively, in mammalian cells) to determine if demonstrated single-base shifts encoding single amino acid substitutions provide the basis for PRS superactivity in these affected patients. IF this is the case, purification and characterization of the recombinant mutant PRS1s will be pursued. Finally, the structural and mechanistic basis of """"""""catalytic"""""""" superactivity of hPRS, in which the translated sequences of hPRS1 and hPRS2 cDNA derived from affected patients are normal, will be sought in studies directed by the results of measurements of PRS transcript levels and PRS isoform contents in cultured cells from these individuals. Our hypothesis is that the DMV is not a diffuse neuronal system that conveys information si
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