Synthesis of 5-phosphoribosyl 1-pyrophosphate (PRPP), a regulatory determinant in the pathways of purine nucleotide synthesis, is catalyzed by PRPP synthetase (PS). Inherited superactivity of PS, an X chromosome-linked trait, results from an array of kinetic alterations in PS and is accompanied by gout with purine nucleotide overproduction. Much is known about the structure and kinetic properties of human PS; knowledge of the organization and control of PS gene expression in man is, however, rudimentary. The proposed Visiting Researcher has recently utilized labeled synthetic oligodeoxynucleotide probes, prepared from knowledge of partial amino acid sequence of human PS, to screen 2 human cDNA libraries for recombinant clones. Clones hybridizing with the probes have been isolated and purified. Some of these recombinants show preferential hybridization to genomic DNA enriched for or restricted to human X chromosomal DNA and appear likely to bear DNA sequences complementary to PS mRNA. The first specific aim of the proposed research is characterization of cDNAs isolated to date in order to determine their usefulness as probes for the PS gene and PS mRNA. Methods to be employed will include Southern and Northern blot analyses, DNA sequencing, and in situ hybridization. The sequences of several overlapping cDNAs will be utilized to derive the full-length sequence of PS cDNA and to deduce the amino acid sequence of PS. This amino acid sequence will be compared with primary structure data obtained by amino acid sequencing of PS peptides. These studies should expedite characterization of the human PS gene. The second specific aim is preparation of cDNA libraries derived from the mRNA of fibroblasts cultured from affected males with superactive forms of PS. Recombinant libraries will be screened for clones containing DNAs complementary to mRNA for superactive forms of PS. Sequencing of cDNA clones should permit identification of precise genetic defects associated with PS superactivity. Additional techniques required for these investigations will include mRNA isolation and cDNA library preparation. Dr. Kelley and his laboratory have extensive experience in the study of the molecular genetics of enzymes of purine metabolism. This experience encompasses the methods relevant to the research proposed. The Host's laboratory will be a unique environment for the Visiting Researcher to extend his long-term investigation of the structure and regulation of normal and aberrant forms of PS to the molecular genetic level.
