Human UMP synthase is a bifunctional protein of approximately 55,000 daltons comprising the two enzymic activities orotate phosphoribosyltransferase and orotidylate decarboxylase in a single polypeptide chain. The activities catalyze the last two steps of de novo uridine-5' monophosphate (UMP) biosynthesis, the conversion of orotic acid to orotidine-5'-monophosphate (OMP) and OMP to UMP. In the human autosomal recessive disease, hereditary orotic aciduria, there is a severe deficiency of both activities of UMP synthase (Type I) or a deficiency of only the decarboxylase (Type II). This disease is the only known human disorder of pyrimidine nucleotide biosynthesis. Fibroblast cell lines are available in culture from three patients with orotic acidura. Using UMP synthase-specific cDNA probes the underlying molecular defects of orotic aciduria will be characterized. UMP synthase DNA, mRNA and protein from normal and deficient cells will be analyzed for alterations in the amount or structure of each component. Using the M13 sequencing methods, the nucleotide sequence of cloned UMP synthase cDNAs will be directly determined and the sequences of the corresponding mRNA and protein deduced. The structure of the UMP synthase gene will be outlined by isolation of DNA fragments from human genomic Gamma libraries and analysis of coding and non-coding regions by restriction mapping and Southern hybridization techniques. These studies into the nature of the mutations in orotic aciduria and the structure of the normal and deficient UMP synthase gene will enhance our understanding of human genetic defects. The techniques will improve our ability to diagnosis and eventually to treat human genetic diseases. The UMP synthase-deficient cells also provide an opportunity to study the regulation of gene expression. When certain drugs are added to the growth media of the deficient cells, the activity for UMP synthase is increased to near normal levels. The mechanism for this increased expression will be studied by determining levels and structures of VMP synthase mRNA in normal and deficient cells following growth in the presence of the drugs. DNA from the cell lines will also be analyzed for possible secondary UMP synthase coding sequences.
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