MOLECULAR-CLONING OF THE HUMAN UMP SYNTHASE GENE AND CHARACTERIZATIONOF POINT MUTATIONS IN 2 HEREDITARY OROTIC ACIDURIA FAMILIES

Uridine monophosphate (UMP) synthase is a bifunctional enzyme catalyzi ng the last two steps of de novo pyrimidine biosynthesis, orotate phos phoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxyl ase (ODC). Loss of either enzymatic activity results in hereditary ero tic aciduria, a rare autosomal recessive disorder characterized by ret arded growth, anemia, and excessive urinary excretion of erotic acid. We have isolated the UMP synthase chromosomal gene from a lambda EMBL- 3 human genomic library and report a single-copy gene spanning similar to 15 kb. The UMP synthase genomic structure encodes six exons rangin g; in size from 115 bp to 672 bp, and all splicing junctions adhere to the canonical GT/AG rule. Cognate promoter elements implicated in glu cocorticoid- and cAMP-mediated regulation as well as in liver-, myeloi d-, and lymphocyte-specific expression are located within the 5' flank ing sequence. Molecular investigation of UMP synthase deficiency in a Japanese erotic aciduria patient revealed mutations R96G (A-to-G trans ition; nt 286) and G429R (G-to-C transversion; nt 1285) in one allele and V109G (T-to-G transversion; nt 326) in the other allele. Expressio n of human UMP synthase cDNAs containing these mutations in pyrimidine auxotrophic Escherichia coli and in recombinant baculovirus-infected Sf21 cells demonstrates impaired activity presumably associated with t he urinary erotic acid substrate accumulations observed in vivo. We fu rther establish the identity of two polymorphism, G213A (v=.26) and 44 0Gpoly (v =.27) located in exons 3 and 6, respectively, which did not significantly compromise either OPRT or ODC function.