Molecular cloning of the human UMP synthase gene and characterization of point mutations in two hereditary orotic aciduria families
- Nagoya City Univ. Medical School (Japan); and others
Uridine monophosphate (UMP) synthase is a bifunctional enzyme catalyzing the last two steps of de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) and orotidine-5{prime}-monophosphate decarboxylase (ODC). Loss of either enzymatic activity results in hereditary orotic aciduria, a rare autosomal recessive disorder characterized by retarded growth, anemia, and excessive urinary excretion of orotic acid. We have isolated the UMP synthase chromosomal gene from a {lambda}EMBL-3 human genomic library and report a single-copy gene spanning {approximately}15 kb. The UMP synthase genomic structure encodes six exons ranging in size from 115 bp to 672 bp, and all splicing junctions adhere to the canonical GT/AG rule. Cognate promoter elements implicated in glucocorticoid- and cAMP-mediated regulation as well as in liver-, myeloid-, and lymphocyte-specific expression are located within the 5{prime} flanking sequence. Molecular investigation of UMP synthase deficiency in a Japanese orotic aciduria patient revealed mutations R96G (A- to-G transition; 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. Expression 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 the urinary orotic acid substrate accumulations observed in vivo. We further establish the identity of two polymorphisms, G213A ({nu} = .26) and 440 Gpoly ({nu} = .27) located in exons 3 and 6, respectively, which did not significantly compromise either OPRT or ODC function. 76 refs., 5 figs., 7 tabs.
- OSTI ID:
- 518536
- Journal Information:
- American Journal of Human Genetics, Vol. 60, Issue 3; Other Information: PBD: Mar 1997
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
BASIC STUDIES
HUMAN CHROMOSOME 3
GENETIC MAPPING
PATIENTS
HEREDITARY DISEASES
ANEMIAS
UMP
STRUCTURE-ACTIVITY RELATIONSHIPS
SPLICING
DNA SEQUENCING
GENE MUTATIONS
DNA-CLONING
ENZYME ACTIVITY
GENE REGULATION
PYRIMIDINES
BIOSYNTHESIS
IN-SITU HYBRIDIZATION
EXONS
PLASMIDS
POLYMERASE CHAIN REACTION
NUCLEOTIDES
RECESSIVE MUTATIONS
OROTIC ACID
ESCHERICHIA COLI