Cloning and expression of a cDNA encoding uridine kinase from mouse brain.
Uridine kinase is the rate-limiting enzyme in the pyrimidine salvage pathway of all mammalian cells. A cDNA for uridine kinase from mouse brain has been isolated, sequenced, and characterized. This is the first report of a complete nucleotide sequence for mammalian uridine kinase. The isolated cDNA is only 95% complete, missing the first 17 codons. The correct 5'-terminus sequence was obtained from high-stringency screening of a mouse liver genomic DNA library. The translated cDNA sequence encodes a protein of 277 amino acids (Mr 31,068). A truncated form of the cDNA was expressed in Escherichia coli. The expressed protein displayed uridine kinase activity and readily formed a tetramer, the most active form of the wild-type enzyme. Analysis of the amino acid sequence identified the three ATP-binding site consensus motifs. The predicted secondary structure for uridine kinase and the sequence comparison with three kinases having known crystal structures are consistent with uridine kinase having an alpha/beta core structure of the nucleotide-binding fold found in many kinases. We have also isolated and cloned a nonfunctional, processed pseudogene from mouse genomic DNA. This pseudogene sequence is 94% identical with the coding DNA.
Ropp
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PA
Department of Biochemistry & Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599-7260, USA.
Traut
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons; D005796:Genes; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014541:Uridine Kinase / Q000235:genetics*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014541:Uridine Kinase