CLONING AND EXPRESSION OF A CDNA-ENCODING URIDINE KINASE FROM MOUSE-BRAIN

Uridine kinase is the rate-limiting enzyme in the pyrimidine salvage p athway of all mammalian cells. A cDNA for uridine kinase from mouse br ain 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 sc reening of a mouse liver genomic DNA library. The translated cDNA sequ ence encodes a protein of 277 amino acids (M(r) 31,068). A truncated f orm of the cDNA was expressed in Escherichia coli. The expressed prote in displayed uridine kinase activity and readily formed a tetramer, th e most active form of the wildtype enzyme. Analysis of the amino acid sequence identified the three ATP-binding site consensus motifs. The p redicted secondary structure for uridine kinase and the sequence compa rison with three kinases having known crystal structures are consisten t with uridine kinase having an alpha/beta core structure of the nucle otide-binding fold found in many kinases. We have also isolated and cl oned a nonfunctional, processed pseudogene from mouse genomic DNA. Thi s pseudogene sequence is 94% identical with the coding DNA. (C) 1996 A cademic Press, Inc.