Cloning and expression of uridine/cytidine kinase cDNA from human fibrosarcoma cells.
Uridine/cytidine kinase which converts uridine and cytidine to their corresponding monophosphates is a rate-limiting enzyme involved in the salvage pathway of pyrimidine synthesis. We isolated cDNA encoding the enzyme from human fibrosarcoma cells, then determined its nucleotide sequence by the 5'-RACE method followed by confirmation employing the human genome DNA library. The isolated uridine/cytidine kinase cDNA (UCK cDNA) consisted of 786 nucleotides encoding 261 amino acids and was found to have approximately 70% homology with mouse UCK cDNA. Northern blot analysis of human leukemia RNAs with labeled UCK gene showed a single band at 1.6 kb to be UCK mRNA, and southern blot analysis of the UCK cDNA after digestion with BamHI, SacI and XbaI enzymes showed four band signals, suggesting the UCK gene to have at least 4 exons. A truncated form of UCK cDNA was expressed as the His-tag conjugated protein in Escherichia coli. The expressed and purified protein specifically converted uridine and cytidine to their corresponding monophosphates and also phosphorylated antitumor nucleosides such as 5-fluorouridine, cyclopentenyl-cytosine and 3'-C-ethynylcytidine. The present results suggest that our cloned human UCK cDNA encodes the correct amino acid sequence for UCK protein, showing high intracellular phosphorylation activity forward natural and synthetic pyrimidine nucleosides.
Koizumi
K
K
The Second Cancer Laboratory, Taiho Pharmaceutical Co., Ltd., 1-27 Misugidai, Hanno-city, Saitama 357-8527, Japan.
Shimamoto
Y
Y
Azuma
A
A
Wataya
Y
Y
Matsuda
A
A
Sasaki
T
T
Fukushima
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005260:Female; D005354:Fibrosarcoma / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D014541:Uridine Kinase / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D014541:Uridine Kinase