Purification, cloning, and expression of murine uridine phosphorylase.
Uridine phosphorylase was purified 10,300-fold from tumors of the murine colorectal adenocarcinoma cell line, Colon-26. Degenerate DNA probes were synthesized corresponding to partial amino acid sequences and used to screen a Colon-26 cDNA library. A cDNA clone of 1327 base pairs that contains a 5' untranslated region, a coding region of 933 base pairs, and a 3' nontranslated region with a polyadenylated tail was identified. The cDNA was confirmed to be uridine phosphorylase by 1) sequence comparison to uridine phosphorylase of Escherichia coli, 2) substrate specificity studies with recombinant protein expressed in COS-7 cells that demonstrated relatively high enzyme activity with uridine as substrate compared low levels when thymidine was used, and 3) inhibition of enzyme activity by the competitive inhibitor 2,2'-anhydro-5-ethyluridine. Northern blot analysis using the cDNA as a probe, demonstrated high levels of mRNA expression in Colon-26. Expression was low in NIH3T3 cells, but high in DMBA-3 and PH-1 cells, which are NIH3T3-derived cells that have been transformed with mutated murine Ha-ras and viral Ha-ras, respectively. Expression of uridine phosphorylase mRNA in these cell lines was further enhanced by treating the cells with the inflammatory cytokines, tumor necrosis factor-alpha, interleukin 1 alpha, and interferon gamma.
Watanabe
S
S
Department of Oncology, Nippon Roche Research Center, Kamakura, Japan.
Hino
A
A
Wada
K
K
Eliason
J F
JF
Uchida
T
T
D000230:Adenocarcinoma / Q000201:enzymology / Q000473:pathology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000473:pathology; D018076:DNA, Complementary / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D004926:Escherichia coli; D015723:Gene Library; D005796:Genes; D011905:Genes, ras; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D051379:Mice / Q000235:genetics*; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D009368:Neoplasm Transplantation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014543:Uridine Phosphorylase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D007375:Interleukin-1; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D014543:Uridine Phosphorylase