PURIFICATION, CLONING, AND EXPRESSION OF MURINE URIDINE PHOSPHORYLASE

Uridine phosphorylase was purified 10,300-fold from tumors of the muri ne colorectal adenocarcinoma cell line, Colon-26. Degenerate DNA probe s were synthesized corresponding to partial amino acid sequences and u sed 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 p airs, and a 3' nontranslated region with a polyadenylated tail was ide ntified. The cDNA was confirmed to be uridine phosphorylase by 1) sequ ence comparison to uridine phosphorylase of Escherichia coli, 2) subst rate specificity studies with recombinant protein expressed in COS-7 c ells that demonstrated relatively high enzyme activity with uridine as substrate compared low levels when thymidine was used, and 3) inhibit ion of enzyme activity by the competitive inhibitor 2,2'-anhydro-5-eth yluridine. Northern blot analysis using the cDNA as a probe, demonstra ted 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 deri ved cells that have been transformed with mutated murine Ha-ras and vi ral Ha-ras, respectively. Expression of uridine phosphorylase mRNA in these cell lines was further enhanced by treating the cells with the i nflammatory cytokines, tumor necrosis factor-alpha, interleukin 1 alph a, and interferon gamma.