Isolation and characterization of 5-fluorouracil-resistant mutants of Chinese hamster ovary cells deficient in the activities of orotate phosphoribosyltransferase and orotidine 5′-monophosphate decarboxylase

A rapid, simple method for isolation of large numbers of Chinese hamster ovary cell (CHO-K1) mutants deficient in the final two enzymes of UMP biosynthesis, orotate phosphoribosyltransferase (EC 2.4.2. 10), and OMP decarboxylase (EC 4.1.1.23) is described. The method takes advantage of the fact that CHO-K1 cells require orotate phosphoribosyltransferase to activate the pyrimidine analog 5-fluorouracil to its active form; hence, mutants lacking this enzyme activity can be isolated simply by selection for cells resistant to 5-fluorouracil. The relevance of these observations to the metabolism of this cancer chemotheraputic agent, to the study of the structure of the protein(s) involved in catalyzing the last two steps of UMP biosynthesis, to the study of the structure of the gene(s) coding for this protein, and to analysis of the human genetic disease orotic aciduria is discussed.Number 5 in the series: Biochemical Analysis of Pyrimidine Biosynthesis in Mammalian Cells.