Pyrimidine pathway variants of cultured mouse lymphoma cells with altered levels of both orotate phosphoribosyltransferase and orotidylate decarboxylase

The isolation and characterization of stable mouse T-lymphoma (S49) cell variants which have altered levels of both orotate phosphoribosyltransferase and orotidylate decarboxylase are reported. These clones were selected for resistance to either 6-azauridine or 5-fluorouracil, two toxic pyrimidine analogs. The cells of one clone, AU-11, are 10-fold more resistant to 6-azauridine than wild type S49 cells and were shown to possess 4 to 6 times higher levels of both orotate phosphoribosyltransferase and orotidylate decarboxylase. Conversely, the cells of a second clone, FU1-2, are resistant to 5-fluorouracil and show a 50% reduction in the levels of both of these enzymes compared to the wild type enzyme levels. This is the first description of the isolation and characterization of mammalian cell variants or mutants with altered activities of the enzymes in the second half of the pyrimidine pathway. The structural and regulatory properties of orotate phosphoribosyltransferase and orotidylate decarboxylase from these clones have been examined. The affinities of orotate phosphoribosyltransferase and orotidylate decarboxylase for autologous substrates, and their heat stabilities, isoelectric points, and feedback inhibition by nucleotides are unaltered in these cell types. In addition, the rate of de novo pyrimidine biosynthesis, the activity of the glutamine-dependent carbamyl phosphate synthetase, and steady state levels of UTP and CTP are essentially unchanged in the variant cells. These presumed mutants provide useful tools for the understanding of the control mechanisms involved in pyrimidine biosynthesis.