Resistance to 5-fluoroorotic acid and pyrimidine auxotrophy: A new bidirectional selective system for mammalian cells

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Abstract
We have isolated a clone of murine erythroleukemic cells which will yield a population when a single cell is suspended in a medium composed of dialyzed sera and small molecules. We report that it is feasible—in one experiment—to screen more than 1011 of these cells for growth under selective conditions. After exposure to a mutagen, descendants of our clone were placed in selective media containing 10−4 M 5-fluoroorotic acid and 10−4 M uridine. Cells capable of sustained growth in such media were eventually recovered. Clones of these cells, unlike clones of the parental population, required uridine for growth and contained only 0.39% as much orotate phosphoribosyltransferase, less than 0.5% as much 5-fluoroorotate phosphoribosyltransferase, and 0.013% as much orotidine-5′-monophosphate decarboxylase activity as the parental clones. The parental and variant clones had similar levels of activity for six other enzymes that participate in pyrimidine metabolism.