SELECTION OF CHINESE-HAMSTER CELLS AUXOTROPHIC FOR THYMIDINE BY 1-BETA-D-ARABINOFURANOSYL CYTOSINE

Thymidine auxotrophs of Chinese hamster ovary cells have been isolated through selection for resistance to the drug 1-β-D-arabinofuranosyl cytosine (ara C). Deoxycytidine and deoxyuridine, but not the other deoxyribonucleosides or ribonucleosides, are also able to supply the nutritional requirement of these mutants. The frequency of isolation of the auxotrophs in mutagenized cultures is about 10-5, and the spontaneous frequency is 5-10 times lower. However, the reversion frequency to thymidine prototrophy is high. Analysis of the revertants suggests that the ara C resistance and the thymidine auxotrophy are the consequence of a single mutation. In hybrid cells, the thymidine auxotrophy can be complemented by wild-type cells or another thymidine-requiring cell line deficient in folate metabolism, while the ara C resistance behaves in a codominant manner. The pool of dCTP in the ara C-resistant thymidine auxotrophs is elevated, accounting for the ara C resistance. Upon thymidine starvation, the pools of dTTP and dGTP decline and the rate of DNA synthesis decreases immediately. We suggest that the complex phenotype and the genetic and biochemical properties of these mutants can be best explained by an inability to reduce UDP to dUDP. © 1979 Plenum Publishing Corporation.