Isolation by tritium suicide of uridine-cytidine kinase-defective mutants in Chinese hamster V79 cells

Abstract

Tritium suicide was shown to be a highly effective method for isolating mutants defective in uridine-cytidine kinase in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of three kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [ 3H]uridine for 10 min, followed by storage of 3H-labelled cells at −70 °C for 4–7 days. Nine clones that survived the third kill cycle were tested for incorporation of [ 3H]uridine and for uridine kinase activity in extracts. Eight of these clones were defective in whole-cell uridine incorporation and in uridine kinase activity. A kinetic study was made on the uridine-cytidine kinase activity of three of the mutants. The apparent V max of the mutants was reduced approx. 10-fold when either uridine or cytidine was used as substrate. In contrast, the apparent K m of uridine was reduced approx. 12-fold in the mutants with only a 2-fold (probably insignificant) reduction in K m's for cytidine or for ATP.