Isolation of hypoxanthine phosphoribosyltransferase-defective mutants in chinese hamster V79 cells by tritium suicide

Abstract

Tritium suicide was shown to be highly efficient method for isolating mutants defective in hypoxanthine incorporation in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of 3 kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [ 3H]hypoxanthine for 5 or 10 min, followed by storage of 3H-labelled cells at −70°C for 4–10 days. 12 clones that survived the 3rd kill cycle were tested for incorporation of [ 3H]hypoxanthine and all were found to be defective. At least 6 of the clones have defective hypoxanthine phosphoribosyltransferase (HPRT) activity. One mutant, H19, chosen for further characterization, had HPRT with a 13-fold elevation in apparent K m for phosphoribosylpyrophosphate (PRPP). Thin-layer chromatography of cell extracts showed that this mutant was incapable of converting intracellular hypoxanthine to IMP or to other purine metabolites. In addition, H19 was resistant to 6-thioguanine.