Isolation of hypoxanthine phosphoribosyltransferase-defective mutants in Chinese hamster V79 cells by tritium suicide.
Tritium suicide was shown to be a 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 degrees 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 lest 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 Km 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 as resistant to 6-thioguanine.
D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D019271:Hypoxanthine; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007042:Hypoxanthines / Q000378:metabolism*; D009154:Mutation; D014316:Tritium
D007042:Hypoxanthines; D014316:Tritium; D019271:Hypoxanthine; D007041:Hypoxanthine Phosphoribosyltransferase