Measurement of spontaneous mutation rates at the Na+/K+ ATPase locus (ouabain resistance) of human fibroblasts using improved growth conditions.
The mutation rate for the Na+/K+ ATPase locus (ouabain resistance, OuaR) in mammalian cells in culture has been reported to be 10-100-fold lower than the mutation rate of other gene loci in culture, such as the hypoxanthine phosphoribosyl transferase (HPRT) locus. Determination of the mutation rate to ouabain resistance is sensitive to culture conditions and the concentration of ouabain used to select mutants. Our improved growth conditions for human cells have permitted absolute cloning efficiencies of 70-90% and population doubling times of 16-17 h with both normal human diploid fibroblasts, KD, and their chemically induced neoplastic derivative, Hut-11A. Ouabain at 10(-7) M was found to be adequate to select for resistant (OuaR) mutants with an absolute recovery efficiency of 54-102%. Under these conditions, the mutation rates to ouabain resistance for human cells were measured and found to be 1-8.5 X 10(-7)/cell/generation for KD cells and 6-13 X 10(-7)/cell/generation for Hut-11A cells. These rates are 5-25 times higher than previously reported for human cells. Improved growth and the use of a lower concentration of ouabain for selection may allow for the increased recovery of OuaR mutants and an improved estimate of the mutation rate at this locus, which is only 2-10-fold less than the mutation rate at the HPRT locus in the same cells.
D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D046508:Culture Techniques; D004351:Drug Resistance; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D009154:Mutation; D010042:Ouabain / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*
D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase