Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase.
Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.
D000659:AMP Deaminase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001377:Azaserine / Q000494:pharmacology; D002460:Cell Line; D003070:Coformycin / Q000494:pharmacology; D006224:Cricetinae; D003470:Culture Media; D005020:Ethyl Methanesulfonate; D005347:Fibroblasts; D005796:Genes; D014644:Genetic Variation; D008168:Lung; D009154:Mutation; D009709:Nucleotide Deaminases / Q000096:biosynthesis*
D003470:Culture Media; D003070:Coformycin; D001377:Azaserine; D005020:Ethyl Methanesulfonate; D009709:Nucleotide Deaminases; D000659:AMP Deaminase