Selection by [3H] amino acids of CHO-cell mutants with altered leucyl- and asparagyl-transfer RNA synthetases

Abstract

Efficient selection procedures, using [3H]amino acids as the selecting agent, were developed for isolating temperature-sensitive (TS) mutations in CHO cells affecting protein synthesis. After chemical mutagenesis, leucyl-tRNA synthetase mutants were obtained when [3H]leucine was used as the selecting agent in two independent experiments. These mutations seem to involve the same genetic locus as the TSH1 mutant described previously (1). A selection with [3H]valine, in which all amino acids except leucine were at low concentration in the selective medium, resulted in a new class of mutants with reduced asparagyl-tRNA synthetase activity. These results were consistent with the finding that all mutants were phenotypically dependent on the concentration of amino acid, specific to the altered synthetase, in the medium. Our observations suggest that although leucyl synthetase mutations are a relatively common class of TS mutations in CHO cells, the spectrum of mutants obtained can be at least partially manipulated through concentrations of amino acids in selective media. The asparagyl-synthetase mutation was shown to be recessive and to complement the leucyl-synthetase mutation in cell-cell hybrids.