A physically altered leucyl-tRNA synthetase complex in a CHO cell mutant

Abstract

CONDITIONALLY lethal mutants in animal cells have been of great value in advancing our knowledge of genetics, biochemistry and molecular biology. Mammalian cells with temperature sensitive (ts) mutant phenotypes have been isolated by several laboratories, however no thorough characterisations of the altered gene products are available¹. Selection procedures have been devised by Thompson et al. to isolate ts aminoacyl-tRNA synthetase variants in Chinese hamster ovary (CHO) cells which behave as missense mutants^2,3. The CHO temperature-sensitive mutant tsH1 has very low in vivo levels of leucyl-tRNA, compared to wild-type levels at non-permissive temperatures; levels of the other 19 aminoacyl-tRNAs are unchanged⁴. This mutant also has an altered leucyl-tRNA synthetase which is thermosensitive in vitro⁴, enzymatically different from wild-type leucyl-tRNA synthetase⁵, and has an altered K_m for leucine⁵. We have now shown the leucyl-tRNA synthetase in tsH1 also has altered physical characteristics. The mutant enzyme exists primarily in a low molecular weight form whereas wild-type leucyl-tRNA synthetase is primarily associated with higher molecular weight particulate forms.