An investigation into the potential of a mammalian temperature sensitive leucyl-tRNA synthetase mutant for mutagenesis studies.
Reversion of the mammalian temperature sensitive leucyl-tRNA synthetase mutant CHO tsH1 [33] has been investigated to determine its potential for mutagenesis studies. The protocol for a mutation assay using the new class of temperature sensitive conditional lethal mutants of somatic cells is presented. While it requires careful control of temperature during revertent selection it promises to provide a system complementary to the existing mutation assays. UV and EMS mutagenesis of tsH1 has confirmed that approx. 3TD of expression time are sufficient for complete expression of induced mutants at low mutagen doses (80% survival following UV and 40-50% with EMS). At a higher UV dose resulting in only 10% survival much longer expression times were required which cannot be explained by growth delays alone. While the reason for this is unknown it suggests that care must be taken in studies which require high mutagen doses. Representative revertants which were isolated show a range of phenotypes between those of tsH1 and WT. They appear to be a promising source of extragenic suppressor mutants [33] with alterations in functions affecting protein synthesis.
D000604:Amino Acyl-tRNA Synthetases / Q000096:biosynthesis*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005804:Genes, Lethal; D005821:Genetic Techniques; D006358:Hot Temperature; D007935:Leucine-tRNA Ligase / Q000096:biosynthesis*; D009154:Mutation; D013696:Temperature; D014466:Ultraviolet Rays
D005020:Ethyl Methanesulfonate; D000604:Amino Acyl-tRNA Synthetases; D007935:Leucine-tRNA Ligase