Selection of temperature-sensitive CHL asparagyl-tRNA synthetase mutants using the toxic lysine analog, S-2-aminoethyl-L-cysteine.
We developed a procedure, using the toxic lysine analog S-2-aminoethyl-L-cysteine (thialysine) as the selective agent, to obtain mutant Chinese hamster lung (CHL) fibroblasts with temperature-sensitive defects affecting protein synthesis. The procedure is efficient, rapid, and simple. Using this selective system, we isolated six temperature-sensitive mutants that fail to grow (plating efficiencies less than 10-5) and synthesize protein at 39 degrees C. The temperature-sensitive phenotype of five of the six mutants is suppressed in vivo by exogenous asparagine. In the mutant studied in the greatest detail, asparagyl-tRNA synthetase activity is extremely thermolabile in vitro. Asparagine-suppressible, temperature-sensitive mutants are induced at a high frequency by the mutagen ethylmethane sulfonate (EMS), and they can be efficiently recovered using the proper selective conditions. Our results suggest that CHL cells have only one functional copy of the gene encoding for asparagyl-tRNA synthetase.
D000604:Amino Acyl-tRNA Synthetases / Q000096:biosynthesis*; D001216:Asparagine / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis; D005796:Genes; D008239:Lysine / Q000031:analogs & derivatives*; D009154:Mutation; D014176:Protein Biosynthesis; D013696:Temperature
D001216:Asparagine; D004247:DNA; D000604:Amino Acyl-tRNA Synthetases; D008239:Lysine