Mutants of yeast with temperature-sensitive isoleucyl-tRNA synthetases.

In order to investigate the mechanisms of macromolecule synthesis and their regulation in a eucaryotic organism, we have undertaken an investigation of temperature-sensitive mutants in yeast. The isolation of temperature-sensitive mutants of Saccharomyces cerevisiae and their characterization with respect to macromolecule synthesis, morphology, and cell division at the nonpermissive temperature has been described previously.1 Such mutants display a variety of phenotypes, including those consistent with a primary lesion, in one of the following processes: protein synthesis, RNA synthesis, DNA synthesis, cell-wall formation, and cell division. We wish to report biochemical and genetic experiments which indicate that two of the mutants from the protein synthesis class, ts-341 and ts-443, have single, allelic temperature-sensitive (ts-) mutations which result in a thermolabile isoleucyl-tRNA synthetase enzyme. Materials and Methods.-Yeast strains: The following heterothallic, haploid strains of Saccharomyces cerevisiae were used in this investigation: A346A (a gal hi7 trl url ad1 ad2 ly2 tyl), X1069-2D (a ad1 hi4 le2 thr4 me2 tr5 ur,), 1505-3B (a ga3 ga7 hi5 ur3 mewo ly2 lyll), and 2262-2A (a gal his url ad1 le2 lyll). All strains were obtained from the collection of Drs. Roman and Hawthorne at the University of Washington, Seattle, except for strain X1069-2D which was obtained from Dr. Mortimer at the University of California, Berkeley. The mutants ts-341 and ts-443 were derived from strain A364A by mutagenesis with nitrosoguanidine.1 Media: The enriched liquid medium, YM-1, was described previously.1 To score marker segregation, synthetic medium was used containing 6.7 gm/liter yeast nitrogen base without amino acids (Difco), 10 gm/liter glucose, 20 gm/liter agar, and whatever biochemical compounds were needed to supplement auxotrophic requirements. Chemicals: Adenosine triphosphate (ATP) and cytidine 5'-triphosphate (CTP) were purchased from Sigma Chemical Company. Yeast tRNA was obtained from General Biochemicals Co. Radioactive amino acids were purchased from Schwarz BioResearch, Inc. C'4 amino acids had a specific activity of 50 mc/mmole and a concentration of 1 mM. H3 methionine from Schwarz was adjusted to 125 mc/mmole, 1 mM concentration. Preparation of aminoacyl-tRNA synthetases: Yeast were grown in medium YM-1 at 230 to a density of 100 Klett units. The cells were harvested by centrifugation, and spheroplast preparations2 were lysed in a 0.01 AM Tris-maleate pH 7.6, 0.0005 M magnesium acetate, 0.02 A3 KCl buffer by homogenization. The extract was centrifuged at 27,000 g for 20 min, and the supernatant was centrifuged at 229,000 g for 70 min. The ribosomal pellet was discarded and the supernatant was dialyzed overnight against a 0.01 M trisacetate pH 7.6, 0.0005 M magnesium acetate, 0.02 AM KCl buffer. Assay of aminoacyl-tRNA synthetase activtity: The formation of aminoacyl-tRNA was assayed on DEAE paper (Whatman DE81).3 Each 100 Mditer incubation contained in a 0.05 M tris-acetate pH 7.6, 0.05 AI magnesium acetate, 0.02 At KCI buffer (buffer A), 0.5 jmole of ATP, 0.05 Mmole of CTP, 5 msmoles of C14 or H3 amino acid, 0.2 mg of yeast tRNA, 0.1 mg of gelatin, and 0.1 mg of enzyme protein. The incubation was carried out for 30 min at the specified temperature. The amount of isoleucyl-tRNA formed is proportional to the enzyme concentration up to concentrations of 0.2 mg of protein per incubation. Protein was determined by the Lowry procedure.4