Current Genetics
Current Genetics (1983) 7:285-288
© Springer-Verlag 1983
A Mutant of Saccharomyces cerevisiae Defective in Arginyl-tRNA-Protein Transferase Margaret Savage,1 Richard L. Soffer,1 and Michael J. Leibowitz 2 1 Departments of Biochemistry and Medicine, Cornell University Medical College, New York, NY 10021, USA 2 Department of Microbiology, UMDNJ-Rutgers Medical School, P.O. Box 101, Piscataway, NJ 08854, USA
Summary. A mutant of Saccharomyces cerevisiae deficient in arginyl-tRNA-protein transferase has been isolated. The responsible mutation designated ate1, was localized near the centromere of chromosome VII. It probably involves the structural gene for the transferase since residual enzyme activity in the mutant is temperature-sensitive. Key words: Arginyl-tRNA-Protein transferase - Yeast Post-translational modification
Introduction
Aminoacyl-tRNA-protein transferases are enzymes catalyzing the transfer of certain amino acids from tRNA into peptide linkage with specific residues of acceptor proteins (reviewed by Deutch et al. 1978; Softer 1974 and 1980). This post-translational addition of amino acids to protein acceptors is believed to play a role in the regulation of the biological activities of these acceptors. The availability of mutants deficient in specific transferase enzymes will be necessary in order to define the function these ubiquitous enzymes play in cellular regulation. Gram-negative bacilli contain leucyl, phenylalanyltRNA-protein transferase, which catalyzes the transfer of leucine and phenylalanine from tRNA into peptide linkage with amino-terminal basic residues of acceptor proteins (Leibowitz and Softer 1970). The complex role of this transferase in bacterial metabolic regulation was demonstrated by studies of an Escherichia coli mutant deficient in this enzyme (Deutch et al. 1977; Deutch and Softer 1975; Scarpulla et al. 1976; Scarpulla and Offprint requests to: M. J. Leibowitz
Soffer'1979; Softer and Savage 1974). A leuyl-tRNAprotein transferase activity with properties different from the bacterial enzyme recently has been discovered in mammalian brain tissue (Laughrea 1982). All eukaryotic cells, including those of plants, mammals, and eukaryotic protists, contain arginyl-tRNAprotein transferase (Deutch et al. 1978; Horinishi et al. 1975; Softer 1980). This enzyme catalyzes the transfer of arginine from tRNA into peptide linkage with aminoterminal dicarboxylic amino acid residues on acceptor proteins (Softer 1970). The conservation of arginyltRNA-protein transferase among all eukaryotic cells, including mammalian cells grown in tissue culture (reviewed by Deutch et al. 1978) suggests that the presence of this enzyme activity confers some selectable advantage. This paper describes the isolation and characterization of a mutant of Saccharomyces cerevisiae which produces thermolabile enzyme. The responsible mutation-(atel) is located near the centromene of chromosome VII and probably represents the structural gene for the enzyme. The availability of this mutant should facilitate the study of the physiologic role of this ubiquitous enzyme of eukaryotes.
Materials and Methods Strains, Media and Mutagenesis. Strains of S. cerevisiae used in this work are shown in Table 1. Growth media included YPAD (rich medium), SD (minimal medium) and others as described (Wickner t974), and genetic methods were those described previously (Mortimer and Schild 1981). Mutagenesis of strain $288C was performed with 3% ethylmethanesuffonate (Wickner 1974), and survivors(0.9%) were isolated on YPAD at 22 °C. Screening of Mutants. Extracts were prepared from cells grown overnight (22 °C) in YPAD (10 ml). The cells were suspended in 0.4 ml of a buffer containing 10 mM Tris-HC1,pH 7.8, and 10
286
M. Savage et al.: ATE1 Locus of Yeast
Table 1. Strains of Saccharomyces cerevisiae Strain
Genotypea
Source or Reference
$288C A364A L594-48A Ate-1 166 191 M522 L574-2C L574-8A S100-3B $100-3D
~ gaI2 [KIL-o] a adel ade2 tyrl lys2 ural his 7 gaH [KIL-k] a trp5 leul ade6 ural [KIL-k] c~gal2 ate1 ts [KIL-o] a leul his5 canrl-l O0 a trpl [KIL-k] ~ his 7 ate1 [KIL-k] ~ ade2 t y r l his7 lys2 ate1 [KIL-k] a ade 2 his 7 ate1 [KIL-k] a A T E 1 [KIL-k] a ate1 [KIL-k]
G. Fink Hartwell et al. 1970 This Work This Work R. Wickner R. Wickner This Work This Work This Work This Work This Work
a The notations [KIL-k] and [KIL-o] refer to the presence or absence of the killer plasmid (Wickner and Leibowitz 1976)
Table 2. Segregation of ate1 a Cross
L574 L580 L582 L583 L617
Number of Tetrads (Ate-1 x A364A) ($288C x L574-8A) (191 x L574-2C) (166 x L574-8A) (M522 x L594-48A)
Total
4+: 0 -
15 8 14 14 38 89
0
3+: 1 -
2+: 2 -
1+: 3 -
1
3 1
14 8 14 11 36
4
83
2
0+: 4 -
1 0
a The predominantly 2+: 2 - segregation pattern is that expected for the segregation of a single chromosomal gene in crosses of the type ate1 x A T E +
mM 2-mercaptoethanol and were broken with glass beads (0.5 mm diam.) in a Braun homogenizer (5 min, with 2 sec liquid CO2 cooling per 20 sec). Beads and debris were removed by centrifugation at 15,000 x g for 3 min, and the supernates were then assayed for ability to catalyze the transfer of [ 14C]-arginine from tRNA into protein (Softer 1970). Reaction mixtures (75 #1) contained 50 mM Tris-HC1, pH 9.0, 0,15 M KC1, 50 mM 2-mercaptoethanol, 0.2 mM puromycin, bovine serum albumin (5 mg/ml) as acceptor, 0.6-2.3 #M [14C]-arginyl-tRNA and extracts in the amounts indicated. Radioactivity insoluble in hot 5% trichloroacetic acid was determined on 50 ul aliquots after incubation as described (Mans and Novelli 1960). Protein concentrations were determined (Lowry et al. 1951) using bovine serum albumin as standard.
Results and Discussion Among the cell extracts made from 399 survivors of mutagenesis, one isolate exhibited very low levels of arginyl-tRNA-protein transferase activity when assayed for 1 hour at 37 °C after a 5 min preincubation at this temperature. This strain (Ate-1) was temperature-sensitive for growth, but in crosses with wild type strains indepen-
dent 2 : 2 segregation was noted for the temperaturesensitivity and the transferase defect. In multiple crosses, the transferase defect segregated upon meiosis as a single chromosomal gene (Table 2), designated a t e 1 . In all crosses, the transferase phenotype was determined by assaying cell extracts for 1 h at 37 °C (after 5 min preincubation) in reaction mixtures containing 25 ~1 of extract and 0.6 ~M [14C]-arginyl tRNA (379 cpm/ pmol). In these assays, extracts of segregants denoted A T E + catalyzed the incorporation of 5 4 4 - 1 , 4 8 8 cpm, wherease those of ate1 segregants catalyzed the transfer o f 1 - 1 0 1 cpm. Extracts of heterozygous diploid cells exhibited a wild type level of enzyme, while homozygous a t e l / a t e l diploids contained enzyme levels similar to those found in a t e 1 haploids, thus suggesting that the ate1 mutation is recessive. Data obtained from the crosses shown in Table 2 indicate that the transferase gene is centromere-linked. As shown in Table 3, the ate1 gene is located 2 centimorgans from l e u l on chromosome VII, and exhibits the expected linkage to the t r p 5 and a d e 6 loci on the same chromosome. Analysis of the data for tetrads showing
287
M. Savage et al.: ATE1 Locus of Yeast Table 3. Genetic linkage of ate1 to trp5 and ade6 on chromosome VIIa
trp5
ate1
leu l
PD NPD T cM PD NPD T cM PD NPD T cM
Table 4. Temperature-sensitivity of arginyl-tRNA-protein transferase in an atel mutant Strain
30 17 0 3 9 18 12 (17.6) b 47 (69.5) b
Growth Temperature
Assay Temperature
Enzyme specific activity a (pmol/min/mg protein)
$100-3B (ATE+)
46 0 2 2
25 °C 25 °C 37 °C 37 °C
25 °C 37 °C 25 °C 37 °C
6.3 26 8.4 27.0
S100-3D (atel)
25 °C 25 °C 37 °C 37 °C
25 °C 37 °C 25 °C 37 °C
atel
leul
31 0 6 8
ade6
18 1 17 32 20 1 17 30 (39.2) b
a For each pair of genes, the number of parental ditype (PD), nonparental ditype (NPD), and tetratype (T) asci is given. These data represent the pooled data from crosses L583 and L617. Each cross showed greater than 96% spore viability. Genetic distances are calculated in centimorgans (eM) by the method of Perkins (1949) b Genetic distances in brackets are from Mortimer and Schild (1980)
recombination in the trpS-leul region confirms a gene order of trpS-atel-leul, and establishes that the ate1 locus is 2 centimorgans centromere-distal to leul. As is indicated in Table 4, the ate1 mutation results in arginyl-tRNA-protein transferase activity which is temperature-sensitive. Enzyme activity in extracts from atel cells grown at 25 °C or 37 °C is reduced relative to wild-type cells, and this residual activity is temperaturesensitive (Table 4). Appropriate mixing experiments (not shown) excluded the possibility that diminished enzyme activity was due to an inhibitor. The temperature-sensitivity of arginyl-tRNA-protein transferase activity in ate1 mutants suggests that this locus corresponds to the structural gene coding for the enzyme rather than a gene whose product regulates the synthesis o f the transferase. The physiologic function of arginyl-tRNA-protein transferase is unknown. Strains of X cerevisiae bearing the ate1 mutation described in this report grow normally at 37 °C, use various carbon and nitrogen sources and vitamins normally and show no defects in respiration or killer plasmid (Wickner 1974; Wickner and Leibowitz 1976) functions. Arginyl-tRNA-protein transferase may therefore not be essential for growth under the physiological conditions examined. It may be that the conditions required to test the functional defect in the mutant depend on the genetic background of the strain, as is the case for the E. coli mutant defective in leucyl, phenylalanyl-tRNA-protein transferase (Deutch and
0.59 0.14 0.50 0.10
Enzyme assays (40 /d) for arginyl-tRNA-protein transferase were performed without preincubation on supernatant fractions (105,000 x g, 90 min) prepared from lystates of the indicated strains (0.3-4.2 mg protein/ml). Substrate [14Cl-arginyl-tNA (345 cpm/pmol) was present at 2.31 #M, and incubations were carried out for 5 min
Softer 1975; Deutch et al. 1977; Scarpulla and Softer 1979). The evolutionary conservation of this specific animoacyl-tRNA-protein transferase in eukaryotes as disparate as animals, plants, yeast and fungi (Deutch et al. 1978; Horinishi et al. 1975; Softer and Deutch 1975) suggests that some selective advantage is conferred by the gene encoding this enzyme. Acknowledgements. This research was supported by a Basil O'Connor Starter Research Grant (No. 5-201) from the National Foundation for Birth Defects of M.J.L. and by NSF Grant BMS-74-23970 to R.L.S.M.J.L is an Alexandrine and Alexander L. Sinsheimer Scholar.
References Deutch CE, Scarpulla RC, Softer RL (1978) Curr Top Cell Reg 13:1-28 Deutch CE, Scarpulla RC, Sonnenblick EB, Softer RL (1977) J Bacteriol 129:544-546 Deutch CE, Softer RL (1975) Proc Natl Acad Sci USA 75: 405-408 HartweU LH, Culotti J, Reid B (1970) Proc Natl Acad Sci USA 66:352-359 Hawthorne DC, Mortimer RK (1960) Genetics 45:1085-1110 Horinishi H, Hashizume S, Seguchi M (1975) Biochem Biophys Res Commun 65:82-88 Laughrea M (1982) Biochemistry 21:5694-5700 Leibowitz MJ, Softer RL (1970) J Biol Chem 245:2066-2073 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) J Biol Chem 193:265-275
288 Mans RJ, Novelli GD (1960) Biochem Biophys Res Commun 3:540-543 Mortimer RK, Schild D (1980) Microbiol Rev 44:519-571 Mortimer RK, Sehild D (1981) In: Strathern JN, Jones EW, Broach JR (eds) The Molecular Biology of the Yeast Saccharomyces, Life Cycle and Inheritance. Cold Spring Harbor Laboratory, New York, pp 11-26 Perkins DD (1949) Genetics 34:607-626 Scarpulta RC, Deutch CE, Softer RL (1976) Biochem Biophys Res Commun 71:584-589 Scarpulla RC, Softer RL (1979) J Biol Chem 254:1724-1725 Soffer RL (1970) J Biol Chem 245:731-737 Softer RL (1974) Adv Enzymol 40:91-139 Softer RL (1980 In: Soil D, Abelson JN, Schimmel PR (eds)
M. Savage et al.: ATE1 Locus of Yeast tRNA: Biological Aspects. Cold Spring Harbor Laboratory, New York, pp 493-505 Softer RL, Deutch CE (1975) Biochem Biophys Res Commun 64:926-931 Soffer RL, Savage M (1974) Proc Natl Acad Sci USA 71:10041007 Wickner RB (1974) Genetics 76:423-432 Wickner RB, Leibowitz MJ (1976) Genetics 82:429-442
Communicated by M. S. Esposito Received March 10, 1983
Current Genetics (1983) 7:285-288
© Springer-Verlag 1983
A Mutant of Saccharomyces cerevisiae Defective in Arginyl-tRNA-Protein Transferase Margaret Savage,1 Richard L. Soffer,1 and Michael J. Leibowitz 2 1 Departments of Biochemistry and Medicine, Cornell University Medical College, New York, NY 10021, USA 2 Department of Microbiology, UMDNJ-Rutgers Medical School, P.O. Box 101, Piscataway, NJ 08854, USA
Summary. A mutant of Saccharomyces cerevisiae deficient in arginyl-tRNA-protein transferase has been isolated. The responsible mutation designated ate1, was localized near the centromere of chromosome VII. It probably involves the structural gene for the transferase since residual enzyme activity in the mutant is temperature-sensitive. Key words: Arginyl-tRNA-Protein transferase - Yeast Post-translational modification
Introduction
Aminoacyl-tRNA-protein transferases are enzymes catalyzing the transfer of certain amino acids from tRNA into peptide linkage with specific residues of acceptor proteins (reviewed by Deutch et al. 1978; Softer 1974 and 1980). This post-translational addition of amino acids to protein acceptors is believed to play a role in the regulation of the biological activities of these acceptors. The availability of mutants deficient in specific transferase enzymes will be necessary in order to define the function these ubiquitous enzymes play in cellular regulation. Gram-negative bacilli contain leucyl, phenylalanyltRNA-protein transferase, which catalyzes the transfer of leucine and phenylalanine from tRNA into peptide linkage with amino-terminal basic residues of acceptor proteins (Leibowitz and Softer 1970). The complex role of this transferase in bacterial metabolic regulation was demonstrated by studies of an Escherichia coli mutant deficient in this enzyme (Deutch et al. 1977; Deutch and Softer 1975; Scarpulla et al. 1976; Scarpulla and Offprint requests to: M. J. Leibowitz
Soffer'1979; Softer and Savage 1974). A leuyl-tRNAprotein transferase activity with properties different from the bacterial enzyme recently has been discovered in mammalian brain tissue (Laughrea 1982). All eukaryotic cells, including those of plants, mammals, and eukaryotic protists, contain arginyl-tRNAprotein transferase (Deutch et al. 1978; Horinishi et al. 1975; Softer 1980). This enzyme catalyzes the transfer of arginine from tRNA into peptide linkage with aminoterminal dicarboxylic amino acid residues on acceptor proteins (Softer 1970). The conservation of arginyltRNA-protein transferase among all eukaryotic cells, including mammalian cells grown in tissue culture (reviewed by Deutch et al. 1978) suggests that the presence of this enzyme activity confers some selectable advantage. This paper describes the isolation and characterization of a mutant of Saccharomyces cerevisiae which produces thermolabile enzyme. The responsible mutation-(atel) is located near the centromene of chromosome VII and probably represents the structural gene for the enzyme. The availability of this mutant should facilitate the study of the physiologic role of this ubiquitous enzyme of eukaryotes.
Materials and Methods Strains, Media and Mutagenesis. Strains of S. cerevisiae used in this work are shown in Table 1. Growth media included YPAD (rich medium), SD (minimal medium) and others as described (Wickner t974), and genetic methods were those described previously (Mortimer and Schild 1981). Mutagenesis of strain $288C was performed with 3% ethylmethanesuffonate (Wickner 1974), and survivors(0.9%) were isolated on YPAD at 22 °C. Screening of Mutants. Extracts were prepared from cells grown overnight (22 °C) in YPAD (10 ml). The cells were suspended in 0.4 ml of a buffer containing 10 mM Tris-HC1,pH 7.8, and 10
286
M. Savage et al.: ATE1 Locus of Yeast
Table 1. Strains of Saccharomyces cerevisiae Strain
Genotypea
Source or Reference
$288C A364A L594-48A Ate-1 166 191 M522 L574-2C L574-8A S100-3B $100-3D
~ gaI2 [KIL-o] a adel ade2 tyrl lys2 ural his 7 gaH [KIL-k] a trp5 leul ade6 ural [KIL-k] c~gal2 ate1 ts [KIL-o] a leul his5 canrl-l O0 a trpl [KIL-k] ~ his 7 ate1 [KIL-k] ~ ade2 t y r l his7 lys2 ate1 [KIL-k] a ade 2 his 7 ate1 [KIL-k] a A T E 1 [KIL-k] a ate1 [KIL-k]
G. Fink Hartwell et al. 1970 This Work This Work R. Wickner R. Wickner This Work This Work This Work This Work This Work
a The notations [KIL-k] and [KIL-o] refer to the presence or absence of the killer plasmid (Wickner and Leibowitz 1976)
Table 2. Segregation of ate1 a Cross
L574 L580 L582 L583 L617
Number of Tetrads (Ate-1 x A364A) ($288C x L574-8A) (191 x L574-2C) (166 x L574-8A) (M522 x L594-48A)
Total
4+: 0 -
15 8 14 14 38 89
0
3+: 1 -
2+: 2 -
1+: 3 -
1
3 1
14 8 14 11 36
4
83
2
0+: 4 -
1 0
a The predominantly 2+: 2 - segregation pattern is that expected for the segregation of a single chromosomal gene in crosses of the type ate1 x A T E +
mM 2-mercaptoethanol and were broken with glass beads (0.5 mm diam.) in a Braun homogenizer (5 min, with 2 sec liquid CO2 cooling per 20 sec). Beads and debris were removed by centrifugation at 15,000 x g for 3 min, and the supernates were then assayed for ability to catalyze the transfer of [ 14C]-arginine from tRNA into protein (Softer 1970). Reaction mixtures (75 #1) contained 50 mM Tris-HC1, pH 9.0, 0,15 M KC1, 50 mM 2-mercaptoethanol, 0.2 mM puromycin, bovine serum albumin (5 mg/ml) as acceptor, 0.6-2.3 #M [14C]-arginyl-tRNA and extracts in the amounts indicated. Radioactivity insoluble in hot 5% trichloroacetic acid was determined on 50 ul aliquots after incubation as described (Mans and Novelli 1960). Protein concentrations were determined (Lowry et al. 1951) using bovine serum albumin as standard.
Results and Discussion Among the cell extracts made from 399 survivors of mutagenesis, one isolate exhibited very low levels of arginyl-tRNA-protein transferase activity when assayed for 1 hour at 37 °C after a 5 min preincubation at this temperature. This strain (Ate-1) was temperature-sensitive for growth, but in crosses with wild type strains indepen-
dent 2 : 2 segregation was noted for the temperaturesensitivity and the transferase defect. In multiple crosses, the transferase defect segregated upon meiosis as a single chromosomal gene (Table 2), designated a t e 1 . In all crosses, the transferase phenotype was determined by assaying cell extracts for 1 h at 37 °C (after 5 min preincubation) in reaction mixtures containing 25 ~1 of extract and 0.6 ~M [14C]-arginyl tRNA (379 cpm/ pmol). In these assays, extracts of segregants denoted A T E + catalyzed the incorporation of 5 4 4 - 1 , 4 8 8 cpm, wherease those of ate1 segregants catalyzed the transfer o f 1 - 1 0 1 cpm. Extracts of heterozygous diploid cells exhibited a wild type level of enzyme, while homozygous a t e l / a t e l diploids contained enzyme levels similar to those found in a t e 1 haploids, thus suggesting that the ate1 mutation is recessive. Data obtained from the crosses shown in Table 2 indicate that the transferase gene is centromere-linked. As shown in Table 3, the ate1 gene is located 2 centimorgans from l e u l on chromosome VII, and exhibits the expected linkage to the t r p 5 and a d e 6 loci on the same chromosome. Analysis of the data for tetrads showing
287
M. Savage et al.: ATE1 Locus of Yeast Table 3. Genetic linkage of ate1 to trp5 and ade6 on chromosome VIIa
trp5
ate1
leu l
PD NPD T cM PD NPD T cM PD NPD T cM
Table 4. Temperature-sensitivity of arginyl-tRNA-protein transferase in an atel mutant Strain
30 17 0 3 9 18 12 (17.6) b 47 (69.5) b
Growth Temperature
Assay Temperature
Enzyme specific activity a (pmol/min/mg protein)
$100-3B (ATE+)
46 0 2 2
25 °C 25 °C 37 °C 37 °C
25 °C 37 °C 25 °C 37 °C
6.3 26 8.4 27.0
S100-3D (atel)
25 °C 25 °C 37 °C 37 °C
25 °C 37 °C 25 °C 37 °C
atel
leul
31 0 6 8
ade6
18 1 17 32 20 1 17 30 (39.2) b
a For each pair of genes, the number of parental ditype (PD), nonparental ditype (NPD), and tetratype (T) asci is given. These data represent the pooled data from crosses L583 and L617. Each cross showed greater than 96% spore viability. Genetic distances are calculated in centimorgans (eM) by the method of Perkins (1949) b Genetic distances in brackets are from Mortimer and Schild (1980)
recombination in the trpS-leul region confirms a gene order of trpS-atel-leul, and establishes that the ate1 locus is 2 centimorgans centromere-distal to leul. As is indicated in Table 4, the ate1 mutation results in arginyl-tRNA-protein transferase activity which is temperature-sensitive. Enzyme activity in extracts from atel cells grown at 25 °C or 37 °C is reduced relative to wild-type cells, and this residual activity is temperaturesensitive (Table 4). Appropriate mixing experiments (not shown) excluded the possibility that diminished enzyme activity was due to an inhibitor. The temperature-sensitivity of arginyl-tRNA-protein transferase activity in ate1 mutants suggests that this locus corresponds to the structural gene coding for the enzyme rather than a gene whose product regulates the synthesis o f the transferase. The physiologic function of arginyl-tRNA-protein transferase is unknown. Strains of X cerevisiae bearing the ate1 mutation described in this report grow normally at 37 °C, use various carbon and nitrogen sources and vitamins normally and show no defects in respiration or killer plasmid (Wickner 1974; Wickner and Leibowitz 1976) functions. Arginyl-tRNA-protein transferase may therefore not be essential for growth under the physiological conditions examined. It may be that the conditions required to test the functional defect in the mutant depend on the genetic background of the strain, as is the case for the E. coli mutant defective in leucyl, phenylalanyl-tRNA-protein transferase (Deutch and
0.59 0.14 0.50 0.10
Enzyme assays (40 /d) for arginyl-tRNA-protein transferase were performed without preincubation on supernatant fractions (105,000 x g, 90 min) prepared from lystates of the indicated strains (0.3-4.2 mg protein/ml). Substrate [14Cl-arginyl-tNA (345 cpm/pmol) was present at 2.31 #M, and incubations were carried out for 5 min
Softer 1975; Deutch et al. 1977; Scarpulla and Softer 1979). The evolutionary conservation of this specific animoacyl-tRNA-protein transferase in eukaryotes as disparate as animals, plants, yeast and fungi (Deutch et al. 1978; Horinishi et al. 1975; Softer and Deutch 1975) suggests that some selective advantage is conferred by the gene encoding this enzyme. Acknowledgements. This research was supported by a Basil O'Connor Starter Research Grant (No. 5-201) from the National Foundation for Birth Defects of M.J.L. and by NSF Grant BMS-74-23970 to R.L.S.M.J.L is an Alexandrine and Alexander L. Sinsheimer Scholar.
References Deutch CE, Scarpulla RC, Softer RL (1978) Curr Top Cell Reg 13:1-28 Deutch CE, Scarpulla RC, Sonnenblick EB, Softer RL (1977) J Bacteriol 129:544-546 Deutch CE, Softer RL (1975) Proc Natl Acad Sci USA 75: 405-408 HartweU LH, Culotti J, Reid B (1970) Proc Natl Acad Sci USA 66:352-359 Hawthorne DC, Mortimer RK (1960) Genetics 45:1085-1110 Horinishi H, Hashizume S, Seguchi M (1975) Biochem Biophys Res Commun 65:82-88 Laughrea M (1982) Biochemistry 21:5694-5700 Leibowitz MJ, Softer RL (1970) J Biol Chem 245:2066-2073 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) J Biol Chem 193:265-275
288 Mans RJ, Novelli GD (1960) Biochem Biophys Res Commun 3:540-543 Mortimer RK, Schild D (1980) Microbiol Rev 44:519-571 Mortimer RK, Sehild D (1981) In: Strathern JN, Jones EW, Broach JR (eds) The Molecular Biology of the Yeast Saccharomyces, Life Cycle and Inheritance. Cold Spring Harbor Laboratory, New York, pp 11-26 Perkins DD (1949) Genetics 34:607-626 Scarpulta RC, Deutch CE, Softer RL (1976) Biochem Biophys Res Commun 71:584-589 Scarpulla RC, Softer RL (1979) J Biol Chem 254:1724-1725 Soffer RL (1970) J Biol Chem 245:731-737 Softer RL (1974) Adv Enzymol 40:91-139 Softer RL (1980 In: Soil D, Abelson JN, Schimmel PR (eds)
M. Savage et al.: ATE1 Locus of Yeast tRNA: Biological Aspects. Cold Spring Harbor Laboratory, New York, pp 493-505 Softer RL, Deutch CE (1975) Biochem Biophys Res Commun 64:926-931 Soffer RL, Savage M (1974) Proc Natl Acad Sci USA 71:10041007 Wickner RB (1974) Genetics 76:423-432 Wickner RB, Leibowitz MJ (1976) Genetics 82:429-442
Communicated by M. S. Esposito Received March 10, 1983
