Received May 23, 1968

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1 -4 MUTANT OF YEAST WITH A DEFECTIVE METHIONY L-tRN A SYNTHETASE CALVIN S. McLAUGHLIN AND LELAND H. HARTWELL2 Department of Molecular and Cell Biology, University of California, Irvine Received May 23, 1968 HE isolation of 40 temperature sensitive (ts-) mutants of yeast and their Tcharacterization with respect to patterns of macromolecular sythnesis at the nonpermissive temperature have been described previously (HARTWELL 1967). TWO mutants which displayed a rapid cessation of protein synthesis at the nonpermissive temperature were shown to have thermo-labile isoleucyl-trna synthetase enzymes (HARTWELL and MCLAUGHLIN 1968). Evidence will be presented in this report that another mutant with the same phenotype is defective in the methionyl-trna synthetase enzyme. Genetic studies are presented which examine the degree of linkage between the structural genes for the isoleucyl and methionyl-trna synthetases and between these two genes and centromere markers. The isoleucyl-trna synthetase gene (ID) has been mapped on chromosome 11. MATERIALS AND METHODS Strains: All strains used in this study are heterothallic haploid strains of Saccharomyces cerevisiae or hybrid diploids derived by mating. The parent strain, A364.4 and the origin of the mutants was described previously (HARTWELL 1967). The genotype of the parent strain was incorrectly reported however and should be: a gal ad, ad, url hi, Zy, tyl. A set of centromere marked strains was kindly provided by DR. ROBERT K. MORTIMER (University of California; Berkeley, California) and other strains were obtained from DR. DO C. HAWTHORNE (University of Washington; Seattle, Washington). Genetic techniques: Techniques for the isolation, sporulation, and tetrad dissection of hybrids and the techniques used for scoring the segregation of various genes including intergenic complementation and the composition of diagnostic media were taken directly from HAWTHORNE and MORTIMER (1960) and MORTIMER and HAWTHORNE (1966). The degree of centromere linkage of the t.r markers was determined from their second division segregation frequencies using well characterized centromere markers and the degree of linkage between two markers was computed from the relative abundance of parental ditype, nonparental ditype, and tetratype asci from dissected tetrads as described by MORTIMER and HAWTHORNE (1966). Only data from complete tetrads are reported. Aminoacy-tRNA msays: The preparation of cell-free extracts and the assay of amino acyltrna synthetases were described previously (HARTWELL and MCLAUGHLJN 1968). RESULTS Protein synthesis in mutant ts- 296: Approximately 20 temperature-sensitive (ts-) mutants of yeast out of our collection of 400 ts- mutants display a rapid This work was supported by Grant No GB 4828 from the Natlonal Science Foundation and by Grant No GM from the National Instltutes of Health, U S Public Health Service Present address Department of Genetics, University of Washington, Seattle Genetics 61: March 1969.

558 C. S. MC LAUGHLIN AND L. H. HARTWELL 0 1 2 0 1 2 H O U R S FIGURE 1.-Time course of protein synthesis in cultures of mutant ts296 and the parent strain, A364A, at 23 C and 36 C.

2 558 C. S. MC LAUGHLIN AND L. H. HARTWELL H O U R S FIGURE 1.-Time course of protein synthesis in cultures of mutant ts296 and the parent strain, A364A, at 23 C and 36 C. Cultures growing in YM-5 media were adjusted to a turbidity of 20 with a Klett-Summerson spectrophotometer by centrifugation and resuspension. After 1 hr of growth at 23 C O.lpC/ml ((24) of reconstituted protein hydrolysate (Schwarz Bioresearch) was added and one half of the culture was shifted to 36 C. Samples were removed at various times and analyzed for the amount of radioactivity incorporated into protein. The composition of the media and the analytical techniques were as described previously (HARTWELL 1967). cessation of protein synthesis following a shift to the nonpermissive temperature. Mutant ts- 296 is one such mutant. Figure 1 shows the time course of protein synthesis in a culture of the ts+ parent strain, A364A, and in a culture of the mutant, ts- 296, at 23 C (the permissive temperature) and at 36 C (the nonpermissive temperature). The mutant grows slightly slower at the permissive temperature than does the parent strain and this fact is reflected in a slightly slower rate of protein synthesis at 23 C. After a shift from 23 C to 36 C the culture of the parent strain, A364A, synthesizes protein at an increased rate while the mutant culture by comparison synthesizes protein at a much reduced rate for about 30 min after which time protein synthesis ceases. Aminoacyl-tRNA synthetase suruey: The rapid inhibition of protein synthesis in cultures of mutant ts- 269 following a shift to the nonpermissive temperature led us to suspect that the mutant might have a lesion in one of the enzymes involved in protein synthesis. Extracts of mutant ts- 296 and the parent strain, A364A, were therefore assayed for 16 different aminoacyl-trna synthetase activities. The extracts were preincubated for 30 min at 35 C to maximize the chance of detecting a thermolabile enzyme and then assayed at 25 C. The specific activities for 15 of the 16 aminoacyl-trna synthetases are approximately the same in the two extracts (Table 1 ). Only one enzymatic activity was strikingly low in the mutant extract, that of the methionyl-trna synthetase. The mutant extract displays less than 3% of the activity of the extract from the parent strain. Methionyl-tRNA synthetase artiuity as a function of protein concentration: In order to further characterize the nature of the defect, extracts were assayed at a series of protein concentrations for the methionyl-trna synthetase activity

METHIONYL-tRNA SYNTHETASE MUTANT 559 TABLE 1 Survey of aminoacyl-trna synthetase activities in extracts of mutant ts-269 and strain A364A mfi moles of aminoacyl-trna formed/mg of proieinfiour at 25 C

3 METHIONYL-tRNA SYNTHETASE MUTANT 559 TABLE 1 Survey of aminoacyl-trna synthetase activities in extracts of mutant ts-269 and strain A364A mfi moles of aminoacyl-trna formed/mg of proieinfiour at 25 C Amino acid A364A is-296 Ratio ts-296/a364a Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine < oo < (Figure 2). The extract from the parent strain, A364A, shows linear activity as a function of protein concentration up to 100 pg of protein in the incubation mixture. The extract from mutant ts- 296 displays very little if any activity at all protein concentrations. A maximal estimate of the residual activity of the mutant extract is about 4% of that of the parent strain. A mixed extract containing equal amounts of protein from the parent and the mutant extract displayed an activity equivalent to that expected from the sum of the two activities (Figure 2). This result indicates that the absence of activity in the mutant extract cannot be due to an inhibitor of enzyme activity or to an enzyme which destroys one of the substrates. Furthermore, the ts- 296 mutation has been shown to be recessive (HARTWELL 1967) ; this fact is also inconsistent with the hypothesis of an inhibitor of the enzyme or destroyer of its substrates. The assays reported in this section were all performed at 25"C, near the permissive temperature, in an attempt to demonstrate some methionyl-trna synthetase activity in mutant extracts; it should also be mentioned that many assays were performed at 36"C, the nonpermissive temperature, and that again no activity was observed. Segregation of the enzymatic defect at miosis: Since these ts- mutants were isolated after heavy mutagenesis with nitrosoguanidine and since the methionyltrna synthetase is defective in extracts at both temperatures, it is essential to establish that the enzymatic defect observed in extracts of the mutant is the same defect which prohibits growth at the nonpermissive temperature. This identity can be established by showing that the enzymatic deiect segregates in tetrads from heterozygous (ts-/ts+ ) diploids with the gene determining temperaturesensitivity of growth. Tetrads from crosses between mutant ts- 296 and tsf strains

4 560 C. S. MC LAUGHLIN AND L. H. HARTWELL 3 To 4 X E n 2 " ug OF PROTEIN FIGURE 2.-Methionyl-tRNA synthetase activity as a function of protein concentration in extracts of mutant ts-296, the parent strain, A364A, and a mixed extract. Extracts containing the designated protein concentrations were incubated at 25% for 30 min. The mixed extract contained equal amounts of protein from the mutant and parent extracts to give a total protein concentration as indicated on the abscissa. segregated ts-:ts+ in approximately 3:l ratios and gave poor spore germination indicating that this mutant had multiple defects. The ts- segregants were outcrossed twice to tsf strains and a ts- segregant was obtained (H14.3.2). A hybrid (H19) between H and A (tsf) gave good spore germination and segregated ts-:ts+ in a 2:2 ratio. Table 2 records the results of marker segregation in four tetrads from hybrid H19. Each of the sixteen haploid segregants was grown up and assayed for methionyl-trna synthetase activity. Temperaturesensitivity segregated in a ratio of 2 -:2 + as did the other markers. Furthermore, the defective methionyl-trna synthetase activity also segregated 2- : 2+ and the pattern of segregation was in all cases identical with the growth response at 36 C. Since a diploid yeast cell has at least fourteen chromosome pairs and a total map length estimated at 3600 centimorgans ( MORTIMER and HAWTHORNE

5 METHIONYL-tRNA SYNTHETASE MUTANT TABLE Analysis of tetrads from hybrid HI9 Methionyl-tRNA Growth at: Requirements synthetase Mating CPM/30 min Tetrad Spore 23OC 36% me le, url lyll type at 25 C/100&g protein Q a f a a a a a 60 - _ + a a a a _ a a ), the linkage of two independently arising mutations is very unlikely. Thus these data provide strong evidence that the inability to grow at 36 C is a result of a mutation in the gene controlling the function of the methionyl-trna synthetase enzyme. The original mutant, ts- 296, displayed a requirement for methionine at the permissive temperature (23"C), although the parent strain from which it was derived did not require methionine. Furthermore, the two ts+ strains used for outcrossing and the ts+ strain, A did not require methionine. Strain H retains this methionine requirement and it segregates in the tetrads from hybrid H19 in an identical fashion to ts-. These data suggest that the methionine requirement is related to the defective methionyl-trna synthetase. Centromere linkage of the methionyl and isoleucyl trna synthetase genes: Forty-seven complete tetrads from hybrid H19 (H a ts-296 me- x A (Y le,) which is heterozygous for the methionyl-trna synthetase defect and 79 complete tetrads from hybrid H7 (A364Aa ts- 341 x A CY lee) which is heterozygous for the isoleucyl-trna synthetase defect were analyzed for marker segregation (Table 3). Both hybrids were heterozygous for le2, a marker closely linked to the centromere of chromosome 111. From the frequency of tetratype asci with respect to the ts- and le, markers and the known second division segregation frequency of le, (13%; MORTIMER and HAWTHORNE 1966) it is possible to calculate the second division segregation frequencies of the ts- markers using

6 5 62 C. S. MC LAUGHLIN AND L. H. HARTWELL TABLE 3 Second division segregation frequencies of the ts-296 and ts-341 mutations Percent second division Gene pair Total asci scored Tetratype asci segregation of ts- marker the equation of PERKINS (1949)3. A centromere-linked marker is expected to display a second division segregation frequency of less than 67%. In forty-five of the forty-seven tetrads from hybrid HI9 le- : le+ segregated 2 : 2 and the ts- 296 mutation (methionyl-trna synthetase) displayed a second division segregation frequency of 63% relative to le; indicating that it is not showing significant centromere linkage. In 45 of the 47 tetrads from hybrid HI9 ts-:ts+ and me-:me+ segregated 2:2 and all were parental ditypes with respect to these two phenotypes. This finding further substantiates the contention that the methionine requirement of this mutant is closely linked to the lesion producing temperature-sensitivity. In one of the exceptional tetrads from hybrid H19 le+ : le- segregated 3:l and in another 1:3; in two other tetrads me+:me- segregated 3:l and 1:3. The latter observation might suggest gene conversion at the me locus without concomitant conversion at the ts locus and indicate therefore that the methionine requirement and temperature sensitivity are due to two closely linked independent mutations. However, the methionine requirement is difficult to score due to its variable expression and further evidence strongly suggests that the methionine requirement and temperature-sensitivity are due to the same mutation. Sixteen ts+ revertants were isolated from strain H All but one of these sixteen revertants also reverted to me+. Four of these revertants were assayed for methionyl-trna synthetase activity; one revertant exhibited wild-type methionyl-trna synthetase activity, a second intermediate activity and the remaining two very low activities. Mutation ts- 341 (isoleucyl-trna synthetase) gave a second division segregation frequency of 53% indicating that it is centromere linked. Test of linkage between ts- 296 and ts- 341: It is important to establish whether or not these two mutations are closely linked on the yeast genetic map since close linkage might imply the existence of an operon controlling the expression of aminoacyl-trna synthetase genes. The fact that ts- 341 shows centromere linkage while ts- 296 does not suggests that the two mutations are not closely linked. However, the extent of linkage between two genes (or a centromere and a gene) can vary from hybrid to hybrid so that it is essential to answer this question in a less ambiguous manner. A hybrid diploid was constnicted which was heterozygous for both the ts- 296 mutation and the ts- 341 mutation. Linkage would be indicated if the tetrads segregating from this hybrid showed a significant egcess of parental ditype relative to nonparental ditype asci. The two markers could be Frequency of tetratype asci = I + y - 3/2 xy where I and y are the second division segregation frequencies of the two markers in question,

7 METHIONYL-tRNA SYNTHETASE MUTANT 563 scored independently by complementation tests of each of the segregating haploid spores and the ts- 296 mutation could be scored as well by the methionine requirement which segregates with this mutation. From 35 complete tetrads which were analyzed 7 parental ditype asci, 6 nonparental ditype asci, and 22 tetratype asci were observed indicating that the two mutations are not closely linked on the yeast genetic map. Map location of ts- 341: Since the ts- 341 mutation (isoleucyl-trna synthetase) displays centromere linkage an attempt was made to find out which of the fourteen chromosomes it was located on by performing crosses with markers showing centromere linkage on each of these chromosomes. The only clear cut evidence for linkage is that between ts- 341 and gal, a centromere-linked marker on chromosome I1 (Table 4). Approximately 55% of the asci in which the two genes were segregating were tetratype and no nonparental ditypes were found among the 42 asci that were analyzed. The centromere linkage data reported in Table 3 together with the data of MORTIMER and HAWTHORNE (1966) indicating that gal has a second-division segregation frequency of 13% lead us to conclude that ts- 341 is further from the centromere than is gal. Linkage was then examined between ts- 341 and markers to the right (lyp) and to the left (ps) of the centromere and the gal marker. A hybrid (H7) heterozygous for ly2 and ts- 341 segregated 18 parental ditype: 9 nonparental ditype: 33 tetratype asci with respect to these two markers indicating that they are not closely linked. Next tetrads were dissected from a hybrid (H33) heterozygous for ts- 341, ps, gal, and two centromere markers on other chromosomes (trl and lea). The patterns of marker segregation were analyzed in 53 complete tetrads from hybrid H33 (Table 5). The second division segregation frequencies indicate that the order of distances from the centromere TABLE 4 Tetrad analysis of mutation ts-341 with respect to fourteen centromere markers Pattern of segregation Parental Nonparental Chromosome Marker ditype &type Tetratype I I1 gal I11 1% IV tr V UT VI hi, VI1 le VI11 ar IX hi, X is XI me XI1 thr, XI11 1Y XIV P

8 5 64 C. S. MC LAUGHLIN AND L. H. HARTWELL TABLE 5 Tetrad analysis of marker segregation in hybrid H33 Division segregation Percent Marker 1st Division 2nd Division 2nd Division ts p gal Pattern of segregation Gene pair Parental ditype Nonparental ditype Tetratype Computed map units is-341-p, pg-ga, ts341-gal is ts- 341 (64)>pg(40)>ga1(8). The values of 40% and 8% second division segregation for the markers pg and gal, respectively, are in accord with the values of 37% and 13% reported by MORTIMER and HAWTHQRNE (1966). Map distances computed from the frequencies of parental ditype: nonparental ditype: tetratype asci establish the gene order ZLS (ts- 341) -p9-gal as shown in Figure 3; the numerical distances should be considered approximate as an insufficient number of tetrads were examined to provide accurate data. We have chosen the symbol ZLS for this gene. The map distance between ps and gal (24 centimorgans) observed in this cross agrees well with the value of 26 reported by MORTIMER and HAWTHORNE (1966). The ts- 341 mutation lies approximately 22 map units to the left of pg. An analysis of crossover patterns in individual tetrads confirms the gene order ZLS-pg-gal as opposed to the other two possibilities (pg-zls-gal and pg-gal-zls), since the latter two orders would have required many more double crossovers to generate the observed data than would the former order. In particular, two tetrads were observed requiring a double crossover pattern for the order ZLS-p,-gal and triple crossover patterns for each of the other two models. This location for the structural gene of the isolencyl-trna synthetase extends the genetic map of Saccharomyces approximately 20 units to the left of previously known markers on chromosome 11. I LS (ti3411 ps gal y2,yi hi7 - I I I I I I I I I FIGURE 3.-The map location of the isoleucyl-trna synthetase gene (ZLS) on chromosome I1 of Saccharomyces. The map locations of markers other than ZLS are froin MORTIMER and HAWTHORNE (1966); not all markers known to lie on this chromosome are included. The map units recorded in this figure are from the data presented in Table 5.

METHIONYL-tRNA SYNTHETASE MUTANT 5 65 DISCUSSION A single mutation reduces the activity of the methionyl-trna synthetase in cell-free extracts of yeast by greater than 95%.

9 METHIONYL-tRNA SYNTHETASE MUTANT 5 65 DISCUSSION A single mutation reduces the activity of the methionyl-trna synthetase in cell-free extracts of yeast by greater than 95%. Thus, a haploid yeast cell pmbably contains a single species of this enzyme which charges all species of methionyl-trna. Furthermore, if a derivative of methionine is the initiator of protein synthesis in yeast as it is in E. coli, (CLARK and R/IARcKER 1966; ADAMS and CAPECCHI 1966; WEBSTER, ENGELHARDT, and ZINDER 1966) and if the blocking of the amino group of methionine occurs after activation as it does in E. coli then it is likely that this same enzyme charges the initiator trna. It is curious that extracts from the mutant have no activity for methionyltrna synthetase even at the permissive temperature. This situation is not without precedence, however, since 'a temperature-sensitive mutant of E. coli defective in the valyl-trna synthetase has been described (EIDLIC and NEID- HARDT 1965) which displays no valyl-trna charging activity in cell-free extracts at the permissive temperature. A reasonable explanation for this behavior is that the conditions used for the enzyme assay in vitro do not reproduce the in vim conditions which permit either the activation or stabilization of the enzyme. The fact that this mutant acquired a requirement for methionine which segregates in tetrads with the temperature-sensitive mutation provides a clue regarding the nature of the defect in this enzyme. It suggests that the mutant form of the enzyme requires high concentrations of methionine,one of its substrates, under in vivo conditions at the permissive temperature either for the activation of the enzyme or for its stabilization. A similar situation has been described for the tryptophanyl-trna synthetase of E. coli (DOOLITTLE and YANOFSKY 1968) ; certain mutants selected as tryptophan auxotrophs have been shown to have tryptophanyl-trna synthetases with reduced affinity for tryptophan. Our finding that the structural genes for the methionyl- and isoleucyl-trna synthetases are not closely linked fails to provide any evidence for an operon controlling the expression of the aminoacyl-trna synthetases. This observation is not, however, surprising since functionally related genes rarely show linkage on the yeast genetic map. Furthermore, the aminoacyl-trna synthetase genes do not appear to be clustered on the genetic niap of E. coli (NEIDHARDT 1966) an organism abundantly endowed with operons. The authors thank DR. HERSCHEL ROMAN for his constructive criticism of this manuscript. SUMMARY A temperature-sensitive mutant of yeast is described which undergoes a rapid cessation of protein synthesis following a shift to the nonpermissive temperature and displays very low activities for the methionyl-trna synthetase in cell extracts. The mutation producing the defective methionyl-trna synthetase segregates in tetrads with the mutation causing temperature-sensitivity of growth and the two are probably therefore identical. The methionyl-rna synthetase gene is not linked to a previously described mutation in the isoleucyl-trna

10 566 C. S. MC LAUGHLIN AND L. H. HARTWELL synthetase gene. The isoleucyl-trna synthetase gene (ZLS) has been located approximately 22 units to the left of the marker pg on chromosome I1 of the yeast genetic map. LITERATURE CITED ADAMS, J. M., and M. R. CAPECCHI, 1966 N-formyl-methionyl-sRNA as the initiator of protein synthesis. Proc. Natl. Acad. Sci. U. S. 55: CLARK, B. F. C., and K. A. MARCKER, 1966 The role of N-formyl-methionyl-sRNA in protein biosynthesis. J. Mol. Biol. 17: DOOLITTLE, W. F., and C. YANOFSKY, 1968 Mutants of E. coli with an altered tryptophanyltransfer ribonucleic acid synthetase. J. Bacteriol. 95: EIDLIC, L., and F. C. NEIDHARDT, 1965 Protein and nucleic acid synthesis in two mutants of Escherichia coli with temperature-sensitive aminoacyl ribonucleic acid synthetases. J. Bacteriol. 89: HARTWELL, L. H., 1967 Macromolecule synthesis in temperature-sensitive mutants of yeast. J. Bacteriol. 93: HARTWELL, L. H., and C. S. MCLAUGHLIN, 1968 Mutants of yeast with temperature-sensitive isoleucyl-trna synthetases. Proc. Natl. Acad. Sci. US. 59: HAWTHORNE, D. C., and R. IC. MORTIMER, 1960 Chromosome mapping in Saccharomyces: centromere-linked genes. Genetics 45: IO. MORTIMER, R. K., and D. C. HAWTHORNE, 1966 Genetic mapping in Saccharomyces. Genetics 53: NEIDHARDT, F. C., 1966 Roles of activating enzymes in cellular physiology. Bacteriol. Revs. 30: PERKINS, D. D., Biochemical mutants in the smut fungus Ustilago maydis. Genetics 34: WEBSTER, R. E., D. L. ENGELHARDT, and N. D. ZINDER, 1966 initiation. Proc. Natl. Acad. Sci. U. S. 55: In vitro protein synthesis: chain

Isoleucine and Valine Metabolism of Escherichia coli

JOURNAL OF BACTERIOLOGY, May 1968, P. 1680-1684 Copyright @ 1968 American Society for Microbiology Vol. 95, No. 5 Printed in U.S.A. Isoleucine and Valine Metabolism of Escherichia coli XVI. Pattern of