SUPPRESSION OF TWO MISSENSE ALLELES OF THE TRPS LOCUS OF SACCHAROMYCES CEREVZSIAEl

ARJUN SINGH2 AND T. R. MANNEY3

Donner Laboratory, University of California, Berkeley, California 94720

Manuscript received February 25, 1974

ABSTRACT

A suppressor SUP101 of alleles trp5-67 and trp5-18 of the trp5 locus of Saccharomyces cereuisiae is described. The two suppressible mutations have been previously classified as missense. The suppression does not result from a physiological bypass of the tryptophan synthetase-catalyzed reaction, since the suppression is allele-specific. IU alleles trp5-70, tryp5-95, and trp5-102; IA alleles trp5-81, trp5-101, and trp5-103; and the ochre alleles trp5-33 and trp5-48 are not suppressed by SUPlOl. SUPlOl does not suppress ochre alleles ade2-I, his5-2, arg4-17, Zysl-I, amber alleles trpl-1, tyr7-I, or unclassified alleles a t a number of other loci. These results indicate SUPlOl is a missense suppressor. Growth on tryptophanless media is dependent upon gene dosage of both the suppressor and the suppressible alleles. Only the diploids homozy- gous both for the suppressor and suppressible alleles produce growth equivalent to growth of the haploids bearing a suppressible allele and the suppressor. Suppressor-bearing strains grow poorly even on tryptophan-supplemented media. In more than 100 asci analyzed partial growth inhibition on the com- plete medium always segregated with the suppressor.

ALLELE-specific and locus-nonspecific suppression is a well characterized phenomenon in bacteria and their viruses (see GORINI 1970 for a recent

review) . Involvement of niutationally altered tRNA’s in this kind of suppression has been shown both for nonsense (CAPECCHI and GUSSIN 1965; ENGLEHARDT et al. 1965) and missense (CARBON, BERG and YANOFSKY 1966; GUPTA and KHORANA 1966) mutations. Studies on super-suppression (HAWTHORNE and MORTIMER 1963) in yeast, which is analogous to nonsense suppression in bac- teria, are well documented (MANNEY 1964; GILMORE 1967; HAWTHORNE and MORTIMER 1968; HAWTHORNE 1968). The super-suppressors cause completion of prematurely terminated protein fragments (MANNEY 1968a; SHAFFER, RYTKA and FINK 1969) and insert specific amino acids at the positions encoded by the nonsense triplets (GILMORE, STEWART and SHERMAN 1968,1971). Evidence sug- gesting that tRNA is involved in super-suppression has been reported (BRUENN and JACOBSON 1972). In contrast there have been only scant reports on missense suppression in yeast. GORMAN and GORMAN (1971) have described two sup- pressors which act on his2-I, a mutation classified as missense on the basis of

1 This work was supported by the U. S. Public Health Service through research grant GM19175 from the National

2 Present address Department of Rehation Biology and Biophysics, School of Mehcine and Dentistry, The University

a Present address: Department of Physics, Kansas State University, Manhattan, Kansas 66506.

Institute of General Medical Sciences

of Rochester, Rochester, New York 14642.

Genetics 77: 661470 August, 1974.

662 A. SINGH AND T. R. MANNEY

leakiness, osmotic remediability and failure of response to two nonsense sup- pressors. A suppressor acting on many nonsense and missense mutation at ade2 locus has been reported (SIMAROV, MIRONOVA and INGE-VECHTOMOV 1971). The mutations were classified as nonsense or missense on the basis of their comple- mentation patterns. Finally, suppression of two alleles at ilvl locus has been con- cluded to be due to missense suppressors because these suppressors did not act on some nonsense alleles and on an allele which can be suppressed by metabolic suppressors ( GUNDELACH 1973).

In this article we describe an allele-specific suppressor which acts on alleles trp5-18 and trp5-67 of the trp5 locus which codes for tryptophan synthetase. These alleles have been classified as missense mutants on the basis of the follow- ing criteria: (1) Both participate in interallelic complementation (MANNEY et al. 1969). (2) None is suppressible by nonsense suppressors (MANNEY 1968a; A. SINGH and T. R. MANNEY, unpublished). (3) trp5-67 is an indole-utilizing (IU) mutant which retains the partial activity indole + serine + tryptophan and trp5-18 is an indole-accumulating (IA) mutant having the partial activity indole-glycerolphosphate +. indole + glyceraldehyde phosphate (MANNEY et al. 1969). (4) Both produce tryptophan synthetase of the same molecular weight as that produced by wild-type strains as judged by gel filtration on sephadex G2OO (M. RUDZIK and T. R. MANNEY, unpublished). This rules out the possi- bility that they could be nonsense mutants; nonsense mutations at the map positions of these alleles would produce detectably smaller proteins (MANNEY 1968~1, MANNEY et al. 1969).

Certain portions of these studies have been briefly reported (SINGH and MANNEY 1971).

MATERIALS AND METHODS

Yeast strains: The origin of partially active trp5 mutants used in these studies have been described (MANNEY et al. 1969). Other strains used were from DR. R. K. MORTIMER'S collection.

Media: Various synthetic culture media described by MANNEY (1964) were used. The routine culture medium was YEPD (1% yeast extract, 2% peptone and 2% dextrose).

Induction and selection of the suppressor: The haploid strain XT1176-S7 ( a trp5-67 ade6 leu1 u r d ) was treated with 3% ethyl methanesulfonate for 1 hour a t 30". The mutagenized culture was then handled as follows: The treated culture was grown in YEPD medium overnight then subjected to nystatin enrichment procedure according to SNOW (1966). A slow-growing colony on tryptophanless agar medium, designated as SC1 from the culture described above was sub- jected to further analysis.

Genetic methods: Standard procedures for construction of diploid strains and their genetic analysis were employed. Segregation of nutritional markers was scored on omission media by replica plating.

After it had been established that the suppressor (detected by its suppression of trp5-67) is not linked to the trp5 locus, the suppression of other trp5 alleles was tested by scoring spore tetrads from trp5-67 SUPIOl/trpS-X diploids. If trp5-X is suppressible, then the two suppressor- bearing spores in each ascus will, in every case, be tryptophan-independent; fiiding only 2+:2- tetrads therefore indicated that trp5-X is suppressible. However, if trp5-X were not suppressible than trp5-X SUP101 spores would be tryptophan-requiring. Consequently I +:3- and O+:+ tetrads respectively would result from tetratype and nonparental ditype segregations of SUP101 and the trp 5 alleles. Later the fact that SUP101 caused partial growth inhibition (see RESULTS)

MISSENSE SUPPRESSION I N YEAST 663

was exploited and the segregation of the suppressor could be scored on the basis of colony size on dissection slabs independently of its effect on the suppressible alleles.

Enzymatic activity: Indole + L-serine 4 L-tryptophan activity was tested in crude extracts of a suppressed trp5-18 mutant as described previously (MANNEY 1968b).

RESULTS

External suppression of trp5-67: A tryptophan-independent colony (strain SCI) obtained from a trp5-67 mutant (strain XTll76-S7) was crossed to the wild-type strain X2180-1B. Among twelve asci having four viable spores, 10 segregated 3:l while 2 segregated 2:2 for growth:nongrowth on tryptophanless medium. If the suppressor were linked to the trp5 locus, 3:l segregations would require at least a single crossover. Therefore, these results indicated that the tryptophan independence of SC1 was due to an external suppressor unlinked to the trp5 locus. The suppressor will hereafter be referred to as SUPIOI.

Allele-specificity of SUPIOI: To determine whether SUPIOI is a metabolic suppressor and therefore acts on al l trp5 alleles or whether it exhibits allele spe- cificity the following analysis was undertaken. A trp5-67 SUP101 segregant from the cross described above was crossed to four IU, four IA, and two ochre trp5 alleles. From 8 to 15 complete tetrads from each cross were analyzed. The data from this analysis are presented in Table 1. A consideration of the relevant geno- types (included in the Table) of the diploids reveals that if the test allele segre- gating in the cross is suppressible by SUP102 only 2:2 tetrads for growth and nongrowth on tryptophanless media should be obtained. On the other hand, if the test allele is not suppressible both 1 :3 and 0:4 in addition to 2:2 tetrads may be obtained. Furthermore, frequent occurrence of 1 :3 and 0:4 tetrads means that the particular test allele is not suppressible. Thus, as shown in Table 1 two alleles, trp5-18 and trp5-67 are suppressible and the remaining 8 alleles are not.

The possibility that trp5-I8 is not suppressible but 2+:2- segregation in all 9 tetrads from XA105 was obtained because SUP101 always segregated with trp5-67 should be considersed. Such a result is unlikely in view of the indepen-

TABLE 1

Analysis of suppressibility by SUP101 of 10 trp5 alleles

Number of tetrads (+ : - phenotypes)

Diploid no. trp5 test allele 0 : 4 1:3 2 : 2

XA105 18 0 0 9 XAllO 33 0 6 2 XA130 48 1 11 3 U 1 0 1 67 0 0 12 XA131 70 1 7 1 XA106 81 0 7 1 XA103 95 1 6 3 XA108 101 1 4 3 XA104 1 02 1 5 2 XA109 1 OB 0 5 3

Relevant genotype of diploids is of the form trp5-67/trp5-X SUPlOi/+.

664 A. S I N G H AND T. R. MANNEY

dent segregation of trp5 and SUP101 described in the previous section. Never- theless, the following results show that SUP101 does suppress trp5-18. trp5-18 is an IA mutation whereas trp5-67, as described before is an IU mutation. Thus the two alleles segregating in XA105 could be scored independently on trypto- phanless media supplemented with indole. The suppressor does not interfere with this scoring as growth of IU mutants is much better on indole than growth due to suppressor on media without indole and tryptophan because the suppression is rather weak (to be described subsequently). It was found that of the 18 spores that grew on tryptophanless medium, 8 carried trp5-67 and 10 carried trp5-18.

These results indicate that the suppression is allele-specific and is not due to any physiological bypass of the tryptophan-synthetase-catalyzed reaction. Fur- thermore, the results €rom diploid XAlOl which is homozygous for the sup- pressible allele trp5-67 show that the suppression is due to a single nuclear gene. The charaicteristics of the two mutations suppressed indicate that SUP101 is a missense suppressors.

A number of nonsense and unclassified mutations of other genes were tested for their susceptibility to SUP101. A trp5-67 SUP101 strain was crossed to strains carrying various mutatiocs and asci derived from them were analyzed. Exclusively 2:2 segregation for a marker indicated that it was not suppressible. In all cases this was confirmed by occurrence of segregants with trp5-67 SUP101 genotype and yet having the mutant phenotype with respect to the mutation being tested. The results, including characteristics of all mutations tested for suppressibility by SUPI01, are summarized in Table 2. The only mutations sup- pressed are trp5-18 and trp5-67. Again, the results are consistent with SUP101 being an allele-specific missense suppressor.

TABLE 2

Allele specificity of SUP101

Most probable Allele mutation type Suppression

trp5-67 IU (missense) + trp5-70 IU (missense) - trp5-95 IU (missense) - trp5-102 IU (missense) - trp5-18 IA (missense) + trp5-81 IA (missense) - irp5-1 01 IA (missense) - irp5-103 IA (missense) - trp5-33 Nonsense (ochre) - trp5-48 Nonsense (ochre) - his5-2 Nonsense (ochre) - arg4-17 Nonsense (ochre) - lysi-l Nonsense (ochre) - ade2-f Nonsense (ochre) - t r p i - f Nonsense (amber) - tyr7-1 Nonsense (amber) -

l e d , lys5, metl, ural unclassified - a d d , ade5,7, adeb, cyh.2, hid,

MISSENSE SUPPRESSION IN YEAST

TABLE 3

Daia on stability of SUP101

665

Strain No. colonies ~

tested No. irp- Percent trp- Percent of trp- colonies colonies colomes that are petite

- sc1 1510 0 0 XA953A 1450 0 0 XA101-4A 1381 1163 84 1.5

-

Stability of the suppressor: GORMAN and GORMAN (1971) uncovered a gene that restricted activity of a suppressor of his2-1, which is apparently a missense mutation, when they investigated instability of the suppressor. One of the inter- esting findings was that all stable his- colonies obtained from suppressed his2-1 strains were also petite. We conducted the following test to determine if this system were subject to similar instability. Three strains which had been main- tained on YEPD for several months were examined for the proportion of cells which still had the suppressed phenotype. As can be seen from Table 3, the suppressor in two strains is quite stable whereas most (84%) cells of the other strain have apparently lost the suppressor. Only 1.5% of the trp- colonies were petite, a percentage similar to petite cells which were still suppressed (2%). We have not crossed the trp- colonies to determine whether or not they contain the suppressor. Nevertheless it is clear that the instability of SUPlOl is not similar to that described by GORMAN and GORMAN ( 1971 ) .

Comparison of growth on tryptophanless medium of strains carrying the sup- pressor and suppressible alleles in uurious combinations: The relative growth of haploid and diploid strains was measured on tryptophanless agar medium. As shown in Table 4 both the suppressor and the suppressible alleles exhibit dosage effect on growth as judged by eye. The diploids which are homozygous both for the suppressor and the suppressible alleles manifest growth which is equivalent to the growth produced by suppressed haploid strains. Heteroallelic diploids carrying a suppressible and a nonsuppressible allele and having the suppressor

TABLE 4

Growth of haploid and diploid strains bearing suppressible and nonsuppressible alleles and SUP101 in various combinations

Strain

XA95-3A XA123 XAlOl XA124 XA103 XA105-3B XA126 XAl27 XA128 XA129

Relevant genotype Relative growth on

tryptophanless media

irp5-67 SUPlOl trp5-67/trp5-67 SUPlOl/SUP101 trp5-67/trp5-67 SUPlOl/+ trQ5-67/trp5-% s ~ P ~ o ~ / s ~ P ~ o ~ irp5-67/trp5-% SUPlOl/+ trp5-18 SUPloi! trp5-18/trp5-18 suPlol/suPlol trp5-18/trpS-l8 SUPlOl/+ trp5-i8/trp5-Y9 SUPlOl/SUPlOl trp5-18/trp5-90 SUPlOl/+

++ ++ + + ++ ++ + +

-

-

(366 A. SINGH AND T. R. MANNEY

in heterozygous condition produce no measurable growth. However, diploids carrying a suppressible and a nonsuppressible trp5 allele and the suppressor in all other combinations produce an intermediate amount of growth. The effect of the suppressor on heteroallelic diploids carrying both suppressible alleles cannot be tested as they complement each other. It should be noted that even the best growth (++) noted in Table 4 is much less than growth of any of the strains listed in Table 4 on tryptophan-supplemented medium or of wild-type strains on tryptophanless medium.

Growth inhibition caused by SUP101 : It was observed during the analysis of allele-specificity of SUP202 that two spores of each complete ascus dissected from the crosses which were heterozygous for SUP202 produced measurably smaller colonies on the YEPD dissection slabs than the other two spores from the same tetrad. Subsequent analysis showed that all segregants which contained SUP102 as scored by suppression of trp5-67 and/or trp5-18 had formed smaller colonies on the dissection slabs compared to other segregants. This indicated that the sup- pressor was inhibiting growth on this medium. The growth inhibition caused by SUP202 was confirmed by correlating the size of colony on the dissection slabs with the segregation of SUP202 (see Figure 1) in a cross where both spores con- taining the suppressor could be independently scored. It appears that the growth inhibition caused by the suppressor is expressed in a variety of genetic back- grounds. In more than 100 asici analyzed from a number of crosses heterozygous for a number of different genes, the growth inhibition always segregated with the suppressor.

A - + - - + - + - + + - - - - + - - - + - + + + + - + + + + + + - - + - + + - + - - - - + - - +

8 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

FIGURE 1 .-Colony size produced by tetrads from a diploid heterozygous for SUPf 01 (A) and n diploid not containing SUPiOi (B). The two parent diploids were isogenic except for the supprrssor contrnt and were homozygous for the suppressible allele irp5-67. The four spores from indivitlual asci are aligned vertically. The photographed was taken after two days' gro-eh on yeast extract-peptone-tlextrose medium at 30". The SUP101 genotypes of the spores, as scored hy its effect on irp5-67, are given at corresponding positions under them (+ = wild-type allele- i.e., no suppressor presrnt; - = SUPfOf-i. e., presence of the suppressor).

MISSENSE SUPPRESSION I N YEAST 667 TABLE 5

Effect on SUPlOl on growth

Growth properties on + Tryptophan - Tryptophan Lag Generation Lag Generation

Strain Relevant genotype period (hrs) time (hrs) period (hrs) time (hrs)

- - XT117gS7 trp5-67 4.5 2.3 sc1 trp5-67 SUP101 8.4 3.5 25 11.8

The loss of the suppressor from inany clones of XAiO1-4A (Table 3) provided the opportunity to test the notion that the growth inhibition was due to the sup- pressor itself and not to a mutation in another linked gene. Five clones each of trp+ and trp- XA1014A were streaked for single colonies on YEPD. In each case it was found that trpf clones produced smaller colonies than did trp- clones.

Results of a quantitative examination of effect of SUP101 on growth are pre- sented in Table 5. SC1 and its parent strain XTli76-S7 were grown in liquid synthetic complete media with and without tryptophan. Growth was measured by plating suitable dilutions on YEPD agar plates at intervals. It is seen that the suppressor causes significant prolongation of lag period and an increase in gener- ation time even on tryptophan-supplemented medium where the suppressor activity is not required for growth.

From the growth properties 02 SC1 on tryptophanless media it is obvious that the suppressor has a poor efficiency as indicated by growth. The enzymatic activity of a trp5-18 SUPlOl strain was measured for the indole *+ L-serine 9 L-tryptophan reaction. No activity could be detected. This method would have detected approximately 5% of the activity of the wild-type strain.

DISCUSSION

Properties such as participation in intracistronic complementation, nonsup- pressibility by nonsense suppressors, partial enzymatic activity, and production of enzyme of wild-type size indicate that trp5-18 and trp5-67 are missense mutants. It should be noted however, that none of these characteristics alone or even in conjunction with some other properties proves that a mutation is mis- sense. Nonsense mutations can have partial enzymatic activity (MANNEY 1968a) and are known to exhibit interallelic complementation at many loci (see MORTI- MER and GILMORE 1968). Although complementation by a nonsense mutation is usually polar, complementing only alleles that map on one side of it, a nonsense mutation showing nonpolar intragenic complementation has been reported (THURIAUX et al. 1971). Furthermore it is very likely that insertion by nonsense suppressors of some amino acids at sites corresponding to the nonsense mutations may not always lead to functional protein, and proteins missing only a few amino acids at the carboxy terminus due to premature chain termination caused by nonsense mutations may not be differentiated from thc wild-type protein by molecular weight estimates from gel filtration. The possession of all these proper- ties by the trp5-18 and frp5-67 mutations nearly rule out the notion that they

668 A. S I N G H A N D T. R. M A N N E Y

are nonsense mutations. An additional fact that adds more weight to the evidence that these mutants produce normal molecular weight tryptophan synthetase is that they are located in approximately the middle 1/3 of the trp5 locus. When considering differentiation between nonsense and missense mutations it should be noted that temperature sensitivity and osmotic remediability are also not always valid diagnostic criteria to distinguish missense from nonsense mutations (JONES 1972).

The allele specificity of SUPlOl indicates that suppression is not physiological; it does not result from a bypass of the tryptophan-synthetase-catalyzed reaction. In this connection it is important to note that the two suppressible alleles produce enzymes wih different activities; trp5 mutants have indole + L-serine -+ L-trypto- phan activity whereas the trp5-18 mutants have indole-3-glycerol phosphate + indole f glyceraldehyde-3-phosphate activity.

In bacterial systems where informational suppression has been studied in great detail both nonsense suppression ( CAPECCHI and GUSSIN 1965; ENGELHARDT et a2. 1965) and missense suppression (CARBON, BERG and YANOFSKY 1966; GUPTA and KHORANA 1966) have been found to involve altered tRNA’s. The super-suppressors of yeast which have been studied in much more detail than missense suppressors, are widely believed to be analogous to nonsense suppressors of bacteria, and involvement of altered tRNA’s has been suggested by many investigators. Biochemical evidence suggesting that altered tRNA‘s are involved in super-suppression has recently been published (BRUENN and JACOBSON 1972). The simplest explanation to explain the codon-specific missense suppression caused by SUP101 would be to assume that its function also is mediated by alteration in tRNA.

It is not unusual that only 2 of 8 possible missense trp5 alleles and none of several other uncharacterized mutations were suppressed even if some of the nonsuppressible mutations are indeed missense.

Suppression effected by SUP101 is quite inefficient if growth on tryptophan- less medium is considered a measure of efficiency. Failure to demonstrate indole 4- L-serine + L-tryptophan activity in the crude extracts of a trp5-18 SUP101 strain corroborates this. However, both these methods are very poor indices of suppression efficiency. Actual efficiency-i.e., percent of total polypeptides con- taining the amino acid inserted by the suppressor-can be quite high and yet the growth may be poor because of low enzymatic activity of the “corrected” protein.

If SUP101 mutation indeed results in a change in some tRNA the growth inhibition caused by the suppressor may be due to either excessive mistranslation or reduction of the corresponding normal tRNA to a deleterious level. Growth inhibition of SUPlOl-carrying strains is in marked contrast to the suppressors of his2-l (GORMAN and GORMAN 1971). Although suppressed, his2-1 strains grew nearly normally on histidineless medium, indicating perhaps high efficiency of suppression, yet none of the suppressors had any significant effect on growth rates. Two different suppressors together in the same cell, however, were defi- nitely deleterious. A similar observation has been reported by GILMORE (1967) for two ochre suppressors in the same cell.

MISSENSE SUPPRESSION IN YEAST 669 We wish to thank DR. R. K. MORTIMER for his hospitality in allowing us to conduct a portion

of this work in his laboratory. A. S. also thanks DR. FRED SHERMAN for his advice and interest during the preparation of the manuscript.

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Corresponding editor: G. R. FINK