GENETIC ANALYSIS OF A GENE REQUIRED FOR THE EXPRESSION OF ALLELE-SPECIFIC MISSENSE SUPPRESSION I N

SACCHAROMYCES CEREVISIAE

JESSICA A. GORMAN AND JOHN GORMAN

Department of Cell Biology, University of Kentucky, Lexington, Kentucky 40506

Received September 22, 1970

HE suppression of a nutritional requirement imposed by a mutant gene by a Tsecond unrelated mutation is a well documented phenomenon in microorgan- isms [see GORINI and BECKWITH (1966) for a review of suppression]. In bacteria, altered transfer ribonucleic acid (tRNA) has been demonstrated to effect mis- sense (CARBON, BERG and YANOFSKY 1966) and nonsense ( CAPECCHI and GUSSIN 1965) suppression. There is increasing evidence that the mechanism of suppres- sion in yeast is analogous to that demonstrated in bacteria (GILMORE, STEWART and SHERMAN 1968; MANNEY 1968), although a direct demonstration of an altered tRNA in suppressed strains is still lacking.

The activity of a suppressor may be altered by mutations in other genes. For example, certain ribosomal mutations, such as streptomycin resistance, result in restriction of the suppression normally exerted by tRNA informational suppres- sors ( LEDERBERG, CAVALLI-SFORZA and LEDERBERG 1964; GORINI 1969; APIRION, PHILLIPS and SCHLESSINGER 1969). The present paper describes the genetic analysis of a mutation in Saccharomyces cerevisiae which affects the expression of allele-specific missense suppressors.

The histidine-requiring mutant employed in this study, his2-I, carries a mu- tation in the structural gene for the biosynthetic enzyme L-histidinol phosphatase (EC.3.1.3.15). Allele-specific suppressors of this mutant have been isolated which allow growth of suppressed strains at nearly wild-type rates. A study of auxo- trophic “desuppressed” mutants was initiated when it was noted that suppressed strains were not stable under certain cultural conditions, and gave rise to mixtures of suppressed and nonsuppressed cells. The relatively high frequency of reversion to auxotrophy indicated that the loss of suppression might involve changes in some cytoplasmic component of the cell rather than gene mutation. A similar type of phenotypic instability in yeast is found in regard to respiratory capacity, where cytoplasmic petites arise spontaneously with high frequency. The observation that the majority of the histidine-requiring revertants isolated from a suppressed strain were also cytoplasmic petites promoted the idea that the suppression might depend on a cytoplasmic component. possibly the mitochondria. One example of a cytoplasmically inherited mutation which ‘Lsuppresses” the activity of a non- sense suppressor has been reported (Cox 1965). Further study of suppressed and “desuppressed” strains might give additional insight into the mechanism of sup-

Genetics 67: 337-352 March, 1971

338 J. A. G O R M A N A N D J. G O R M A N

pression in yeast. The results of genetic studies of three missense suppressors and a back revertant of one of these are presented here.

MATERIALS A N D M E T H O D S

Strains: The histidine-requiring mutant primarily employed, strain 4766C (crhis2-2) has been previously described (GORMAN and H u 1969). This allele, as well as several others used to examine suppressor specificity were derived from strains of the Berkeley stock collection, as listed in Table 1 . The ochre (trp5-48, arg4-17, lysl-1, l e d - 1 , and his5-2), amber (try7-l and t r p l - I ) , and leul-19 mutants were kindly provided by Dr. FRED SHERMAN. All other mutants were iso- lated following mutagenesis of the wild-type strain D273-1OB with ethyl methanesulfonate or nitrous acid.

Media The following media were employed, either as liquid or solidified with 2% agar for plates.

YPG: Difco yeast extract 1 %, Difco Bacto-Peptone 1 %, and 2% dextrose. This medium was used for routine propagation of cultures and stock maintenance.

YP-glycerol: Similar to YPG, with dextrose reduced to 0.1% and supplemented with 2% glycerol. This medium differentiates respiratory-sufficient (grande) and respiratory-deficient (petite) colonies.

YNB: Difco yeast nitrogen base (without amino acids) and 2% dextrose, used to score nutritional requirements. The various supplements (histidine, uracil, tryptophan, etc.) were added as needed to give a final concentration of 20 pg/ml.

YNB-glycerol: YNB with 2% glycerol instead of glucose. SP: Sporulation medium, containing 1 % potassium acetate, 0.25% yeast extract, and 0.1 %

dextrose. Zsolation of suppressors: All suppressors were isolated by selecting for spontaneous revertants

to prototrophy for a given nutritional marker in haploid strains. The ochre and amber suppressors were isolated from revertants of a his4-l and a tyr7-1 mutant, respectively. Of the alleles tested, the ochre suppressor, which was designated SUP-H4, suppresses all the ochre mutants listed in Table 5, while the amber suppressor, designated SUP-T2, suppresses trpl-l and tyr7-1, the only two amber alleles tested.

While they have not been mapped or tested for allelism with the 22 different nonsense sup- pressors described by HAWTHORNE and MORTIMER (1968), these two suppressors are not linked to the his2 gene.

Classification of mutanfs: All the mutants listed in Table 5 were examined for leaky growth, osmotic-remedial growth on minimal medium supplemented with IM KC1 (HAWTHORNE and FRIIS 1964.), temperature-sensitive growth, and sensitivity to suppression by the nonsense sup- pressors SUP-H4 and SUP-TI. On the basis of characteristics summarized in Table 1, strains can be roughly grouped into three classes: (a) nonsense mutants, which are suppressed by SUP-H4 or SUP-HZ; (b) osmotic-remedial, temperature-sensitive, or leaky mutants, which do not appear to be supersuppressible; (c) mutants which show neither (a) nor (b) characteristics. The properties of the group (b) mutants are generally indicative of missense mutations (HAWTHORNE and FRIIS 1964) and not characteristic of nonsense mutations (HAWTHORNE 1969).

While it has been suggested that the leaky growth of his2 mutants may be the result of non- specific alkaline phosphatase rather than a partially active histidinol phosphatase, two obser- vations argue against this. (1) A his2 mutant, his2-5, has been isolated which is not leaky and has normal levels of nonspecific alkaline phosphatase activity, and (2) a his2-l mutant which is also devoid of nonspecific alkaline phosphatase activity has been isolated (GORMAN, unpublished results). The double mutant retains the property of leaky growth on histidine-deficient medium. Until data to the contrary are obtained, it is reasonable to consider his2-l to be a missense mutation.

Genetic methods: Diploids were constructed by mass mating the parent haploids on YPG and isolating individual zygotes by micromanipulation. Sporulation, dissection, and tetrad analysis

RESTRICTION O F SUPPRESSION IN YEAST

TABLE 1

General characteristics of the mutants employed

339

Allele

- h i s 4 his5 h i s 2 - 1

h i s 6

h i s 8

h i s 1

h i s10

u r a l

- - - - -

Strain of origin

S -1795 -A s - 8 2 7 - ~

S-732-C

X- 901 -3 5 C S -123 7- B

S -395 -D JB-96 S-733-A

New Mutants

h i s k - 1 h i s4 -3

h i s2 -2 h i s 2 - 1

his2-3

h i s 2 - 4

h i s 5 - 4 h i s 6 - 2

h i s l o - 2 h i s ? - 1

his?-3 h i s 8 - 1

h i s2 -5

Growth characteristics

Osmotic- Temperature. Leaky remedial sensitive

- i

1.

t

c

t +

- + 3 3 O C + 3 3 O C

Suppression by SUP-HI SUP-T2

- *

* -These alleles were not tested for suppression by SUP-TZ.

followed standard genetic techniques (HAWTHORNE and MORTIMER 1960). Nutritional require- ments of diploids and haploid segregants were scored on appropriately supplemented YNB agar plates. The unlinked markers ural and leul-12 were employed in crosses to score for false tetrads. As these two alleles are not affected by any of the suppressors studied, the segregation of these markers is not included in the table. Only tetrads giving regular segregation of all markers are included in the analysis, unless otherwise noted.

Terminology: The terminology employed follows the proposed system of genetic nomen- clature for yeast genetics presented in the Yeast Genetics Supplement to Microbial Genet. Bull. No. 31, 1969.

RESULTS

Suppressors of his2- 1 : Three suppressors were derived from independently isolated spontaneous revertants of strain 486-6C (his2-I) . Each of the prototrophic revertants, 9-R, F-R, and L-1, was shown to contain a dominant suppressor of the his2-1 allele by appropriate genetic crosses (Table 2). Each revertant gave segre- gation ratios of 2 histidine-independent (his + ) to 2 histidine-requiring (his-)

340 J. A. GORMAN A N D J. GORMAN

TABLE 2

Segregation data demonstrating the presence of unlinked suppressor genes

Cross no. Parent strains

Segregation ratios (his+ : his-)

4 : O 3 : l 2:2

JK 142 9-R x his2-l 0 0 30 JK 144. F-R x his2-I 0 0 22 JK 100 L-l x his2-1 0 0 28 JK 137 9-R x wild type 7 9 7 JK 145 F-R x wild type 3 10 6 JK 106 L-I x wild type 9 12 10

spores in each tetrad when crossed to a his2-l auxotroph, and ratios of 4:0, 3: 1, and 2:2 (his4:his-) when crossed to wild type, indicating the segregation of an unlinked suppressor gene. To confirm the presence of each suppressor, a his+ spore from a 2:2 tetrad of each of the crosses JK 137, JK 145, and JK 106 was crossed back to strain 476-6C (Table 3). The segregation ratios for histidine re- quirement were those expected if the his+ parent carried the wild-type HIS2 allele and an unlinked suppressor gene. The three suppressors have been desig- nated SUP-HI, SUP-H2, and SUP-H3, as indicated in Table 3. The wild-type allele of each suppressor is either not indicated or indicated by a -1- when needed for clarity. Centromere linkage of SUP-HI and SUP-H3 is indicated by the segre- gation ratios shown in Tables 2 and 3. Employing data from all crosses involving a suppressor and a known centromere-linked gene leu1 or trpl , second-division segregation frequencies were calculated for each suppressor (HAWTHORNE and MORTIMER 1960). The second-division segregation frequencies of SUP-HI and SUP-H3 are significantly lower than 0.667, indicating centromere linkage. The values are approximately 0.16 for SUP-HI and 0.12 for SUP-H3. There is no indication that SUP-H2, with a second-division segregation frequency of 0.55, is centromere linked.

Crosses to determine allelism of suppressors: To determine if the three sup- pressors involve mutations in the same gene, suppressed strains were crossed to each other; the results are given in Table 4. The recovery of his- haploid spores in crosses JK 211 and JK 325 indicated that SUP-H2 was not allelic to either SUP-HI or SUP-H3. However, cross JK 338 indicated that SUP-HI and SUP-H3 are probably allelic, for only parental ditype asci were recovered. While the

TABLE 3

Crosses demonstrating suppressors in his+ segregants

Segregation ratios Parent strains (his+ : his-) Genotype of

his+ parent Cross no. his+ his2-I 4.0 3: l 2:2

JK 155 137-3C x 476-6C 7 9 9 SUP-HI JK 327 145-7D X 476-6C 2 10 7 SUP-H2 JK 229 106-2A X 4766C 7 5 4 SUP-H3

RESTRICTION O F SUPPRESSION IN YEAST

TABLE 4

Crosses to determine the allelism of suppressors

341

Cross no. Parent strains

Segregation (his+ : his-)

4 : O 3:l 2:2

JK 211 hisZ-I SUP-HI x his2-I SUP-HZ 6 10 0 JK 325 hisZ-l SUP-H2 x hi&-I SUP-H3 5 11 4 JK 338 his2-I SUP-HI X his2-I SUP-H3 35 0 0

germination of JK 325 and JK 338 was excellent, it was relatively poor for JK 21 1. Out of 32 asci dissected, only 16 complete tetrads were recovered. In each tetrad segregating 3 his+ to 1 his-, one his+ segregant grew very poorly and had a dis- tinctive granular colonial morphology. These spores probably contain both sup- pressors SUP-HI and SUP-H2, which might account for the poor growth. This has been found to be the case for certain combinations of super suppressors ( GIL- MORE 1967). Attempts to directly verify this were unsuccessful, as crosses of these segregants to his2-I strains failed to germinate. As the ascus type ratios for the cross involving SUP-H3 (which appears to be allelic to SUP-HI) and SUP-H2 gave no indication of linkage, it is unlikely that linkage of SUP-HI to SUP-H2 can account for the lack of recovery of 2:2 tetrads in JK 21 1.

Allele specificity of suppressors: The three suppressors are highly allele speci- fic. failing to suppress any other mutants tested (Table 5). The inability to sup- press other his2 mutants ruled out the possibility that the suppression involved a bypass of this enzymatic step in histidine biosynthesis. Several mutants listed in the table are osmotically remedial, i.e., are capable of slow growth in media with- out histidine when the osmotic pressure of the medium is increased by the addition of KCl or organic solutes. Therefore, suppression of only the his-2-1 allele indicat- ed that the suppression does not involve a genetically controlled physiological change in the cellular environment, causing a1 teration in the tertiary configur- ation of certain proteins. Such a mechanism has been postulated to explain the osmotic-remedial growth of many missense mutants (HAWTHORNE and FRIIS 1964).

On the basis of the different nonsense alleles tested, it can be concluded that these suppressors do not fall into any of the ten phenotypic classes of nonsense suppressors described by HAWTHORNE and MORTIMER (1968). The highly re- stricted suppression favors the interpretation that these suppressors represent missense suppressors analogous to the nonsense suppressors of yeast, involving mutations in genes whose products are associated with protein translation (see DISCUSSION).

Eficiency of suppression: If the growth rate in minimal medium is used as an index of the efficiency of each suppressor in restoring histidinol phosphatase ac- iivity, SUP-Ill and SUP-H3 are very efficient, for the rate of growth is only slightly lower than that of the wild type (Table 6). A significantly reduced rate under conditions requiring suppression is evident only for the his2-l strain sup- pressed by SUP-H2. In histidine-supplemented medium, where growth should

342 J. A. G O R M A N AND J. GORMAN

TABLE 5

Allele specificity of suppressors

- SUP-Ill SUP-€12 SUP-H3

Nonsense muta t ions

t ryp5-48 a r o 4 - 1 1

l eu2-1 lysl-1

t r p l - 1 t y r 7 - 1

h i s 4 h i d - 3 * h i s & - 1 h i s 5 - 2 *

Pos,s ible mis sense muta t ions

h i s 2 - 1

h i s 2 - 4

h i s 2 - 2 h i s 2 - 7

h i s 2 - 3 his6 his8 h i s 6 - 2 h i s 3 - 1 h i s 8 - 1

h i s 5 - 4 h i s lD-2 h i s3 -3

U n c l a s s i f i e d

h i s 1

l e d - 1 2 & h i s 2 - 5

-

+ + +

Y

* * Y lr

* ic

* it

i c *

A plus (+) indicates suppression; a minus (-), no suppression; an asterisk (*), not tested.

not be limited by the efficiency of histidine suppression, the growth rate is only slightly, if at all, reduced by the presence of a single suppressor, indicating that total protein synthesis is not significantly affected by the suppressor gene. In strains carrying two suppressors, however, there is a noticeably reduced growth rate. As these rates are the same in minimal and histidine-supplemented media, this would appear to reflect a general effect on overall growth rather than a re- duced efficiency of histidine suppression.

Instability of suppressed strains: Strains of the three suppressors appear to be unstable under certain conditions. When various cultures were streaked onto minimal medium after being maintained on YPG stock slants, the initial growth was frequently poor and two to four days were required to obtain adequate growth. The appearance of single colonies in heavily streaked areas indicated that only a portion of the cells were actually growing. To test for reversion back to histidine auxotrophy, stock cultures were diluted and plated on YPG. then replica- plated onto YNB with and without histidine.

Variable proportions of his- colonies were found in the different stocks. ranging from about 1 % up to 90%. No correlation between the specific suppressor tested

RESTRICTION O F SUPPRESSION IN YEAST

TABLE 6

Growth rates of strains carrying suppressors

343

Genotype YNB 4- histidine YNB

+ + his2-I + f SUP-HI + SUP-H2 + SUP-H3

his2-I SUP-HI h id - I SUP-HZ his2-I SUP-H3 hid-I SUP-HI SUP-H2 his2-I SUP-HZ SUP-H3

108 120 125 114 136 130 128 135 165 210

110

125 115 138 130 195 135 168 21 0

>20 hours

Each value given represents the average of three experiments. Differences of 15 min or less are not considered significant, as variation was found between individual experiments using the same strain under identical conditions. Each strain was precultured in YNB-histidine to provide the inocula for the two experimental media. All experiments were at 28°C. Cultures were grown with constant aeration by shaking in a Psycrotherm incubator shaker.

and the amount of instability was evident. When individual colonies were picked and retested, the his+ colonies gave no indication of instability. The majority of colonies selected as his- also had regained the ability to grow without histidine. Apparently they had only become phenotypically his- during a long stationary phase on YPG. Between 0.1 and 5% of the his- colonies tested were stable auxo- trophs. The interesting finding was that all of the stable his- colonies derived from strains of SUP-H3 were also petite. A second experiment, involving his2-l SUP-H3 segregants from three different crosses gave the same result: all stable his- colonies were petite. The apparent correlation between the loss of suppression and loss of respiratory sufficiency in these cells indicated a possible role of func- tional mitochondria in suppressor activity, and invited further study of these his- “desuppressed” isolates.

Induction of cytoplasmic petites: Cytoplasmic petites were induced by two methods in strains suppressed by each of the three suppressors: (1 ) overnight growth at 37°C in liquid YPG, and (2) growth in YNB containing 100 pg/ml acriflavin for 4 hr. None of the petites obtained by either procedures had lost the ability to grow without histidine. Thus petiteness per se does not appear sufficient to cause a loss of suppression.

Genetic analysis of an auxotrophic revertant: A stable his- colony isolated from L-1 (his2-1 SUP-H3) was selected for genetic study. This strain, designated L1-D1, is a his- cytoplasmic petite. Diploids were constructed to see if the presence of the suppressor could be demonstrated in grande diploids. If the loss of suppres- sion were related to the loss of a cytoplasmic element, the grande parent should contribute this element and restore the activity of the suppressor. Fortuitously, L1-D1 was a suppressive petite, [rho-(85%s)] , so that petite as well as grande diploids were produced ( EPHRUSSI, DE MARGERIE-HOTTINGUER and ROMAN

344 J. A. GORMAN A N D J. GORMAN

TABLE 7

Genetic analysis of the his- strain L l -Dl

Phenotype of Segregation ratios’ diploids (his+ : his-)

Grandc Petite 4:0 3: l 2:2 1:3 0:4 -

Cross no. Cross

JK281 Ll-D1 [rho-(s)] x his2-lSUP-HS [rho+] his+ his+ 0 0 19 0 0 JK280 L1-D1 [rho-(s)] x his2-I + [rho+] his+ his+ 0 0 7 4 10 JK282 L1-D1 [rho-(s)] x + SUP-H3 [rho+] his+ his+ 8 10 9 0 0

* All segregants are grande, [rho+].

1955; EPHRUSSI, JAKOB and GRANDCHAMPE 1966). As shown in Table 7, the diploids JK 280 (Ll-Dl [rho-] / his2-1 [rho+]) and JK 281 (Ll-Dl[rho-] / his2-1 SUP-H3 [rho+] ) all had the suppressed histidine phenotype regardless of respiratory capacity. This made it highly unlikely that the loss of suppression was directly related to defective mitochondria.

Sporulation and tetrad analysis of a grande JK 281 diploid demonstrated 2:2 segregation for histidine requirement, possibly indicating that L1 -D1 had lost the suppressor. However, JR 280 showed that this was not the case. First. the two his- parents yielded a his+ diploid, suggesting that the suppressor was still present although inactive in L1-D1. This was confirmed by the recovery of his+ haploid segregants. The segregation ratios were best accounted for by postulating that a second nuclear gene, necessary for suppressor activity. was segregating in a regu- lar manner. This gene was designated sin1 (suppressor-interacting gene). This mutant allele will be referred to as sinZ-.1, while the wild-type allele will be in- dicated by SIN1 or +. In the suppressed strain L1, the sin1 gene is present in the wild-type allelic form, which allows suppression to be manifest. while in the auxotrophic “desuppressed” revertant L1-D1, it is present in a mutant form which does not allow suppression. Thus all spores carrying the combination his 2-1 SUP-H3 SIN1 are histidine independent, while spores of the genotype his2-1 SUP-H3 sinl-1 as well as his2-1 SIN1 and his2-1 sinl-1 are histidine re- quiring. The JK 282 cross shown in Table 7 gave segregation ratios compatible with independent segregation of his2-1 and sinl-1. This cross ruled out the pos- sibility that sinl-1 was a second histidine mutation not suppressible by SUP-H3; if it were, all segregation would be 2: 2 for histidine.

While in cross JK 281, which is heterozygous only for sing, the 2:2 segregation of SIN1 and sinl-l was clear, this pattern could not be confirmed directly for crosses involving segregation of both his2-1 and sinl-1 (JK 282) or SUP-H3 and sinl-1 (JK 280). for in each case, three different genotypes are phenotypically identical. To determine the genotype in regard to SUP-H3 and sin1 in cross JK 280, all his- segregants were mated to two tester strains: (1 ) his2-1 SIN1 to detect the presence of the suppressor; and (2) his2-1 SUP-H3 sing-1 to see if those segre- gants not containing the suppressor were SIN1 or sinl-1. The diploids were scored for ability to grow without histidine. This complementation analysis is outlined in Table 8. The genotypes deduced in this manner confirmed the 2: 2 segregation of sinl-1 and its independent segregation from SUP-H3.

RESTRICTION O F SUPPRESSION I N YEAST

TABLE 8

345

The use of diploids to distinguish different genotypes of the h i s segregants of JK 280

Phenotype of

tetrad hid-l +

S I N 1 his2-l SUP-H3

sinl-I

Genotype of

tetrad

Type I ( 6 tetrads tested) his+ + + his2-l SUP-H3 f + his2-I SUP-H3 +

- his2-I f sinl-I his- - his- - - hid-l + sinl-I his+ +

Type I1 (4 tetrads tested) + his2-l SUP-H3 + - his2-I SUP-H3 sinl-I

his- - - h i d - I + sinl-I - his2-I SUP-H3 sinl-I - h i d - I SUP-H3 sinl-I

his+ + his- + his- - + his2-I + + his-- + his- + his- - + his2-I f $. his- - + h i d - I + +

Type I11 ( I O tetrads tested)

A plus indicates the ability of the diploid to grow without histidine; a minus indicates a re-

Histidine-independent segregants were tested in one tetrad of each type as a control. quirement f or histidine.

In cross JK 282, which was heterozygous for his2-l and sinl, the three different genotypes giving a his+ phenotype could not be distinguished by this technique. The differential growth of diploids heterozygous for sinl, described below, was used to distinguish the his2-l SUP-H3 SINl segregants from those carrying the wild-type HIS2 allele. All 4: 0 tetrads were shown to contain two his2-l SUP-H3 S INl segregants and two HIS2 segregants, while 3: 1 tetrads contained only one his2-1 SUP-H3 SIN1 spore, as expected. No method for determining the sin1 genotype in the absence of the his2-I allele has yet been devised. This analysis helped to confirm the independent segregation of his2-1 and sinl-2.

“Conditional” dominance of SIN1 : The dominance of the suppressed pheno- type in diploids heterozygous for sin1 is in some manner dependent on the carbon source employed. As shown in Table 9, all diploids dependent on suppressor ac- tivity for growth in the absence of histidine grow well on YNB (glucose), but cannot grow on YNB-glycerol if sin1 is heterozygous. Homozygous SIN1 diploids and all SIN1 haploids grow equally well on both media. Further investigation of this phenomenon will be the subject of a separate publication.

This differential growth allows his2-1 SUP-El3 SIN1 segregants to be distin- guished from segregants containing the wild-type HIS2 allele by mating them to a his2-I + sin1 strain. The diploids will not grow on YNB-glycerol if the his+ parent is of the genotype l~is2-I SUP-H3 SINZ, while the diploids that are hetero- zygous for his2-l will grow on this medium.

Reciprocal crosses: Segregants of genotypes his2-1 SUP-H3 sinl -I , his2-l + sinl-1, and HIS2 SUP-H3 sinl-l were mated to L-1 (his2-1 SUP-H3 S I N l ) to

346 J. A. GORMAN AND J. GORMAN

TABLE 9

The differential growth of diploids on YNB-glucose and YNB-glycerol

D i p l o i d Glucose G l y c e r o l Glucose Glycerol

YNB + YNB + YNB YNB h i s t i d i n e h i s t i d i n e

+ + + - h i s 2 - 1 SUP-HJ sinl-1 h i s 2 - 1 + +

h i s 2 - 1 SUP-K3 s i n l - 1 f + + -

h i s 2 - 1 + sinl-1 h i s 2 - 1 SUP-H3 +

+ + f -

h i s 2 - 1 SUP-HJ sinl-l

h i s 2 - 1 + sinl-1 + + - -

h i s 2 - 1 SUP-m + h i s 2 - 1 + +

+ + + +

+ + + + h i s 2 - 1 SUP-KJ sinl-1 + + + or sinl-1

+ + SUP-KJ sinl-1 + + + h i s 2 - 1 + + or sinl-1

h i s 2 - 1 + + h i s 2 - 1 + + or sinl-1

f + -

Diploids were scored for growth on agar plates on day 4. A minus indicates no visible growth after 4 days of incubation at 30°C.

give the reciprocal crosses of JK 281, JK 280, and JK 282. I n all cases, the segre- gation ratios were similar to those of the original three crosses, confirming the independent segregation of his2-I, SUP-H3, and sin-I.

The segregation ratios of sinl-l respective to SUP-H3 and his2-1, as seen in JK 282 and JK 280, indicate that sinl-I is centromere linked. Employing addi- tional data on segregation of sinl-l in relation to the centromere-linked gene arg4, an approximate second-division segregation frequency of 0.22 has been calculated. While this is highly indicative of centromere linkage, further crosses will be necessary to confirm this.

RESTRICTION O F SUPPRESSION IN YEAST

TABLE 10

The effect of sinl-1 on SUP-H1 and SUP-H2

347

Cross no. cross

Segregation ratios

phenotype 2:2 1:3 0:4 Diploid (his+ : his-)

JK 309 his2-1 SUP-H2 x his2-1 sinl-1 his+ 5 27 7 JK 339 his2-1 SUP-HI x hisZ-1 sinl-1 his+ 9 6 5

The effect of sinl-1 on SUP-H1 and SUP-H2: Crosses were made to examine the effect of the sinl-l mutation on the other his2-1 suppressors. A strain sup- pressed by each suppressor was crossed to a his2-l sinl-2 strain. The suppressed parent in each cross would have to be SZNI i f the sinl-I mutation caused “de- suppression” of the suppressor, while it could be either SZNI or sin1 if there were no interaction. In the latter case, all segregation would be expected to be 2:2 for histidine, regardless of the sin2 allele. The deviation from this 2:2 segregation found in both crosses (Table 10) demonstrated that the sinl-I mutation affected both SUP-HI and SUP-H2 in the same manner as with SUP-N3; that is, segre- gants of the genotype his2-I SUP-HI sinl-l or his2-I SUP-H2 sinl-I require histidine. The effect of this mutation on other suppressors is currently being tested.

The efect of sin1 on growth rate: As shown in Table 11, the sinl-I mutation has no effect on the growth rate of either wild type or his 2-1. While the differ- ences in strains carrying a suppressor are not dramatic, in every case slightly faster growth is displayed by the sinl-l strain than by the comparable SZNl strain. As all strains used for determining these growth rates were isogenic for all markers except his2-2, the suppressors, and sinl, the differences cannot be at-

TABLE 1 1

The influence of sin-1-1 on growth rate

Genotype Double time

(minutes)

+ + + + + sinl-l + SUP-H3 sinl-l

his2-l + + his2-l + sinl-l hid-1 SUP-Hl + his2-1 SUP-Hl sinl-l his2-1 SUP-H2 + hisZ-1 SUP-HZ sinl-f his2-l SUP-H3 f his2-1 SUP-H3 sinl-1

+ SUP-H3 $. 108 110 138 115 120 1 20 130 102 132 105 135 105

All cultures were started from cloned isolates to remove possible heterogeneity. The cultures were grown in YNB-histidine at 28°C with constant aeration by shaking in a Psycrotherm incu- bator shaker. Each value represents the average of two separate experiments.

348 J. A. G O R M A N A N D J. G O R M A N

tributed to unrelated strain heterogeneity. The observation that sinl-l has no effect on growth unless coupled with a suppressor with which it interacts may be helpful in understanding the nature of the SZNl gene.

DISCUSSION

In order to understand the interaction between these suppressors and the sinl-l mutation, it will first be necessary to establish the mechanism of the suppression itself. The allele specificity exhibited by all three suppressors rules out the pos- sibility of indirect suppression and indicates that a codon-specific mechanism is involved.

While both nonsense and missense suppressors exhibit codon specificity, both the missense characteristics of the his2-l mutant and the lack of suppression of any other mutants make it unlikely that these are nonsense suppressors. Consid- ering the nature of nonsense and missense mutations, it might be expected that fewer amino acid substitutions could restore enzyme activity to a protein inac- tivated by a single amino acid substitution than to a protein defective due to pre- mature chain termination. The likelihood that a given suppressor could suppress an identical mutant codon in a different gene should be far less for a missense suppressor than for a nonsense suppressor. The large number of possible different missense codons further reduces the probability of detecting multiple missense mutants suppressible by a single suppressor. It is not surprising that none of the other mutants tested to date is suppressed by the suppressors of his2-I if they are missense suppressors. Such specificity is not typical of nonsense suppressors or super suppressors. which are characterized by the ability to suppress a variety of different amber or ochre mutants. While it is possible that the his2-l suppres- sors represent a new atypical class of nonsense suppressors with a very limited spectrum of activity, it presently appears more reasonable to consider these sup- pressors as typical missense suppressors.

Codon specificity implies that one of the components of protein synthesis di- rectly involved in translating the altered codon is responsible for suppression. This would most likely involve an altered tRNA or activating enzyme. The ele- gant work on bacterial suppressors has demonstrated that missense suppression is the result of an altered tRNA (CARBON, BERG and YANOFSKY 1966; GUPTA and KHORANA 1966) and that the suppressor loci are structural genes for specific tRNA subspecies (CARBON, SQUIRES and HILL 1969). The nonsense suppressors of yeast show properties analogous to the bacterial tRNA nonsense suppressors, and it has been proposed that the mechanism involved is the same in the two sys- tems (MORTIMER 1969). While there is no direct evidence to substantiate the involvement of an altered tRNA in yeast suppression, such involvement is con- sistent with the present data and is useful in directing further investigation.

Of the three suppressors studied, SUP-HI and SUP-H3 impart similar pheno- typic characteristics, and appear to be either allelic or closely linked. Further study will be necessary to distinguish these two possibilities. The suppressor SUP-H2, which involves a mutation in a different genetic locus, is also distin-

RESTRICTION OF SUPPRESSION IN YEAST 349

guished by a lower efficiency of suppression. This difference between SUP-H2 and SUP-H3, reflected in the growth rate of suppressed his2-2 mutants in the absence of histidine, may be interpreted in two ways, if it is assumed that the sup- pressors involve altered tRNA. First, the two loci could be associated with two different species of tRNA, so the amino acid substitution effected by SUP-H2 gives an enzyme with less activity than the enzyme synthesized under the in- fluence of SUP-H?. Alternatively, the suppressors may involve multiple copies of the same species (or subspecies) of tRNA which are present in different quan- tities or have variable activity depending on the suppressor gene involved. An analysis of the enzyme synthesized in the presence of each of the three suppres- sors will be necessary to determine if the substitution involves the same or differ- ent amino acids. This analysis has been hampered by the inability to demon- strate enzyme activity in vitro for any of the suppressed strains, employing the standard procedure of enzyme extraction and assay (GORMAN and Hu 1969). This probably reflects a greatly reduced stability of the suppressed enzyme mole- cule.

In light of the minor effect of each suppressor on cell growth, it is apparent that the normal product of the suppressor gene is not indispensable. The existence of redundant tRNA in yeast ( SOLL, CHERAYIL and BOCK 1967) governed by dif- ferent genetic loci (GILMORE, STEWART, and SHERMAN 1968) allows a mechanism whereby the loss of one of several redundant species could occur without deleteri- ous effects on general protein synthesis.

Missense suppression has the inherent problem of possible misreading of nor- mal codons by the altered tRNA. One would expect to find a direct relationship between efficiency of suppression and reduction of growth and viability. This has generally been found to be the case in missense suppression. However, the sup- pressors studied here do not severely restrict growth, yet they allow strong sup- pression. If the suppression involves altered tRNA, the changes must be in a minor species of tRNA which is in some way preferentially used to translate the missense codon.

The presence of two nonallelic suppressor mutations would be expected to have a more severe effect if the two involve the same species of tRNA, for the level of the nonmutant tRNA might become limiting. This explanation may account for the substantial reduction of growth rate found in strains carrying two suppres- sors. The combination SUP-H2 SUP-H3 leads to even greater growth reduction than does the combination SUP-HI SUP-H2. This may indicate that the two mutations SUP-HI and SUP-H3 are not identical, possibly involving different minor species or subspecies of tRNA.

Several examples of unstable suppressed phenotypes have been reported in bacterial systems ( BRODY and YANOFSKY 1965; LEDERBERG, CA’ITALLI-SFORZA and LEDERBERG 1964). In several cases, the instability has been shown to be the result of partial heterozygosity for the suppressor locus, so that the suppressor may be segregated out with the extrachromosomal factor with relatively high frequency or integrated into the chromosome to form a stable suppressed colony ( SCHWARTZ 1965; HILL et al. 1969). The instability reported here is of interest in light of the

350 J. A. GORMAN AND J. GORMAN

apparent correlation between loss of suppression and loss of the cytoplasmic factor [rho]. The genetic analysis of the suppressors of his2-1 rules out the possibility that these suppressor genes are located on autonomous cytoplasmic elements. The lack of alteration of the suppressed phenotype in acriflavin-treated cells also argues against a direct involvement of functional cytoplasmically determined factors in the suppression. As the sinl gene is clearly transmitted as a chromo- somal marker in all crosses, the mutational event creating the his- phenotype cannot be the result of cytoplasmic instability, in which a cytoplasmically in- herited element carrying the sinl gene has a high probability of being lost.

Certain pleiotropic genic mutations have been reported which result in both nutritional auxotrophy and loss of respiratory sufficiency. For example, certain lysine-requiring mutants of yeast, Zys6 and lys8, invariably lose the cytoplasmic factor [rho] and are unable to grow on nonfermentable substrates such as glycerol (SHERMAN 1963). In the situation reported here, the conversion to petiteness does not appear to be a direct result of the sin2-1 mutation, for stable grande sinl-1 colonies are recovered from the cross of a sinl-1 [rho-(s)] isolate to a respiratory sufficient [rho+] strain. However, the association seems to be more than fortui- tous, for all sinl mutants isolated to date have been cytoplasmic petites. If there is indeed any connection, it is more likely to be the result of some unknown pleiotropic effect of the suppressor SUP-H3, possibly only occurring in stationary phase when the synthesis of mitochondria is derepressed.

It is unnecessary to speculate at length on the nature of the sinl gene, or the mechanism of its interaction with the suppressor, in light of the limited informa- tion yet obtained. The observation that growth is better when the suppressor is accompanied by the sinl-1 allele rather than the SIN1 allele indicates that the synthesis or activity of the suppressor gene product is affected by this mutation. If the suppressor, a source of deleterious effects on general protein synthesis, is no longer available or in an inactive form, this would be reflected in an improved growth rate.

The effect of the sinl-l mutation also appears to involve a certain degree of specificity for the suppressor, for there is not observable reduction of growth rate in the absence of suppressor in sinl-1 cells. If the suppressor is assumed to be al- tered tRNA, for example, a similar interaction with the normal species of tRNA should produce a deficiency of that tRNA for translation of a given codon, which should affect total protein synthesis.

The restriction of suppression of at least two genetically different suppressor genes either could indicate a complex specificity of this interaction or may only reflect a redundancy of genes for one specific tRNA species. It will therefore be of interest to determine if this mutation can affect the suppression of other mis- sense and nonsense mutations where different tRNA species are involved.

In msny respects, the sinl-1 mutation confers properties similar to those asso- ciated with certain drug-resistant mutations in bacteria. These bacterial muta- tions, which involve changes in ribosomal subunits, restrict missense suppression by tR!NA suppressors, yet do not appear to affect growth in general (APIRION, PHILLIPS and SCHLESSINGER 1969; GORINI 1969). It is conceivable that sinl-1 re-

RESTRICTION O F SUPPRESSION IN YEAST 35 1

sults in some ribosomal alteration so that tRNA suppressors of the his2-l missense codon can no longer be efficiently used for translation of this mutant codon. How- ever, alternative explanations cannot be excluded, and it is premature to postulate any specific mechanism until more is known about the sinl gene.

The authors are indebted to Miss MARY KATHLEEN MOORE for her outstanding technical assistance. This research was supported by U. S. Public Health Service Research Grant 5R01 GM14745-03.

SUMMARY

Three allele-specific suppressors have been isolated from revertants of a histidine-requiring missense mutant his2-l in Saccharomyces cereuisiae. Genetic analysis demonstrated the independent segregation of each suppressor in relation to his2-1, and two of the suppressors have been shown to be nonallelic. The three suppressors are specific for the his2-l allele, failing to suppress any other his2 mutations or unrelated mutations, including several amber and ochre lesions. Suppressed strains grow nearly as well as wild type in the absence of histidine, indicating that the suppression is very efficient, and that the suppressors have little effect on total cell growth. The suppressors appear to be relatively instable during prolonged stationary phase maintenance on complete medium, and give rise to mixed cultures of histidine-independent and histidine-requiring cells. As all the stable revertants of a suppressed strain were found to be cytoplasmic pe- tites, genetic analysis of a histidine-requiring revertant was undertaken to see if there would be any correlation between the loss of suppression and the loss of respiratory sufficiency. The crosses demonstrated that the loss of suppression was not due to the loss of the suppressor, but rather to the presence of a second muta- tion which restricts the suppressed phenotype. This gene, designated sinl (sup- pressor interacting), segregates independently of both his2-1 and each suppressor. Suppression occurs if the wild-type allele SZNl is present, but not if the suppressed strain has the mutant allele s in l - l . No explanation for the apparent correlation between sinl-l and petiteness in the original isolates can be given, as both the suppressors and sinl have been shown to be nuclear genes, and all segregants of crosses involving the sinl-l allele are stable grandes. The sinl-1 mutation does not appear to have any effect on cell growth or nutritional requirements. All three allele-specific suppressors are affected by sinl-2 in the same manner. Sup- pressors of other auxotrophic mutants are currently being tested for susceptibility to restriction by sinl-1 in order to test the specificity of this interaction. Possible mechanisms of interaction between these missense suppressors and sinl -1 are dis- cussed.

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