Copyright 0 1990 by the Society of America

Isolation and Characterizationof Omnipotent Suppressorsin the Yeast

L. Paul Wakem* and Fred Sherman**t

Departments of * and +, School of Medicine and Dentistry, Rochester, New York 14642 Manuscript received September 5, 1989 Accepted for publication November 15, 1989

ABSTRACT Approximately 290 omnipotent suppressors, which enhance translational misreading, were isolated in strains of the yeast Saccharomyces cerevisiae containing the $+ extrachromosomal determinant. The suppressors could be assigned to 8 classes by their pattern of suppression of five nutritional markers. The suppressorswere further distinguished by differences in growth on paromomycin medium,

hypertonic medium, low temperatures (10 O), nonfermentable carbon sources, a-aminoadipic acid medium, andby their dominance andrecessiveness. Genetic analysis of 12 representativesuppressors resulted in the assignment of these suppressors to 6 different loci, including the three previously described loci SUP?5 (chromosome ZV),SUP45 (chromosome IZ) and SUP46 (chromosome IZ), as well as three new loci SUP42 (chromosome ZV), SUP43 (chromosome XV) and SUP44 (chromosome VZZ). Suppressorsbelonging to the same locus had a wide range of different phenotypes.Differences between alleles of the same locus and similarities between alleles of different loci suggest that the omnipotent suppressorsI. encode proteins thateffect different functions and that altered formsof each of the proteins can effect the same function.

HE omnipotent suppressorsof Saccharomyces cere- BERTSON, 1988),and mutations in the CRL genes T visiae suppress certain UAA, UAG and UGA (cycloheximide resistant temperature sensitive lethal), nonsense mutations, as well as certain frameshift and which resemble both gcn (general control of amino other types of mutations (INGE-VECHTOMOVand AN- acid biosynthesis) and omnipotent suppressor muta- DRIANOVA 1970; HAWTHORNEand LEUPOLD1974; tions, also cause increased levels of translational mis- GERLACH1975; CHATTOOet al., 1979a; CULBERTSON, reading (MCCUSKERand HABER1988a, b). GABERand CUMMINS1982). In addition to suppres- We have isolated an extensive number of omnipo- sion, the omnipotent suppressors exhibit a wide range tent suppressors, characterized them phenotypically of pleiotropic effects, including, cycloheximide de- and genetically mapped 12 of the suppressors in an pendency, respiratory deficiency, sensitivity to high attempt to identify new suppressor loci. In order to and low temperatures,high osmotic pressure and obtain a broader range of suppressors at new loci, aminoglycoside antibiotics (GERLACH1975; MASURE- suppressors were isolated in strains containing the ++ KAR et al. 1981 ; SURGUCHOV et al. 1984). Recessive cytoplasmic determinant. The 4' factor enhances the omnipotent suppressors have been isolated at several activity of UAA and certain frameshift suppressors different loci, including; SUP35 (also denoted SUP2, (Cox 1965, 1971; CULBERTSONet al. 1977; LIEBMAN SUF12, SUPPand SAL3), SUP45(also denoted SUPl, and SHERMAN1979) as well as enhancing the pheno- SUPQ and SAL4) (INGE-VECHTOMOVand ANDRI- typic suppression of nonsense mutations by paromo- mycin (PALMER, WILHELMand SHERMAN1979a). Sim- ANOVA 1970; HAWTHORNEand LEUPOLD1974; GER- ilarly, ALL-ROBYNet al. (1990) recently isolated om- LACH 1975; COX 1977; CULBERTSON, GABERand nipotent suppressors in (eta') strains and showed CUMMINS1982), suplll, sup112 and sup113 (ONO et 7' that q+ increased the level of suppression of certain al. 1982, 1984).Dominant suppressors have been nonsense mutations by some omnipotent suppressor isolated at the single locus, (ONO, STEWART SUP46 alleles. Our collection contains dominant or semidom- and SHERMAN198 1). The suppression by omnipotent inant alleles of the previously described suppressors, suppressors is due to increased levels of misreading, SUP35, SUP45and SUP46,plus alleles of three previ- as measured by in vitro translation assays (MASUREKAR ously unidentified suppressors, which we have named et al. 1981; SURGUCHOVet al. 1984; EUSTICEet al. SUP42, SUP43and SUP44. These new suppressors 1986). In addition, mutationsin the TEF2 gene, which behave similarly to SUP35, SUP45and SUP46. codes for elongation factor-1a (SANDBAKENand CUL- MATERIALSAND METHODS The publication costs of this article were partly defrayed by the payment of page charges.This article must therefore be hereby marked"advertisement" Genetic methods:The yeast strains used in this study are in accordance with 18 U.S.C. 5 1734 solely to indicate this fact. described in Table 1. Since the majority of our suppressor

Genetics 124 515-522 (March, 1990) 516 L. P. Wakem and F. Sherman

TABLE 1 Yeast strains

Strain Genotype Source Dl 142-1A $' MATa lys2-187met8-1leu2-Iaro7-1his4-166 ura3-l cycl-72 This study D1150-1D $+ MATa lys2-187met8-1 leu2-I aro7-1 his4-166 canl-100 trpl-289 cycl-72 This study B-7316 $' MATa SUP35lys2-187 met8-1 leu2-Iaro7-1 his4-166 ura3-1 cycl-72 This study B-7331 J.'MATa SUP35lys2-187 met8-1 leu2-1 aro7-1 his4-166 canl-100 trpl-289 cycl-72 This study B-7329 $' MATa SUP42lys2-187 met8-1 leu2-1 aro7-1 his4-166 canl-100 trpl-289 cycl-72 This study B-7332 $' MATa SUP42lys2-187 met8-1 leu2-1 aro7-1 his4-166 canl-100 trpl-289 cycl-72 This study B-7317 $' MATa SUP43lys2-187 met9-I leu2-I aro7-l his4-166 ura3-1 cycl-72 This study B-7334 $' MATa SUP43lys2-187 met8-l Eeu2-1 aro7-1his4-166 canl-100 trpl-289 cycl-72 This study

€3-7319 J.+ MATa SUP44lys2-187 met8-1 leu2-I aro7-1 his4-166 ura3-lcycl-72 This study B-7323 $' MATa SUP45lys2-187 met8-1leu2-I aro7-1 his4-166 ura3-1 cycl-72 This study B-7324 J.'MATa SUP46lys2-187 met8-1leu2-I aro7-1 his4-166 ura3-1 cycl-72 This study B-7325 J.'MATa SUP46lys2-187 met8-lleu2-I aro7-1 his4-166 ura3-1 cycl-72 This study B-7326 J.'MATa SUP46lys2-187 met8-lleu2-I aro7-1 his4-166 ura3-1 cysl-72 This study B-7327 J.'MATa SUP46lys2-187 met8-1leu2-I aro7-1 his4-166 ura3-1 cycl-72 This study B-7924 MATa his7trp4arolCade8 ura4 ma3ade5leu2-3 lys2 Pet- This study B-7925 MATatrp4arolCade8ura4 his7 ma3arg4leu2-3 lys2 Pet- This study cdc28-I glcl lys2-187 tyrl cycl-72 B-7926tyrl lys2-187 glclMATO cdc28-I This study aro7-1 trpl-289 lys2-187 cdc37-IB-7927 lys2-187 trpl-289MATa aro7-1 This study ade5 lys5 met13 cyh2 trp5 leul trp5 cyh2B-7928 met13 lys5MATa ade5 This study B-7929met13 lys5MATa ade5 leul cyh2 trp5 This study SK672-1 MATa cdc37-I ural Stock center K381-15C trplpet17 MATalys2 met14 asp5 ura3aro7 arg4 ade6 S. KLAPHOLZ K381-10Atrpl pet17 lys2 met14 asp5 aro7MATa arg4 ade6 ura3 S. KLAPHOLZ K382-23A MATacanl ura3ham3 his7cyh2 ade2 spoll S. KLAPHOLZ K382-19D MATacanl ura3 tyrlhom3 cyh2his7 ade2 spa1 I S. KLAPHOLZ SL8 18-2C MATa SUP39met8-1 leu2-Iaro7-1 trpl-1 ade3-26 ilvl-1 his5-2 Lys- S. LIEBMAN isolates at the six different loci described here were either RESULTS dominant or semidominant for suppression, we have used the upper case designation for mutant alleles of SUP35, Isolation and characterizationof omnipotent sup SUP42, SUP43, SUP44,and SUP45SUP46 throughout this paper. Media and genetic procedures including crosses, pressors: A total of 294 omnipotent suppressors were sporulation, dissection, tetrad analysis and scoring nutri- isolated by selecting for revertants of strains Dl 142- tional markers have been described (SHERMAN,FINK and 1A and D 1 1 50-1 D (Table 1) on ninetypes of omission HICKS1987). Suppressors were selectedon various omission media, each lacking one of the following combinations media, each lacking a different combination of two amino of amino acids: lysine and methionine; lysine and acids required by the strains. Suppression of suppressible tyrosine; lysine and leucine; lysine and histidine; me- markers was tested by spotting on the appropriateomission medium and incubating at 30" for 5 days. Sensitivity of the thionine and leucine; methionine and histidine; tyro- suppressors to various agents or conditions was measured sine and leucine; tyrosine and histidine; leucine and on the following media: a-aminoadipate medium described histidine. These conditions selected for the isolation by CHATTOOet al. (1 979b);high osmotic media containing of omnipotent suppressors, since revertants must con- YPD (1% Bacto-yeast extract, 2% Bacto-peptone and 2% comitantly suppress two different types of the follow- dextrose) and 1.O to 1.5 M ethylene glycol; cold tempera- met8-1 tures on YPD medium incubated at 4-10"; and paromo- ing mutations: (UAG), aro7-1 (UAG); leu2-I mycin (a gift from Warner Lambert)media containing YPD (UAA); his4-166 (UGA) and lys2-187 [not a nonsense and 0.5 to 5.0 mg/ml paromomycin. The parental strains mutation (CHATTOOet al. 1979a)l. Revertants were D1142-1A and D1150-1D grew at concentrations of 2.5 isolated as spontaneous isolates (50%) or induced mg/ml paromomycin but were inhibited at 5.0 mg/ml. In using either ultraviolet light (500 ergs rnm-') (20%) contrast, the growthof hypersensitive suppressors was inhib- or X-ray (15 kR) treatments (30%).The suppressors ited at 0.5 to 1.0 mg/ml and resistant suppressors grew at 5.0 mg/ml. were subcloned, then tested on omission media for Chromosomalassignment of suppressors: The chro- their ability to suppress all five suppressible markers. mosomal assignment of some of the dominant paromomy- While all isolates suppressed lys2-I87 and met8-I, they cin-sensitive suppressors was achieved using the benomyl differed in their ability to suppress the other three method described by WOOD (1982b). Diploid strains were mutations and could be divided intoeight classes treated with benomyl (a gift from E. I. Dupont deNemours) based on differences in their pattern of suppression and samples were plated on YPD medium containing 2.5 mg/ml paromomycin. Paromomycin resistant colonies were (Table 2). The majority of the isolates suppressed isolated and tested on omission media for the expression of either all five suppressible markers or all the markers auxotrophic markers. except for his4-166 (classes I and 11). The remainder Om nipotent SuppressorsOmnipotent in Yeast 517

TABLE 2 Phenotypic characterizationof omnipotent suppressors

Paromo- Cold& Suppression" Dominance' mycind

met8-1 leu2-1 aro7-1 his4-166 aro7-1 leu2-1 met8-1 a-aab Osm. Resp. Class lys2-187 (UAG) (UAA) (UAG) (UGA) No. (-) Dom Sd Rec sens. res. sens.' def! (-) (+)

~ ~~~ I + + 96 20 39 55 2 30 23 22 2 11 9 11 + - 116 13 53 43 20 28 27 25 1 20 6 111 + - 29 0 21 6 2 13 4 11 9 9 1 IV + - 18 0 15 1 2 1 2 3 0 0 0 V + - 18 4 12 6 0 2 9 2 1 5 1 VI + + 12 1 8 1 1 2 3 3 2 2 0 VI1 + + 4 0 2 1 1 0 0 0 0 0 0 0 VI11 + +-1 -0 -0 -1 -0 -0 -0 -0 -0 - -0 Totals 294117 38 150 27 7615 68 66 47 17

a Suppression of lys2-187, met8-1 (UAG), leu2-I (UAA), aro7-1 (UAG) and hid-166 (UGA) was measured by spotting isolates on the appropriate omission media and incubating for 5 days at 30" (+ = suppression, - = no suppression). .. .. L. 'b Isolates unable to utilize or-aminoadipate as sole nitrogen source. ' Dominance and recessiveness of suppression of lys2-187, met8-I, leu2-1, aro7-1 and his4-166: dominant (Dom), semidominant (Sd), recessive (Rec). Isolates hypersensitive (sens.) (0.5-1.0 mg/ml) or resistant (res.) (5.0 mg/ml) to paromomycin. e Isolates sensitive to high osmotic pressure (1.0-1.5 M ethylene glycol). 'Isolates which are respiratory deficient (measured as the inability to utilize glycerol as sole carbon source). Isolates with diminished (-) or enhanced (+) growth at cold temperatures (5-10"). of the isolates were groupedinto 6 classeswhich The isolates were tested for dominance or reces- differed in their ability to suppress leu2-1, aro7-1 and siveness of suppression by crossing themto either his4-166. Analysis of the number of isolates in each Dl 142-1A or Dl 150-1D and checking the suppres- class allowed us to rank the suppressible markers in sion spectrum of the diploids. Most isolates were either the following order, from most suppressible to least dominant (150) or semidominant (1 17) for suppres- suppressible: lys2-187 = met8-1 > leu2-1 > aro7-1 > sionwith only a small number (27) being recessive his4-166. (Table 2). Isolates were classed as semidominant if The different patterns of suppression reflect either they suppressed lys2-187 and met8-1 dominantly but different levels of suppression or different aminoacid suppressed one or moreof leu2-1, aro7-1and his4-166 replacements. Variations in the levels of suppression recessively. There was no correlationbetween the of the Zys2-187 mutation can be tested on a medium class of suppression spectrum of the isolates and their supplemented with lysine and containing a-aminoad- dominance or recessiveness. ipate as the principal nitrogen source. In contrast to As shown in Table 2, the isolates exhibited a variety LYS2+ normal strains, Zys2- strains are able to utilize of pleiotropic effects in addition to suppression. Due a-aminoadipate as a principal nitrogen source (CHAT- to the large number of mutants isolated in this study, TOO et al. 1979b). Apparently the anabolism of high most strains were characterized without any genetic levelsof a-aminoadipate throughthe biosynthetic backcrossing. These strains may containsecondary pathway of lysine causes the accumulation of a toxic mutations which may account forsome of the presum- intermediate andZys2 mutants block the formation of this intermediate (ZARET and SHERMAN1985). Fur- ably pleiotropic effects that were uncovered. How- thermore, the degree of growth on a-aminoadipate ever, in the 12 strains which were mapped by tetrad medium is related to the degree of the lys2 block. In analysis (Table 4), those pleiotropic phenotypes pres- contrast to the lys2-187 parental strain, 38 of the 294 ent in each strain segregated with the suppressor. In suppressors were unable to grow on a-aminoadipate addition, other workers have reported similar pleio- medium, suggesting thatthe levelsof suppression tropic effects for various omnipotent suppressors werehigher in thesestrains. However, the isolates (GERLACH 1975;MASUREKAR et al. 1981 ; SURCUCHOV which suppress the Zys2-187 mutation best, do not all et al. 1984). show a similar high level of suppression of the other About 25% of the isolates were hypersensitive to suppressible mutations since about half of the a-ami- the aminoglycoside, paromomycin. Paromomycin and noadipate sensitive isolates do not suppress one or other aminoglycosides increase the level ofmisreading more of the suppressible markers: leu2-1, aro7-1 and in in vitro translation assays using yeast ribosomes his4166 (see Table 2). Nevertheless the efficiency of (PALMER,WILHELM and SHERMAN, 1979b; SINGH, omnipotent suppression is specific for each of the URSICand DAVIES1979) and can also phenotypically suppressible mutations. suppress some nonsense and certain other mutations 518 L. P. Wakem and F. Sherman suppressible by omnipotent suppressors (CHATTOOet Recessive auxotrophic mutations on the same chro- al. 1979a; PALMER,WILHELM and SHERMAN,197%; mosome will also be expressed if the suppressor-bear- SINGH,URSIC and DAVIES1979). Paromomycin hy- ing chromosome is lost. Suppressors were assigned to persensitive alleles of SUP35, SUP45 and SUP44 have a chromosome by testing diploid isolates which were been isolated previously (MASUREKARet al. 1981 ; MI- able to grow at paromomycin concentrations of 2.5 RONOVA et al. 1982).However, in addition tothe mg/ml for the concomitant expression of any auxo- paromomycin hypersensitive isolates, we also uncov- trophic mutations. A total of 22 of the suppressors ered anequal number of isolates which were resistant were assigned to 4 different groupsusing this method. to paromomycin. Whereas the normalstrains grew A numberof the suppressors expressed either thehis7 and were inhibited on media containing 2.5 mg/ml (five suppressors) or trp4 (six suppressors) mutation and 5.0 mg/ml of paromomycin respectively, the hy- and were assigned to chromosome 11 and ZV, respec- persensitive isolates were unable to grow at paromo- tively. This result was expected because both SUP45 mycin concentrations of 0.5 to 1.O mg/ml, and resist- and SUP46 map on chromosome ZZ and SUP35 maps ant isolates grew at levels of 5.0 mg/ml. on chromosome IV. However, the rest of the suppres- Allelesof SUP35 and SUP45 have also been de- sors were not assigned to either chromosome ZZ or ZV scribed which are respiratory deficient, and sensitive but could be divided into two more groups on the to high osmotic pressure, and to high or low temper- basisof theirchromosomal assignment. These two atures (GERLACH 1975; SURCUCHOVet al. 1984). We groups of suppressors appeared to be two new sup- also uncovered a small number of isolates that were pressors. One group of seven suppressors was erro- respiratory deficient, as determined by their inability neously assigned to chromosome XZZ since they ex- to utilize glycerol as acarbon source. In addition, pressed the ura4 mutation and the other group of some isolates were sensitive to high osmotic pressure four suppressors could not be assigned to any chro- (1 .O-1.5 M ethylene glycol) and cold temperatures (5- mosome using the benomyl procedureand various 10'). None of our isolates were sensitive to growth at mapping strains. The two new suppressors were de- high temperatures; however, a small number grew noted SUP43 (unassigned) and SUP44 (chromosome better at 5-10' than the original strains. XU). However, our assignment of SUP44 to chromo- There was no relationship between the pattern of some XZZ by the benomyl mapping method was incor- suppression and the different phenotype characteris- rect. Meiotic mapping showed no linkage between tics. Isolates ranged from those which exhibited no SUP44 and various genes on chromosome XZI (data other phenotype besides suppression to isolates with not shown); however, there was linkage between the any number of different combinations of the pheno- SUP44 allele in B-73 19 andgenes on chromosome VZZ types shown in Table 2. (Table 3). The inaccurate assignment of SUP44 to Chromosomal assignment of suppressors: The chromosome XZI may have been dueto the high suppressors could not be unambiguously assigned to frequency of multiple chromosome losses induced by complementation groups by testing heterozygous dip- benomyl (WOOD1982a). loids for their suppression capabilities, since even the Meiotic mapping of suppressors: Twelve suppres- mostrecessive mutationsexhibited some degree of sors were mapped by crossing to several strains and dominance. Therefore, isolates with the easily select- assaying for the suppressors by either paromomycin able dominant phenotype of paromomycin hypersen- sensitivity or by suppression of suppressible mutations. sitivity were chosen and subdivided into groups using The genetic mapping of these 12 suppressors defined the rapid chromosomal assignment method described six differentsuppressor loci (Table3). Of the five by WOOD (1982b).This method relies on mitotic isolates assigned to chromosome 11, one (B-7323) chromosome loss induced by benomyl to uncover mapped at the SUP45 locus (centromere proximal to recessive auxotrophic markers. Isolates were crossed both tyrl and cdc28), while four mapped at SUP44 to mapping strains (B-7924 and B-7925) containing (centromere distal to cdc28 and tyrl, but closely linked auxotrophic markers on chromosomes I1 (his7) and to tyrl) (Tables 3 and 4). This mapping data shows IV (trp4), aswell as other chromosomes. Diploids that the order of genes on chromosome ZZ is SUP45, were heterozygous fora suppressor mutation and cdc28,tyrl and SUP46. This corresponds well with retained their dominant phenotypeof hypersensitivity previously reported mappingdata for SUP45 and to paromomycin. These diploids were treated with SUP46 (ONO,STEWART and SHERMAN 198; ON01 et benomyl, then grown on medium containingparo- al. 1984). Isolates assigned to chromosome ZV mapped momycin (2.5 mg/ml) to select for strains which had at two different loci; two of the suppressors mapped lost the chromosome carrying the suppressor muta- at SUP35 (linked to cdc37) and two mapped at a new tion. Loss of the suppressor mutation results in loss of locus unlinked to cdc37 but linked instead to trpl. the paromomycin hypersensitivity of the diploid since This new suppressor was denoted SUP42. the recessive wild-type gene (sup') is now homozygous. Two of the SUP43 isolates, which could notbe Om nipotent SuppressorsOmnipotent in Yeast 519 TABLE 3 linkage between SUP42 and SUP39. Tetrad analysis Meiotic mappingof omnipotent suppressors of this cross showed that these two suppressors are not linked (2P:9N:14T). No. of Tetradsd Our mapping data shows that the omnipotent sup- alleles Chromo- pressor loci SUP42, SUP43 and SUP44 do not corre- StrainsSuppressor" mappedb some' Genepair P N T cM' spond to the SUP35, SUP39, SUP45 or SUP46 loci. In B-7323 SUP45 1 11 SUP45-cdc281 2 017 11 addition, they do not map near sup1 11, sup112 or SUP45-tyrl 19 1 9 26 supll3, which mapon chromosome VZZZ, the right cdc28-tyrl 33 0 9 7 arm of chromosome VZZ and chromosome XZZZ, re- B-7327 SUP46 4 I1 SUP46-tyr1 40 0 3 4 spectively (ONOet al. 1986). SUP42 and SUP43 are SUP46-cdc28 34 0 1415 cdc28-tyrl 36 0 9 10 new suppressor loci while SUP44 maps close to and may be allelic to SUP38 (B. ONO,unpublished data) B-7316 SUP35 IV 2 SUP35-cdc37 10 0 6 18 (MORTIMERand SCHILD1985). SUP35-trpl 5 5 1 1 cdc37-trpl 4 7 9 The phenotypes of 12 suppressors,mapped to 6 B-7332 SUP42 2 1V SUP42-trpl 14 0 9 20 different loci are summarized in Table 4. Different SUP42-cdc37 3 5 15 alleles can have very different suppression spectra, cdc37-trpl 7 7 13 suppression efficiencies and various other phenotypes. B-7319 SUP44 VI1 1 SUP44-met13 22 0 3 6 While these different phenotypic characteristics are SUP44-cyh2 23 1 6 20 allele-specific, they appear to be a common trait of metl3-cyh2 23 6 120 mutants isolated at all six omnipotent suppressor loci B-7334 SUP43 XV 2 SUP43-pet17 6 0 18 37 described here. SUP43-ade2 8 7 30 Suppressors werecrossed tothe following strainsfor tetrad DISCUSSION analysis: SUP45 and SUP46 to B-7926; SUP35 and SUP42 to B-7327 and SR672-I; SUP44 to B-7328 and B-7329; and SUP43 to K381- Many different factorsare involved in ensuring the 15C, K381-10A, K382-19Dand K382-23A. accurate translationof mRNA in yeast. Mutations with a The suppressor contained in each of the strains mapped. ' The number of alleles which we have mapped to each of the increased levelsof misreading can arise in various suppressor loci. genes including theomnipotent suppressor genes. ' The chromosome on which each of the suppressor loci map. The results of our tetrad analyses (P = parental, N = nonpar- Our search for omnipotent suppressors has expanded ental and T = tetratype). the number of genes involved in translational fidelity ' The distance (x) in centiMorgans between various genes, cal- by three new genes; sup42, sup43 and sup44. These culated by new omnipotent suppressor genes do not map at any T + 6N previously described loci and are located on chromo- somes ZV, XV and VZZ, respectively. assigned to a chromosome by the benomyl mapping In other studies of omnipotent suppressors, reces- method, were foundto map on chromosome XV. sive alleles of SUP35 and SUP45 or dominant alleles Tetrad analysis showed that SUP43 was linked to petl 7 of SUP46 are the predominant or exclusive isolates (INGE-VECHTOMOVand ANDRIANOVA 1970; HAW- but not to ade2, which is centromere distal to petl 7. THORNE and LEUPOLD1974; GERLACH1975; CUL- This locates SUP43 centromere proximal to petl 7. BERTSON, GABERand CUMMINS 1982; ONO, STEWART A suppressor isolated by ALL-ROBYNet al. (1 990)is and SHERMAN198 1). The exception is a study by ONO allelic to our SUP44 isolate, B-7319, since crosses et al. (1982) designed to isolate inefficient suppressors between B-73 19 and their suppressor yielded only inwhich recessive alleles of suplll,sup112 and parental ditype tetrads (16/16) (ALL-ROBYNet al. supll3 were isolated. In contrast, our study resulted 1990). These authors assigned their SUP44 isolate to inmostly dominantand semidominant alleles of chromosome VI1 by OFAGE mapping of a cloned SUP35,SUP45, SUP46, SUP42, SUP43 and SUP44. SUP44 gene. Our SUP44 isolate (B-73 19) also mapped The dominance or recessiveness of omnipotent sup- on chromosome VZI, between met13 and cyh2, but pressors doesnot appear to belocus-dependent as closely linked to met13 (Table 3). This corresponds to previously suggested (ONO et al. 1984), since both the map position of a previously isolated suppressor, dominant or semidominant and recessive alleles of SUP38 (B. ONO,unpublished results) (MORTIMERand SUP35 and SUP45 have been isolated. In addition SCHILD1985), suggesting that SUP44 and SUP38 may dominant andsemidominant alleles of SUP42, SUP43, be allelic. SUP44 and SUP44 have also been isolated. The differ- Another unmapped suppressor, SUP39, isolated by ences in the rangeof suppressor loci isolated in various ALL-ROBYNet al. (1990) doesnot map at SUP35, studies and the predominance of either dominant or SUP43, SUP44, SUP45 or SUP46 (ALL-ROBYNet al. recessive alleles is probably due to differences in the 1990). B-7329 was crossed to SL818-2C to test for initial strains. Some of these differences include the 520 L. P. Wakem and F. Sherman

TABLE 4 Phenotypes of mapped suppressors

~ ~~ ~~ Suppression" Strain Suppressor Dorn.' Resp. Osnl. Co,dg sens.''"" a-aad def.' sed lys2-187 met8-1 led-1 aro7-1 his4-166 B-7316 SUP35 + + + + Sd S S + + + B-7331 SUP35 + + - - Dom S + + + 0 B-7329 SUP42 + + + + Doni S + + S 0 B-7332 SUP42 + + + + Sd S + + + 0 B-7317 SUP43 + + + + Sd S + + + 0 B-7334 SUP43 + + + + Sd S + + + 0 B-7319 SUP44 + + + + Dom S + + + 0 B-7323 SUP45 + + + + Sd S + + + 0 B-7324 SUP46 + + + + Dom S + + S - B-7325 SUP46 + + + + Dom S S + + - B-7326 SUP46 + + + + Sd S S + i. - B-7327 SUP46 + + + - Sd S + + + -

a Suppression of lys2-187, metd-1 (UAG), leu2-1 (UAA), aro7-1 (UAG) and his4-166 (UGA) was measured by spotting isolates on the appropriate omission media and incubating for 5 days at 30". (+ = suppression, - = no suppression). Dominance and recessiveness of suppression: dominant (Dom), semidominant (Sd). ' Hypersensitivity to paromomycin at 0.5-1.0 mg/ml. Suppressors unable to utilize a-aminoadipate as sole nitrogen source (+ = growth, S = no growth). e Suppressors which are respiratory deficient (measured as the inability to utilize glycerol as sole carbon source). 'Suppressors which are sensitive to high osmotic pressure (1.0-1.5 M ethylene glycol). Suppressors with enhanced (+) or diminished (-) growth at 5-10'' (0 = growth similar to parental strains). isolation of suppressors in either #- or #+ strains and SUP45 and SUP46 mutants are also similar biochemi- the use of different suppressible mutations to select cally. In in vitro translation assays, using poly(U) as for suppressors. Since we used $J'strains and 5 sup- template and ribosomal components isolated from pressible mutations with a rangeof suppressibility (see various mutants, the misreading defect of these sup- Results), we were able to select for a broader range pressors was localized to the 40 S subunit (EUSTICEet of suppressors with presumably different efficiencies al. 1986). Thisresult plus other datasuggests that the of suppression. sup35, sup44, sup45 and sup46 genes may code for There is a range of allele-specific pleiotropic phe- ribosomal proteins (40s subunit) or for enzymes which notypes which appears to be common to all omnipo- modify ribosomal proteins. However, SUP43 mutants tent suppressor loci. However, some of these pleio- differ from the other suppressors. Ribosomes from tropic phenotypes, such as respiration deficiency may two alleles of SUP43 did not show increased levels of be due to secondary mutations in some of the sup- misreading of the poly(U) template in nitro (EUSTICE pressor isolates. The omnipotent suppressor mutants et al. 1986). This differencemay be due todifferences described in this study can exhibit very different in specificity, or the sup43 gene may not code for suppression spectra and suppression efficiencies, as either a ribosomal protein or for an enzyme which wellas being dominant, semidominant or recessive modifies a ribosomal protein. In vitro translation data for suppression. In addition, mutants can be respira- for sup42 mutants is not available since this suppressor tion deficient, show sensitivity to high osmotic pres- was only recently identified. sure andcan exhibit a rangeof sensitivities and resist- Recent cloning and sequencing data show that the ance to aminoglycoside antibiotics and diminished and sup35 and sup45 genes have openreading frames enhanced growth at cold temperatures. These pleio- which are too large to be ribosomal proteins (BREIN- tropic effects do not appear to be locus-specific since ING, SURCUCHOVand PEIPERSBERC1984; HIMMEL- different alleles can exhibit very different phenotypes FARB, MAICASand FRIESEN1985; BREININCand PEI- (Tables 2 and 5). In addition, the different suppres- PERSBERG 1986; KUSHNIROVet al. 1988; WILSONand sion and phenotypic patterns can not be explained CULBERTSON, 1988). In addition, the sup35 gene se- simply by differences in the efficiency of suppression. quence has some homology to elongation factor la This diversity of phenotypes of different alleles at the (KUSHNIROVet al. 1988; WILSON and CULBERTSON six omnipotent suppressorloci described here suggests 1988). This suggests that these two genes code for that these omnipotent suppressors code for proteins translation factors ratherthan ribosomal proteins. that effect different functions and that altered forms Since the misreading defect of mutants in all these of each of the proteins can effect the same function. genes is localized to the 40s subunit, these factors In a previous study, we showed that SUP35, SUP44, appear to be closely associated to this subunit. Addi- O m nipotent SuppressorsOmnipotent in Yeast 521 tionally, certain alleles of SUP35 have a40s ribosomal determinant suppresses nonsense mutations in yeast. J. Bacte- riol. 139 1068-1071. protein with an altered electrophoretic mobility on MASUREKAR,M., E. PALMER,B. ONO, J. M. WILHELMand F. two-dimensional acrylamide gels (EUSTICEet al. 1986). SHERMAN,1981 Misreadingof the ribosomal suppressor As more of theomnipotent suppressorgenes are SUP46 due to an altered 40s subunitin yeast. J. Mol. Biol. 147: cloned and sequenced,we should gain further insight 381-390. MCCUSKER,J. H., andJ. E. HABER, 1988a Cycloheximide-resistant into the complex mechanism which ensures the accu- temperature-sensitive lethal mutations of Saccharomyces cerevis- rate translation of mRNA into protein in yeast. iae. Genetics 119 303-315. MCCUSKER,J. H., and J. E. HABER, 1988b crl mutants of Saccha- This investigation was supported by the U.S. Public Health romyces cerevisiae resembleboth mutants affecting general Research grant GM12702 from the National Institutes of Health. amino acid biosynthesis andomnipotent translational *sup- We wish to thank Ms. Sandra Consaul for assistance in the isolation pressor mutants. Genetics 119: 317-327. and testing of the suppressors. MIRONOVA,L. N., N. A. PROVOROV,M. D. TER-AVANESYAN,S. G. INGE-VECHTOMOV,V. N. SMIRNOVand A. P. SURGUCHOV, LITERATURECITED 1982 The effect of paromomycin on the expression of ribo- somal suppressors in yeast. Curr. Genet. 5: 149-152. ALL-ROBYN,J. A., D. KELLY-GERAGHTY,E. GRIFFIN,N. BROWN MORTIMER,R., and D. SCHILD,1985 Genetic map of Saccharomy- and S. W.LIEBMAN, 1990 Isolationof omnipotentsuppres- ces cerevisiae, edition 9. Microbiol. Rev. 49: 181-212. sors in an [eta'] yeast strain. Genetics 124: 505-514. ONO,B., J. W. STEWART andF. SHERMAN, 1981 Serine insertion BREINING,P., and W. PIEPERSBERG, 1986Yeast omnipotent sup- caused by the ribosomal suppressor SUP46 in yeast. J. Mol. pressor supl (sup45):nucleotide sequence of thewild type and Biol. 147: 373-379. a mutant gene. Nucleic Acids Res. 14: 5187-5197. ONO,B., M. TANAKA,M. KOMINAMI,Y. ISHINOand S. SHINODA, BREINING,P., A. P. SURGUCHOVand W. PIEPERSBERG,1984 1982 Recessive UAA suppressors of the yeast Saccharomyces Cloning and identification of a DNA fragment coding for the cerevisiae. Genetics 102: 653-664. supl gene of Saccharomyces cerevisiae. Curr. Genet.8: 467-470. ONO,B., N. MORIGA,K. ISHIHARA,J. ISHICURO,Y. ISHINOand S. CHATTOO, B. B., E. PALMER,B. ONOand F. SHERMAN, SHINODA,1984 Omnipotent suppressorseffective in $+ 1979a Patterns of genetic and phenotypicsuppression of lys2 strains ofSaccharomyces cerevisiae: recessiveness and dominance. mutations in the yeast Saccharomyces cerevisiae. Genetics 93: 67- Genetics 107: 219-230. 79. ONO,B., Y. ISHINO-ARAO,M. TANAKA,I. AWANO andS. SHINODA, CHATTOO,B. B., F. SHERMAN,D. A. AZUBALIS,T. A. FJELLSTEDT, 1986 Recessive nonsense suppressors in Saccharomyces cerevis- D. MEHNERT andM. OGUR, 1979b Selection of lys2 mutants iae: action spectra, complementation groups and mappositions. of the yeast Saccharomyces cerevisiae by the utilizationof a- Genetics 114 363-374. aminoadipate. Genetics 93: 51-65. ONO, B., M. TANAKA,1. AWANO, F. OKAMOTO,R. SATOH, N. Cox, B. S., 1965 A cytoplasmic suppressor of super-suppressor in YAMAGISHI and Y. ISHINO-ARAO,1990 Two new loci that yeast. Heredity 20: 505-521. give rise to dominant omnipotent suppressors in Saccharomyces COX,B. S., 197 1 A recessive lethal super-suppressor in yeast and cerevisiae. Curr. Genet. (in press). other $ phenomena. Heredity 26: 21 1-232. PALMER,E., J. M. WILHELMand F. SHERMAN, 1979aVariation of Cox, B. S., 1977 Allosuppressors in yeast. Genet. Res. 30: 187- phenotypic suppression due to the$+ and $- extrachromosomal 205. determinants in yeast. J. Mol. Biol. 128: 107-1 10. CULBERTSON,M. R.,R. F. CABERand C. M. CUMMINS, PALMER,E., J. M. WILHELM andF. SHERMAN,197913 Phenotypic 1982 Frameshiftsuppression in Saccharomyces cerevisiae. V. suppression of nonsense mutants in yeast by aminoglycoside Isolation and genetic properties of nongroup-specific suppres- antibiotics. Nature 277: 148-150. sors. Genetics 102: 361-378. SANDBAKEN,M. G., and M. R. CULBERTSON,1988 Mutations in elongation factor EF-la affect the frequency of frameshifting CULBERTSON,M. R., L. CHARMAS,M. T. JOHNSON and G. FINK, and amino acidmisincorporation in Saccharomyces cerevisiae. 1977 Frameshifts and frameshift suppressors in Saccharomyces Genetics 120: 923-934. cerevisiae. Genetics 86: 745-764. SHERMAN,F., G. R. FINK and J. B. HICKS,1987 Methodsin Yeast EUSTICE,D. C., L. P. WAKEM,J. M. WILHELMand F. SHERMAN, Genetics. Cold Spring Harbor Laboratory,Cold Spring Harbor, 1986 Altered 40s ribosomal subunits in omnipotent suppres- N.Y. sors of yeast. Mol. Biol. 188: 207-214. J. SINGH, A.,D. URSICand J. DAVIES,1979 Phenotypic suppression GELUGNE,J., and J. B. BELL,1988 Modifiers of ochre suppressors and misreading in Saccharomyces cerevisiae. Nature 277: 146- in Saccharomyces cerevisiae thatexhibit ochre suppressor-de- 148. pendent amber suppression. Curr. Genet.14: 345-354. SURGUCHOV,A. P., V. N. SMIRNOV,M. D. TER-AVANESYAN andS. GERLACH,W. L., 1975 Genetic properties of some amber-ochre G.INGE-VECHTOMOV, 1984 Ribosomalsuppression in eukar- supersuppressors in Saccharomyces cerevisiae. Mol. Gen. Genet. yotes. Physiochem. Biol. Rev. 4: 147-205. 138: 53-63. WILSON, P. G., and M. R. CULBERTSON,1988 SUFI2 suppressor HAWTHORNE,D. C., and U. LEUPOLD,1974 Suppressor mutations protein of yeast: a fusion protein related to the EF-1 family of in yeast. Curr. Genet. 64: 1-47. elongation factors. J. Mol. Biol. 199: 559-573. HIMMELFARB, J.,H. E. MAICASand J. D. FRIESEN,1985 Isolation WOOD, J. S., 1982a Genetic effects of methyl benzimidazole-2-yl- ofthe sup45 omnipotentsuppressor gene of Saccharomyces carbamate on Saccharomyces cerevisiae. Mol. Cell. Biol. 2: 1064- cerevisiae and characterization of its gene-product. Cell Biol. 5: 1079. 816-822. WOOD,J. S., 1982b Mitotic chromosome loss induced by methyl INCE-VECHTOMOV,S. G., and V. M. ANDRIANOVA, 1970Recessive benzimidazole-2-yl-carbamateas a rapid mapping method in super-suppressors in yeast (in Russian). Genetika 6: 103-1 15. Saccharomyces cerevisiae. Mol. Cell. Biol. 2: 1080-1087. KUSHNIROV,V. V., M. D. TER-AVANESYAN,M. V. TELCKOV,A. P. ZARET,K. S., and F. SHERMAN, 1985 a-Aminoadipateas a primary SURGUCHOV,V. N. SMIRNOVand S. G.INGE-VECHTOMOV, nitrogen source for Saccharomyces cerevisiae mutants. J. Bacte- 1988 Nucleotide sequence of the sup2 (sup35) gene of Sac- riol. 162: 579-583. charomyces cerevisiae. Gene 66: 45-54. Communicating editor: M. CARLSON LIERMAN,S. W.,and F. SHERMAN,1979 Extrachromosomal $+ 522 L. P. Wakem and F. Sherman

ADDENDUM Another similar study of omnipotentsuppressors describes SUP139 (ONOet al. 1989). Table 5 summarizesthe different theisolation and mapping of thetwo suppressors, SUP138 andomnipotent suppressors and their alleles.

TABLE 5 Omnipotent suppressors and their alleles

References

SUP35 SUP2, SUPP, SUP36, SUF12, SAL3 INGE-VECHTOMOVand ADRIANOVA(1970), HAWTHORNE and LEUPOLD(1974), GERLACH(1975), Cox (1977), CULBERTSON, GABERand CUMMINS (1982), ONO et al. (1984) Sl'P39 ALL-RORYNet al. (1 990) SUP42 This study SLP4 3 This study SL'P44SL'P38, SUP138 This study, ALL-ROBYNet al. (1990). ONOet al. (1 990) WP45 SLIPI, SUPQ, SUP47,SAL4, IMOSI INGE-VECHTOMOVand ADRIANOVA(1970), HAWTHORNEand LEUPOLD(1974), GERLACH (1975),Cox (1977), CULBERTSON, GABERand CUMMINS (1982), ONO et al. (1984), GELUCNEand BELL(1 988) SUP46 STEWARTONO, and SHERMAN(1 98 1) sup1 11 ONOet al. (1986) sup1 12 ONOet al. (1986) sup113 ONOet al. (1 986) SUP139 ONOet al. (1 990)