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515.Full.Pdf Copyright 0 1990 by the Genetics Society of America Isolation and Characterizationof Omnipotent Suppressorsin the Yeast Saccharomyces cerevisiae L. Paul Wakem* and Fred Sherman**t Departments of *Biochemistry and +Biophysics, University of Rochester 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). Suppressors belonging 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.
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