Frameshift Suppression in Saccharomyces Cerevzszae

Suppressors of ICR-induced mutations that exhibit behavior similar to bacterial frameshift suppressors have been identified in the yeast Saccharomyces cerevisiae. The yeast suppressors have been divided into two groups. One of these groups (Group 11: SUFI, SUF3, SUF4, SUP5 and SUF6) appears to include a set of informational suppressors in which the vehicle of suppression is glycyl-tRNA. Some of the genetic properties of Group I1 suppressors are described in this communication.-Corevertants of the Group I1 frameshift mutations his4-519 and leu2-3 have been characterized to determine the spectrum of reversion events induced by the frameshift mutagen ICR-170. Seventythree ICR-induced corevertants were analyzed. With the exception of one corevertant, which carried an allele of SUFI, all carried alleles of SUF3 or SUF5. SUFI, SUPS, SUF4 and SUP6 were represented among spontaneous and UV-induced corevertants. In the course of these experiments one of the suppressors was mapped. SUPS, the probable structural gene for tRNAQLY1, is located between ade2 and a h 9 on chromosome XV.-SUFI, SUF4 and SUF6 have novel properties and comprise a distinct subset of suppressors. Although these suppressors show no genetic linkage to each other, they share several common features including lethality in haploid pairwise combinations, reduced tRNAQLYs isoacceptor activity and increased efficiency of suppression in strains carrying the cytoplasmically inherited [PSI] element. In addition, strains carrying SUFI, SUF4 or SUP6 are phenotypically unstable and give rise to mitotic Suf + segregants at high frequency. These segregants invariably contain a linked, second-site mutation that maps in or adjacent to the suppressor gene itself. Strains carrying any of these suppressors also give rise to mitotic segregants that exhibit enhanced efficiency of suppression; mutations responsible for this phenotype map at two loci, upjI and upj2. These genes show no genetic linkage to any of the Group I1 suppressors.-Methods that permit positive selection for mutants with decreased or enhanced efficiency of suppression have been devised in order to examine large numbers of variants. The importance of these interacting mutants is underscored by their potential utility in studying suppressor function at the molecular level. * To whom reprint requests should be addressed Genetics 95: 833-853 August, 1980. 834 M. R. CULBERTSON AND K. M. UNDERBRINK MUTATIONS induced by the acridine half-mustards (ICR compounds) have been studied extensively in Salmonella typhimurium (AMES and WHITFIELD 1966; YOURNO and HEATH 1969; YOURNO 1971). A large proportion of the mutants contain +I G/C insertions in monotonous runs of G/C base pairs. These types of mutations shift the reading frame of the message out of phase beyond the point of insertion and result in the production of a nonfunctional protein. ICR-induced revertants of 4-1 G/C insertions in the Salmonella histidiae operon frequently carry mutations mapping at sites external to the operon that confer a His+ phenotype (RIDDLE and ROTH 1970). These external suppressors map at sites on the bacterial chromosome known to contain tRNA genes, and altered forms of tRNA have been shown to mediate framesehift suppression by reading a four-base codon. Strains of Salmonella carrying the frameshift suppressor sufD produce a glycyl-tRNA with the nucleotide quadruplet CCCC at the anticodon position, instead of CCC normally found in wild type (RIDDLE and ROTH 1972a,b; RIDDLE and CARBON 1973). The addition of this extra base is presumed to permit recognition of the four-base code word GGGN and thereby correct the reading frame. A second class of frameshift suppressors was shown to alter the chromatographic behavior of prolyl-tRNA (RIDDLE and ROTH 1972b). These suppressors are also believed to act by reading a four-base code word. These results demonstrate that the acridine half-mustards derive their powerful mutagenic activity in part from an ability to promote G/C base pair insertions in DNA. Two groups of external suppressor mutations, Group I1 and Group 111, have been identified among revertants of ICR-induced mutations at the his4 locus in Saccharomyces mrevisiae ( CULBERTSON et al. 1977). Their properties suggest that they may be analogous to bacterial frameshift suppressors. Elution profiles obtained by co-chromatography of tRNA €rom wild-type strains and strains carrying the suppressors suggest that one group (Group 11: SUFI, SUF3, SUF4, SUE75 and SUF6) may be mutations in the structural genes for glycyl-tRNAs. Some of the genetic properties of these suppressors are described in this communication, Corevertants of the Group I1 frameshift mutations his4-519 and leu2-3 have been characterized to determine the spectrum of reversion events induced by the frameshift mutagen ICR-170. ICR-induced reversion of these mutations results in suppressors that map at the SUF3 and SUFS loci. By contrast, spontaneous and UV-induced revertants carry mutations that map at SUFI, SUF3, SUF4 and SUF6. Similar results were obtained in a genetic study of Group I11 suppressors (CUMMINS et al. 1980). ICR-induced revertants of the Group I11 mutation hid-713 were shown to carry alleles of the SUFZ locus; whereas, spontaneous and UV-induced revertants carry mutations that are distributed among six Group I11 suppressor loci. These studies demonstrate that only three of the 11 suppressor genes obtained by reversion of ICR-induced his4 mutations are targets for mutagenesis by ICR-170. FRAMESHIFT SUPPRESSORS IN YEAST 835 Strains carrying the Group 11 suppressor, SUF5, have been shown to produce a chromatographically altered species of tRNAGLY1, suggesting that SUFS is the structural gene for this tRNA (CULBERTSON et al. 1977). In the course of this study, SUFS was mapped on chromosome XV between ade2 and ade9. Three of the five Group I1 suppressors, SUFI, SUF4 and SUF6, result in reduced isoacceptor activity of tRNAGLPs and have unusual properties that are described in this communication. The genetic map positions of these suppressors have not yet been determined, but painvise crosses show that these suppressors represent three unlinked genes. SUFI, SUF4 and SUF6 are lethal in combination with each other; double-mutant spores carrying two suppressors cannot be recovered after meiosis. In addition, it has been shown that the efficiency of suppression by SURI, SUF4 or SUP6 is increased in strains carrying the cytoplasmically inherited [PSI] element (CULBERTSON et al. 1977). [PSI] also increases the efficiency of the serine-inserting ochre suppressors SUP17 and SUQ5 (SUQ5 = SUPIS = SUPId), and autonomously suppresses certain ochre mutations (e.g., trp5-48) (Cox 1965; LIEBMAN, STEWART and SHERMAN 1975; LIEBMAN and SHERMAN 1979; ONO, STEWART and SHERMAN 1979). In this study we show that strains carrying SUFI, SUF4 or SUF6 are phenotypically unstable and give rise to mitotic Suf+ segregants at high frequency. These segregants invariably contain a mutation that maps in or adjacent to the suppressor gene itself. Mitotic and meiotic recombination analysis shows that they are second-site mutations, rather than back-mutations to wild type. A method is described in which second-site revertants of the suppressors can be isolated by positive selection for canavanine resistance in cells carrying a suppressor and a Group I1 suppressible can1 mutation. In addition, mutations conferring enhanced efficiency of framesehift suppression have been isolated as mitotic segregants from strains carrying SUFI, SUP4 or SUF6. These mutations, designated upf for “up-frameshift suppressor,” can be recovered from strains carrying a suppressor and the suppressible his# allele his#-38. These strains are temperature sensitive for growth on minimal medium. Up-suppressor mutations confer the ability to grow at the restrictive temperature. Mutations of this type map at two loci that show no genetic linkage to each other or to the Group I1 suppressor loci. Unlike the [PSI] element, which also confers growth at the restrictive temperature in strains carrying his4-38 and SUFI, SUR4 or SUF6, upf mutations are chromosomally inherited. The molecular basis of temperature-sensitive growth associated with suppression of his4-38 is not yet understood. Two models consistent with the observations are discussed in relation to the interaction between frameshift suppressors and upf mutations. The similarities of SUFI, SUF4 and SUF6 suggest that these suppressors may represent redundant forms of the same gene. We anticipate that the peculiar genetic properties of these suppressors will eventually be resolved at the molecular level and will provide information on the synthesis and function of the suppressing tRNA. 836 M. R. CULBERTSON A N D K. M. U N D E R B R I N K MATERIALS A N D METHODS Yeast strains and genetic methods: All strains used in this study are derivatives of the wild-type laboratory strain S288Ca. The isolation of frameshift mutations, frameshift suppressors and the characterization of strains used in this study have been described (CULBERTSON et al. 1977). Genetic methods and nomenclature are those described in the Cold Spring Harbor Yeast Course Manual (SHERMAN, FINK and LAWRENCE 1971). Media: The following types of media were used: YEPD, which contains 2% Bactopeptone, 1% yeast extract, 2% glucose and 2% agar; minimal medium, which contains 6.7 g/l Difco Yeast Nitrogen Base, 2% glucose and 2% agar; KAC (sporulation medium), which contains 1% potassium acetate, 0.1% glucose, 1.25 g/1 yeast extract and 2% agar. When required, purines, pyrimidines, or amino acids were added to minimal medium at concentrations given in the Cold Spring Harbor Yeast Course Manual (SHERMAN, FINK and LAWRENCE 1971). Coreuersion of frameshift mutation: His+ Leu+ corevertants of a strain carrying the frameshift mutations his4-519 and leu2-3 were isolated as follows: Single colonies were isolated on YEPD plates. Cells from individual colonies were picked and suspended in culture tubes containing 3 ml of YEPD and grown overnight with shaking at 30" to a density of 2 x lo8 cells/ml. In this procedure, the cloning of independent lines prior to mutagenesis insured that mutants obtained from different culture tubes were of independent origin. The cells were centrifuged, washed twice with sterile water and concentrated 10-fold by resuspension in 0.3 ml of water. 0.1 ml aliquots were spread on minimal plus leucine plates to select for His+ revertants. Plates were either incubated without mutagenesis, irradiated with UV for 25 sec at a dose that gave 80% survival, or treated with ICR-170 (2-methoxy-6-chloro-9-[-3(ethyl-2-chloroethyl) aminopropylamino] acridine.ZHC1 (Polysciences, Inc.). ICR-I 70 mutagenesis was performed according to the method of CULBERTSON et al. (1977) with the following modification: cells were mutagenized directly on minimal plus leucine plates containing 0.2 ml YEPD. The small amount of YEPD added to the synthetic medium is insufficient to supplement the His and Leu auxotrophies, but is absolutely required for the mutagenic activity of ICR-170. The reason for the YEPD requirement is unknown. Following the appearance of His+ revertants, the cells were replicaplated to minimal medium to detect His+ Leu+ corevertants. The corevertants were analyzed in genetic crosses described in RESULTS. Genetic mapping: Linkage of SUF5 to markers on chromosome XV was determined in standard crosses, and linkage distances were calculated in centimorgans (CM) using the equation X (CM) = 50 [tetratype asci + 6(nonparental-ditype asci)]/total asci (PERKINS 1949). The ade9 mutation used in this mapping study was identified in a collection of ICR-170-induced auxotrophs and is not suppressed by SUFS (GABER, EDELMAN and CULBERTSON, unpublished). Isolation of suppressible canauanine-resistant mutants: Mutations conferring resistance to canavanine were induced by ICR-170. Ten of a 1 mg/ml solution of ICR-170 was placed on a YEPD plate that had first been covered with a lawn of canavanine-sensitive cells. The strain to be mutagenized carried the group I1 mutations his4-519 and leu2-3 so that canavanineresistant mutants could be analyzed in a coreversion test. Cells were grown in the presence of ICR-I70 overnight at 30" on YEPD medium and then replica-plated to minimal medium supplemented with histidine, leucinine and 60 mg/l L-canavanine sulfate (Sigma). The plates were incubated for 5 days. Colonies Forming a ring around the drop of ICR-170 were picked, purified and tested as described in RESULTS. Estima,tion of cell-doubling time: The doubling time of a strain carrying SUFI was compared to that of a Suf+ revertant of this suppressor in order t o determine whether the apparent high frequency of reversion observed for SUFI is due to a selective growth advantage of Suf+ segregants. A strain carrying his4-519 leu2-3 canl-I01 SUFI was plated on media containing canavanine to select for Suf+ revertants. The canl-IO1 mutation is suppressible by SUFI (see RESULTS). Thus, the his4-519 leu2-3 canl-101 SUFI strain is canavanine sensitive due to suppression, whereas Suf+ revertants of this strain are canavanine resistant due to loss of suppressor function. Suf+ revertants selected in this way were purified and tested for loss of suppressor function by replica-plating to minimal media lacking histidine and leucine. A HisLeuCan= FRAMESHIFT SUPPRESSORS IN YEAST 837 revertant was used to compare the doubling time of the revertant with the isogenic SUFI parental strain from which it was derived. Cultures of the two strains were prepared by inoculating YEPD medium at a starting density of 1 x 106 cells per ml. The SUFI strain was precultured in minimal medium to ensure that 100% of the cells in the primary culture initially carried the suppressor. ?*he Suf+ strain was precultured in minimal medium supplemented with histidine and leucine. The cultures were monitored at 2 hr intervals by diluting and plating aliquots on YEPD medium to determine the number of viable cells per ml. It was necessary to test the SUFI culture at each time point for the appearance of Suf+ revertants by comparing the plating efficiency of the culture on YEPD medium and minimal medium. In addition, colonies that grew on YEPD medium were replica-plated to minimal medium, and any colony that failed to grow on minimal medium was scored as a Suf+ revertant. This procedure permitted a determination of the percentage of cells at each time point that had maintained a functional suppressor. The doubling time of the SUFl strain was calculated using a culture in which more than 99% of the cells had retained the SUFI phenotype at the end of the logarithmic phase of growth. The doubling times of the SUFI and Suf+ strains could then be accurately compared by plotting log cell concentration us. time of growth. As an additional control, a reconstitution experiment was performed in which SUFI cells were mixed with Suf+ revertant cells at a ratio of 4 to 1, and the ratio of the 2 types of cells was monitored throughout the growth of the culture, using the methods described above.