JOURNAL OF BACTERIOLOGY, JUlY 1985, p. 8-14 Vol. 163, No. 1 0021-9193/85/070008-07$02.00/0 Copyright © 1985, American Society for Microbiology Characterization of Saccharomyces cerevisiae Mutants Supersensitive to JOACHIM F. ERNST* AND RUSSELL K. CHAN Department ofMicrobiology, Biogen S. A., CH-1227 Geneva, Switzerland Received 14 January 1985/Accepted 25 March 1985 We describe mutants of Saccharomyces cerevisiae that are more sensitive than the wild type to the aminoglycoside antibiotics G418, , destomycin A, and X2. In addition, the mutants are sensitive to , kanamycin B, lividomycin A, neamine, , , and tobramy- cin-antibiotics which do not inhibit wild-type strains. Mapping studies suggest that supersensitivity is caused by mutations in at least three genes, denoted AGS), AGS2, andAGS3 (for aminoglycoside sensitivity). Mutations in all three genes are required for highest antibiotic sensitivity; ags1 ags2 double mutants have intermediate antibiotic sensitivity. AGS1 was mapped 8 centimorgans distal from LEU2 on chromosome III. Analyses of yeast strains transformed with vectors carrying antibiotic resistance genes revealed that G418, gentamicin X2, kanamycin B, lividomycin A, neamine, and paromomycin are inactivated by the Tn903 phosphotransferase and that destomycin A is inactivated by the hygromycin B phosphotransferase. ags strains are improved host strains for vectors carrying the phosphotransferase genes because a wide spectrum of aminoglycoside antibiotics can be used to select for plasmid maintenance.

Several approaches have led to the discovery of dominant procedures were used for crosses and scoring of phenotypes selectable transformation markers in the yeast Sac- (26). The Ags- phenotype was routinely scored on 1% yeast charomyces cerevisiae. First, procaryotic antibiotic resis- extract-2% peptone-2% glucose-2% Bacto-agar (Difco Lab- tance genes have been expressed in yeast, and it has been oratories, Detroit, Mich.) (YPD) agar (26) containing 20 jig of demonstrated that certain aminoglycoside antibiotics select G418 per ml or 20 ,ug of hygromycin B per ml; in addition, for the growth of strains expressing the corresponding resis- zones of inhibition surrounding antibiotic-containing filter tance gene. The kanamycin phosphotransferase (APH) en- disks on yeast lawns were determined in some cases. Con- coded by a gene of transposon Tn9O3, which inactivates the centration-dependent inhibition by antibiotics was deter- antibiotic G418 (8, 10, 32), and the gene encoding mined by inoculating 5 ml of liquid YPD containing antibiot- hygromycin B phosphotransferase (HPH) (7, 12) have been ics with 50 ,ul of a saturated yeast culture grown in minimal used in this fashion. Second, certain proteins have been medium (26); starter cultures of transformed strains were overproduced in yeast by inserting the encoding genes on grown selectively (without uracil) in minimal medium. After high-copy-number vectors; yeast strains transformed with incubation at 30'C for 72 h on a rotating wheel, the optical these vectors are resistant to specific metabolic inhibitors of density at 600 nm was recorded. Sensitivity to chlorampheni- the overproduced enzymes. Overproduction of enzymes (15, col (4 mg/ml), cycloheximide (0.5 jig/ml), or (2 22), copper chelatin (11), and a ribosomal protein (5) ren- mg/ml) was determined on YP agar (26) containing 4% glyc- dered yeast resistant to the corresponding specific inhibitors. erol, sensitivity to oligomycin (5 jig/ml) was determined on Whereas the overproduction approach usually requires YP agar containing 3% glycerol, and sensitivity to dequali- the use of high-copy-number vectors, the gene encoding nium chloride (5 ,ug/ml) was determined on YPD agar. Plat- APH renders yeast resistant to G418 even at low copy ing efficiencies were determined on YPD agar for whole cells numbers (32). In addition, the genes encoding APH and and on YPD agar containing 1 M sorbitol for spheroplasts. HPH are expressed in S. cerevisiae, as well as in Escherichia The ability of ags mutants to suppress nonsense mutations coli (7, 8, 32), allowing for antibiotic selection in both was assessed by crossing an ags strain with a strain carrying organisms with yeast shuttle vectors (2). However, the nonsense mutations. The resulting diploid was sporulated, relatively high concentrations of G418 and hygromycin B (7, and tetrads were dissected. The presence of ags spores that 32) needed in selective growth media have hampered the use expressed the phenotype of the unsuppressed marker was of phosphotransferase genes as dominant selectable markers taken as evidence that ags did not suppress that mutation. in yeast. We describe here mutants of S. cerevisiae that are Plasmids. Standard procedures were used for the con- particularly sensitive to G418 and hygromycin B. These struction and amplification of plasmids (14). For construc- strains are also sensitive to a series of other aminoglycoside tion of pEX-2, the CYCI terminator region was subcloned antibiotics, many of which are inactivated by the Tn9O3 first. The 270-base-pair HaeIII-to-HindIII fragment contain- phosphotransferase. Thus, in our approach the modification ing the terminator (29, 33) was isolated and inserted between of the yeast host allows optimal use of the existing phospho- the HincII and HindIII sites of bacteriophage M13mp8 (14). transferase genes. The EcoRI-to-HindIII terminator fragment was isolated MATERIALS AND METHODS from double-stranded phage DNA and ligated with the following two fragments to construct pEX-1: (i) the 4.85- Strains and growth conditions. The yeast strains used in kilobase HindIII-to-BamHI fragment of plasmid pAB107 (S. this study are listed in Table 1. Standard yeast genetic Baim and F. Sherman, unpublished results)-pAB107 con- tains the 0.85-kilobase EcoRI-to-HindIII ARSI fragment of * Corresponding author. YRp7 (2) inserted between the EcoRI and HindIII sites of 8 VOL. 163, 1985 S. CEREVISIAE MUTANTS SUPERSENSITIVE TO ANTIBIOTICS 9

TABLE 1. Yeast strains than were other laboratory strains. This phenotype was denoted Ags-, for aminoglycoside antibiotic sensitivity. Strain Genotype Origin Comparisons of wild-type and Ags- strains for antibiotic BJ1991 MATaz pep4-3 prbl-1122 E. Jones, sensitivity demonstrated that Ags- strains are 10- to 20-fold ura3-52 leu2 trpl Carnegie- mnore sensitive to G418 and hygromycin B than are wild-type Mellon strains (Fig. 2). In complex growth medium, 50% inhibition University HR12S-llb MATa leu2-3 leu2-112 G. Sprague, was obtained at 1 to 2 ,ug/ml. Ags- strains were also more trpl ura3-52 his3 his4 University of sensitive to gentamicin X2 and destomycin A-two antibiot- Oregon ics that inhibit growth of wild-type strains (Fig. 3, Fig. 4). MB50OC-T1OC MATa his7 met2 tup7 gall L. F. Bisson (1) To verify that the Ags- strain could be used as host for ags yeast vectors carrying the known phosphotransferase genes RC1678 MATa ura3-52 trpl tup7 HR125-llb x (7, 10), we constructed plasmids that carry either the HPH- ags MB50OC-TlOC encoding gene (pLG89) or the Tn9O3 APH-encoding gene RC1705 MATa ura3-52 trpl leu2 BJ199i x RC1678 (pEX-4) or both (pGH41) (Fig. 1). All constructed plasmids prbl-1122 carry the yeast URA3 gene in addition to the antibiotic RC1707 MATa ura3-52 trpl tup7 Same tetrad as pep4-3 prbl-1122 ags RC1705 resistance marker(s). An Ags+ ura3 strain and an Ags- ura3 strain were transformed with plasmids pEX-2, pLG89, or pGH-1, selecting for uracil prototrophy. Quantitative aniti- biotic sensitivity tests were performed on the untransformed YIp5 (2); (ii) the 1.1-kilobase BamHI-to-EcoRI fragment of and transformed strains (Fig. 3, Fig. 4). As expected, an plasmid pAB63 carrying the cycl-13 promoter (Baim and Ags+ transformant expressing APH (encoded on pEX-4) was Sherman, unpublished results). The cycl-13 mutation is a resistant to G418 at all antibiotic concentrations tested. single nucleotide change altering the ATG translation start Similarly, an Ags- strain expressing APH was more resis- codon to ATA (27). The sequence of the junction cycl-13 tant to G418 than the untransformed Ags- host and the Ags- promoter-M13 sequences-CYCI terminator is given in Fig. host carrying plasmid pLG89. However, at G418 concentra- 1A. pEX-2 was constructed by insertion of the 1.47-kilobase tions greater than 20 ,ug/ml, inhibition by G418 was seen HincII fragment carrying the origin of replication of the even when the Ags- strain contained pEX-4. The presence yeast 2,u circle (8) into the single PvuII site of pEX-1. The of plasmid pLG89 rendered Ags+ and Ags- strains more construction of pLG89 from pEX-2 has been described resistant to hygromycin B (Fig. 3, Fig. 4). As described previously (7). pGH-1 and pEX-4 were constructed by previously (7), hygromycin B resistance was incomnplete at insertion of the 1.7-kilobase PvuII fragment of plasmid pAJ50 (10) carrying the APH-coding gene of Tn9O3 (16) into the filled-in SalI sites of pEX-2 and pLG89, respectively (Fig. 1B). The Sall sites were restored in these construc- A EcoRI SMO I BOmHI tions; in pGH-1, a 0.6-kilobase Sall fragment containing TTAATAIJACTGAATTC CCGGGGATCCGTC CCCCTTTTCC upstream sequences of the cycl-13 promoter and pBR322 cyc/-I3 promoter ------Ml3mp8----- *-_CYC/ terminator sequences was deleted during the construction. Transforma- tion of yeast strains with plasmids was performed by the spheroplast method (26) or by the salt method (9). B Reagents. G418 sulfate (Geneticin) was obtained from ARSI GIBCO Europe Ltd., Paisley, United Kingdom, and from Schering Corp., Bloomfield, N.J. Hygromycin B was ob- PEX-2 G89 R tained from Eli Lilly & Co., Indianapolis, Ind. B B I Hyy sulfate (95% kanamnycin A, 5% kanamycin B), neomycin 9.3kb 310.3kb 2,p cyc/- 3 B sulfate (90 to 95% neomycin B), , chlorampheni- oni col, cycloheximide, tetracycline, oligomycin (65% oligomycin A, 20% oligomycin B, 15% oligomycin C), ami- URA3 smR S kacin, and dequalinium chloride were obtained from Sigma Chemical Co., St. Louis, Mo. Paromomycin was obtained from Farmitalia, Milano, Italy. Neamine (91%), (58.4%), apramycin (86%), , lividomycin A, genta- micin X2, kanamycin B, destomycin A, and were gifts fronm J. Davies. The enzymes used in plasmid pEX-4 B pH- HygR constructions were from New England Biolabs, Beverly, 11i kb A11.4kb Hy Mass., and were used according to the recommendation of the manufacturer.

KmR RESULTS FIG. 1. Plasmid constructions. (A) sequence of the promoter- Mutants with enhanced sensitivity to anti- terminator junction in pEX-2. The boxed nucleotides indicate the amninoglycoside ATG start codon of the CYCI gene, which is changed to ATA by the biotics. Wild-type strains of S. cerevisiae are relatively cycl-13 mutation (27). (B) construction of pEX-4 and pGH-1. The resistant to the aminoglycoside antibiotic G418 (8, 18). construction of pLG89 has been described previously (7). Yeast During routine strain constructions we discovered that strain sequences are drawn as heavy black lines, and pBR322 sequences MB500C-T1OC (1) and some of its outcrossed progeny were are drawn as thin lines. Antibiotic resistance genes are drawn as more sensitive to G418 and other aminoglycoside antibiotics open boxes. Arrows indicate the direction of transcription. 10 ERNST AND CHAN J. BACTERIOL. high antibiotic concentrations; in our experiments an Ags+ pLG89 transformant was fully resistant up to 50 jig/ml and an Ags- pLG89 transformant was resistant up to 10 pugIml. We demonstrated that antibiotics gentamicin X2 and destomycin A have properties similar to G418 and hygromycin B, respectively. Both antibiotics acted on wild- type strains; gentamicin X2 was inactivated by APH, and destomycin A was inactivated by HPH in Ags+ and Ags- cells (Fig. 3, Fig. 4). New antibiotic sensitivities of Ags- strains. The Ags- strain was sensitive to antibiotics that did not act on wild-type strains. Apramycin, kanamycin B, lividomycin A, neamine, E * 4. neomycin, paromomycin, and tobramycin inhibited the 0 growth of the Ags- strain (Table 2). , gentamicin o 200 400 c loo 200 D B, kanamycin A, netilmicin, ribostamycin, and sisomicin 0D showed no inhibitory effect on Ags+ or Ags- strains. Growth tests in liquid YPD medium (Fig.- 5) demonstrated that neamine, kanamycin B, lividomnycin A, and paromomycin 1.0 are inactivated by APH, since Ags- strains transformed with pEX-4 grew to higher densities as compared with the untransformed strain. The difference in growth was small for neamine and for kanamycin B; it was virtually absent for 0.I

200 400 40 80 Concentration (Mug/mi) FIG. 3. Inhibition of untransformed and transformed RC1705 1.0 (Ags+) by (A) G418, (B) hygromycin B, (C) gentamycin X2, or (D) destomycin A. Cells were untransformed (D) or transformed with plasmid pEX-4 (A), encoding kanamycin phosphotransferase, or with plasmid pLG89 (0), encoding hygromycin B phosphotransfer- ase. The assay was as described in the text. OD, Optical density.

0.1 neomycin. Therefore, it was of interest to determine whether these antibiotics could be used to select for yeast transform- ants expressing the aminoglycoside-inactivating enzyme. To test this possibility, we grew an Ags- strain transformed with plasmid pGH-1 (which carries the genes encoding APH and HPH) in the presence of antibiotics for approximately 48 c generations (Table 2). Generally, the concentration of anti- 0 0 biotics was chosen such as to allow maximal growth of a o trapsformant expressing the resistance gene, while inhibiting tD maximally the untransformed Ags- host. Concentrations for neamine, kanamycin B, and neomycin were chosen to allow sufficient growth. After growth, single colonies were tested 1.0 for the maintenance of the URA3 gene also present on the yeast vector, The results show that all antibiotics that are inactivated in yeast as shown by the growth tests also will select for plasmid maintenance. This result was also ob- tained with an Ags- strain transformed with pEX-4 or with pLG89, indicating that the presence of both the APH- and 0.I the HPH-encoding genes on pGH-1 does not influence the selection for either resistance gene. The percentage of plas- mid maintenance varied, since the antibiotic concentration during the growth period was not optimized. Surprisingly, neomycin appears to select for plasmid maintenance, even though the selective growth advantage of a yeast strain 20 40 expressing APH is negligible. As expected, tobramycin and apramycin conferred no selective advantage for plasmid Concentration (,ug/ml ) maintenance, since neither antibiotic is inactivated by either FIG. 2. Inhibition of RC1705 (Ags+) (A) and RC1707 (Ags-) (O) APH or HGH (4). by G418 (A) and hygromycin B (B). The assay was as described in Genetic characterization of ags. To determine the genetic the text. OD, Optical density. basis for the Ags- phenotype we crossed strains BJ1991 VOL. 163, 1985 S. CEREVISIAE MUTANTS SUPERSENSITIVE TO ANTIBIOTICS 11

a phenotype with intermediate sensitivity to aminoglycoside antibiotics. Mutants of this intermediate phenotype showed enhanced sensitivities to G418, hygromycin B, gentamicin X2, destomycin A, and paromomycin, with 50% growth inhibition at concentrations of 8, 8.5, 45, 8.5, and 80 ,uwg/ml, respectively. These enhanced sensitivities, as compared with the wild-type, were not associated with the additional antibiotic sensitivities of the Ags- mutant (Table 2). The intermediate and the Ags- phenotypes could be conven- iently scored on YPD agar containing different concentra- tions of G418; whereas the original Ags- mutant was sensi- tive to 20 jig of G418 per ml, a mutant with the intermediate e 80~ phenotype was resistant to 20 jig of G418 per ml, but oDto0 40 80o 20 40 sensiti've to 40 ,ug of G418 per ml.

0 Analyses of the cross described above revealed that 25 of 30 tetrads showed 2:2 segregation for Ags+:Ags- when the criterion for Ags- was sensitivity to 40 jig of G418 per ml; of the remaining tetrads four segregated 3:1 and one segregated 4:0. The presence of 4:0 and 3:1 segregation suggested that sensitivity is determined by more than one gene. If the criterion for Ags- was sensitivity to 20 ,ug of G418 per ml, 0.I then 14 out of 30 tetrads showed 2:2 segregation for Ags+:Ags-; of the 16 tetrads that were not 2:2, 10 were 3:1 and six were 4:0. The simplest explanation for our segregation results is that the original Ags- mutant phenotype is determined by muta- 40 80 4 8 tions in three genes. Two genes, denoted agsl and ags2, are required for sensitivity to 40 jig of G418 per ml (intermediate Concentration (Mg/mi) phenotype); at least one more mutation, denoted ags3, is FIG. 4. Inhibition of untransformed and transformed RC1707 required for the increased sensitivity of the Ags- strain. (Ags-) by antibiotics. Panels are as in Fig. 3. Cells were Segregation of genetic markers and sensitivity to 40 ,ug of untransformed (l) or transformed with plasmid pEX-4 (A), enco4- G418 per ml showed that at least one ags mutation is ing kanamycin phosphotransferase or with plasmid pLG89 (0), centromere linked; further analysis revealed that this muta encoding hygromycin B phosphotransferase. The assay was as described in the text. OD, Optical density. (Ags+) and RC1678 (Ags-). The diploid showed the Ags+ _~~~~~~~~~~~~ phenotype, demonstrating that antibiotic sensitivity is reces- sive. The diploid was sporulated and dissected, and the segregation of antibiotic sensitivity was determined in the progeny. Three phenotypes were found in the progeny: the two parental phenotypes, Ags+ and Ags-, and, in addition,

TABLE 2. Properties of antibiotics E Antibiotic Inhibition Inactivationb Selectionc (00 160 2100 C260 D Ags+ Ags- pEX-4 pLG89 (CLognc) Ura+/total 0 Apramycin 130 - - 172 4/40 10 Destomycin A 30 3 - + 5 40/40 G418 30 2 + - 20 40/40 Gentamicin X2 250 10 + - 50 39/40 Hygromycin B 25 1 - + 10 40/40 Kanamycin B 210 + - 350 15/40 Lividomycin A 70 + - 280 38/40 Neamine 360 + - 455 21/40 Neomycin 60 - - 38 30/40 Paromomycin 120 + - 120 36/40 , . , , ,. Tobramycin 140 - - 234 1/40 200 400 600 200 400 a Concentrations required for 50% inhibition (72 h) of RC1705 and RC1707. Concentration ( mg/mI) b Growth difference between RC1707 transformed with pEX-4 (expressing kanamycin phosphotransferase) or with pLG89 (expressing hygromycin B FIG. 5. Inhibition of untransformed and transformed RC1707 phosphotransferase) and untransformed RC1707. (Ags-) by (A) paromomycin, (B) lividomycin A, (C) neamine, or RC1707 transformed with pGH-1 grown 48 generations in YPD plus (D), kanamycin B. Synmbols are as in Fig. 4. The assay was as antibiotics; 40 single colonies were tested for plasmid maintenance (Ura+). described in the text. OD, Optical density. 12 ERNST AND CHAN J. BACTERIOL. tion maps 8 centimorgans from LEU2 on chromosome III TABLE 4. Plating efficienciesa (Table 3). We arbitrarily define agsl as the mutation that is Plating efficiencyb most closely linked to LEU2. Two arguments suggest that G418 concn Whole cells Spheroplasts agsl is located distal to LEU2 on the left arm of chromosome III. First, agsl shows a greater frequency of second-division Ags+ Ags- Ags+ Ags- segregation than LEU2, as deternmined with the tightly linked 0 1.0 1.0 1.0 1.0 centromere marker tup7. Second, the distance between agsl 2 1.1 0.9 0.65 0.3 and the MAT locus on the other side of the centromere is 5 1.0 0.5 ND 7 x 1o-4 greater than the distance between LEU2 and MAT. Finally, 10 1.1 <4 x 10-5 0.2 <2 x 10-4 the high proportion (25 of 30) of 2:2 tetrads suggests that 25 0.9 <2 x 10-7 0.2 <2 x 10-4 ags2, the second gene required for the intermediate mutant 40 0.43 <2 x 10-7 ND ND x <2 x 10-7 ND ND phenotype, is linked to agsl. 100 2.7 10-4 Reversion tests (Table 4) demonstrated that Ags- strains a RC1705 (Ags+) and RC1707 (Ags-) were used. ND, Not determined. have a plating efficiency of less than 2 x 10-7 on YPD plates b Cells grown/cells plated. containing 25 and 40 ,ug of G418 per ml. Table 4 also shows that spheroplasts are more sensitive to G418 than are intact cells; the significant difference in antibiotic sensitivity be- tween Ags+ and Ags- observed inl intact cells, however, is neomycin, paromomycin, and lividomycin A-antibiotics also observed with spheroplasts, albeit at lower antibiotic which do not inhibit wild-type strains. In addition, enhanced concentrations. Thus, although the cell wall may act as a sensitivity of Ags- strains was observed for G418 and barrier to aminoglycoside antibiotics, an altered cell wall hygromycin B, antibiotics which have been used previously appears not to be the reason for the Ags- phenotype. for selection, but to which wild-type cells are relatively Since sensitivity to paromomycin is a phenotype that has resistant (7, 8, 18). Another new finding described here is been reported for other mutants (13, 20, 21, 30), we decided that the antibiotics gentamicin X2 and destomycin A can be to check the Ags- strains for some of the properties associ- used like G418 and hygromycin B, respectively. ated with those previously described mutants. As deter- Rank et al. (20, 21, 23) previously described a mutation mined by replica plating, Ags- strains are not more resistant designated pdrl, which causes collateral sensitivity to sev- to oligomycin, cyclohexi'mide, , or tetra- eral agents, including neomycin and paromomycin, but cycline thanl wild-type strains, nor are they more sensitive to which causes also a pleiotropic resistance to a number of dequalinium chloride than wild-type strains. Although the other compounds including chloramphenicol, cyclohexi- original MB50OC-TlOC strain does not grow on glycerol mide, or tetracycline. In addition, pdrl mutants were shown plates at 37°C, ags segregants can be found that do grow on to be particularly sensitive to increased osmolarity, pH, and glycerol plates at 370C. Ags- strains are also not osmotically temperature; also, pdrl caused partial respiratory deficiency sensitive, since they can grow on plates containing 1 M (20, 21). None of the pdrl phenotypic traits that we tested sorbitol. Crosses of Ags- strains to strains carrying ochre was found to be associated with the ags mutation described mutations revealed that the Ags- phenotype does not in this paper. Ags- strains were not more resistant to cosegregate with suppression of ade2-1, leu2-1, trp548, oligomycin, chloramphenicol, cycloheximide, or tetra- tyrl-I, lysJ-L, hisS-2, ura4-1, can1-100, and trpS-2. cycline than were Ags+ strains and also showed no enhanced sensitivity to osmnotic pressure, pH, and temperature. Most importantly, Ags- strains were fully respiratory proficient. DISCUSSION Furthermore, we have mapped one of the genes involved, agsl, 8 centimorgans distal to LEU2 on chromosome III, Any mutants of S. cerevisiae with enhanced aminoglyco- whereas pdrl has been mhapped to chromnosome VII. side antibiotic sensitivity are potentially improved host Strains carrying certain omnipotent suppressors and strains for vectors expressing appropriate resistance genes. antisuppressors have also been reported to be more sensitive In particular, the ags mutants we describe in this paper to neomycin and paromomycin (13, 30). Some of these significantly broaden the applicability of the two strains show additional deficiencies, such as osmotic sensi- phosphotransferases, APH (10, 16) and HPH (7, 12). We tivity, temperature sensitivity, and respiratory deficiency show with Ags- host strains that the kanamycin phospho- (31). Altered ribosomal proteins are associated with mutants transferase will inactivate neamine, kanamycin B, carrying omnipotent suppressors (25). Since phenotypic suppression of nonsense and missense mutations can be shown with neomycin and paromomycin in wild-type strains (17, 28), it appears plausible that the increased sensitivity of TABLE 3. Mapping of agsla omnipotent suppressor strains to these antibiotics is due to Marker pairs PD NPD TT cM %SDS enhanced mistranslation. However, the ags mutation did not suppress trp548, ade2-1, or leu2-J, (all ochre mutations that agslb and leu2 21 0 4 8 agslb and tup7 12 9 4 16 are suppressed by known omnipotent suppressors) and agslb and MAT 12 1 12 36 caused none of the above-mentioned additional deficiencies Ieu2 and MAT 25 0 10 14.3 of omnipotent suppressor mutations. Furthermore, no leu2 and tup7 14 19 2 6 known omnipotent suppressor gene maps in the vicinity of agsl (8 centimorgans distal to LEU2 on chromosome III). a Tetrad analysis of diploid formed by crossing RC1678 with BJ1991. PD; parental ditype; NPD, nonparental ditype; TT, tetratype; cM, distance in SUP53, a gene encoding a leucine inserting UAG suppressor centimorgafis as calculated by the equation of Perkins (19); %SDS, percentage (25), is probably the closest known gene to agsl. of second-divisiotn segregation. The mechanism by which the ags mutations enhance b Ags- was scored as failure to grow after replication to YPD plates sensitivity to certain aminoglycoside antibiotics is not containing 40 F.g of G418 per ml; only tetrads where Ags+ and Ags- segregated 2:2 are included. known. Conceivably, mutations of the target site of anti- VOL. 163, 1985 S. CEREVISIAE MUTANTS SUPERSENSITIVE TO ANTIBIOTICS 13 biotic action, i.e., the ribosome (3, 6, 25), or mutations expression of foreign genes in Saccharomyces cerevisiae. Curr. facilitating access of the antibiotics to the target site (or both) Top. Microbiol. Immunol. 96:119-144. may lead to the Ags- phenotype. However, enhanced sen- 9. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transfor- mation of intact yeast cells treated with alkali cations. J. sitivity is limited to certain aminoglycoside antibiotics. Bacteriol. 153:163-168. Therefore, a general permeability increase of the yeast cell 10. Jimenez, A., and J. Davies. 1980. Expression of a transposable membrane is probably not the cause of the Ags- phenotype. antibiotic resistance element in Saccharomyces. Nature (Lon- Our data also show that an alteration of the cell wall is don) 287:869-871. probably not involved in the Ags- phenotype. 11. Karin, M., R. Najarian, A. Haslinger, P. Valenzuela, J. Welch, As we have shown, the multigenic composition of the and S. Fogel. 1984. Primary structure and transcription of an Ags- phenotype makes it difficult to analyze genetically. A amplified genetic locus: the CUPI locus of yeast. Proc. Natl. better approach to identify all of the genetic components of Acad. Sci. U.S.A. 81:337-341. the Ags- phenotype would be to clone the genes by comple- 12. Kaster, K. R., S. G. Burgett, R. Nagaraja Rao, and T. D. Ingolia. mentation. With the it might 1983. Analysis of a bacterial hygromycin B resistance gene by availability of the cloned genes, transcriptional and translational fusions and by DNA sequenc- also be desirable to construct nonrevertable (null) ags muta- ing. Nucleic Acids Res. 11:6895-6911. tions in vitro and to insert them into the genome by 13. Liebman, S. W., and M. Cavenagh. 1980. An antisuppressor that transplacement (24). Toward this goal we have recently acts on omnipotent suppressors in yeast. Genetics 95:49-61. cloned at least one of the ags genes (Ernst, unpublished 14. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular results). cloning. A laboratory manual. Cold Spring Harbor Laboratory, Ags- mutants have obvious applications for research and Cold Spring Harbor, N.Y. biotechnology. The amount of antibiotic required to select 15. Miyajima, A., I. Miyajima, K. Arai, and N. Arai. 1984. Expres- for a yeast strain carrying a vector with an inactivating sion of plasmid R388-encoded type II dihydrofolate reductase as In addition, a dominant selective marker in Saccharomyces cerevisiae. Mol. phosphotransferase gene is greatly reduced. Cell. Biol. 4:407-414. easily available antibiotics such as paromomycin, kanamy- 16. Oka, A., H. Sugisaki, and M. Takanami. 1981. Nucleotide cin B, and neomycin can be used for selection in complex sequence of the kanamycin resistance transposon Tn9O3. J. growth media. If yeast is used as host to produce mammalian Mol. Biol. 147:217-226. proteins it may be desirable if an antibiotic not toxic for 17. Palmer, E., J. W. Wilhelm, and F. Sherman. 1979. Phenotypic mammalian cells is used during fermentation. Mammalian suppression of nonsense mutations in yeast by aminoglycoside cells are not sensitive or only slightly sensitive to kanamycin antibiotics. Nature (London) 277:148-150. B, neomycin, and lividomycin A (M. Hirschi, unpublished 18. Panchal, C. J., G. K. Whitney, and G. G. Stewart. 1984. results). Furthermore, the strategy described in this paper, Susceptibility of Saccharomyces spp. and Schwanniomyces to first establish host strains with enhanced aminoglycoside spp. to the aminoglycoside antibiotic G418. Appl. Environ. Microbiol. 47:1164-1166. antibiotic sensitivity that then can be used in conjunction 19. Perkins, D. D. 1949. Biochemical mutants in the smut fungus with the existing phosphotransferases, may well be ap- Ustilago maydis. Genetics 34:607-626. plicable to species other than S. cerevisiae. 20. Rank, G. H., J. H. Gerlach, and A. J. Robertson. 1976. Some physiological alterations associated with pleiotropic cross re- sistance and collateral sensitivity in Saccharomyces cerevisiae. ACKNOWLEDGMENTS Mol. Gen. Genet. 144:281-288. 21. Rank, G. H., A. J. Robertson, and K. L. Phillips. 1974. Modifi- The initial experiments for the construction of pEX-2 were per- cation and inheritance of pleiotropic cross resistance and col- formed in the laboratory of F. Sherman, University of Rochester, lateral sensitivity in Saccharomyces cerevisiae. Genetics Rochester, N.Y. We thank D. Pietras for help with M13 construc- 80:483-493. tions and M.-F. Planche and R. Guenin for skillful technical assis- 22. Rine, J., W. Hansen, E. Hardeman, and R. W. Davis. 1983. tance. We acknowledge J. Davies for useful discussions and for Targeted selection of recombinant clones through gene dosage generously supplying antibiotics for this study. effects. Proc. Natl. Acad. Sci. U.S.A. 80:6750-6754. 23. Saunders, G. W., and G. H. Rank. 1982. 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Cold Spring ence of hygromycin B with translocation and with aminoacyl- Harbor Laboratory, Cold Spring Harbor, N.Y. tRNA recognition. Eur. J. Biochem. 87:21-27. 26. Sherman, F., G. Fink, and J. Hicks. 1981. Methods in yeast 4. Davies, J. E. 1983. Resistance to : mechanisms genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, and frequency. Rev. Infect. Dis. 5:S261-S267. N.Y. 5. Fried, H. M., and J. R. Warner. 1981. Cloning of yeast gene for 27. Sherman, F., and J. W. Stewart. 1982. Mutations altering trichodermin resistance and ribosomal protein L3. Proc. Natl. initiation of translation of yeast iso-1-cytochrome c; contrasts Acad. Sci. U.S.A. 78:238-242. between the eukaryotic and prokaryotic initiation process, p. 6. Gonzales, A., A. Jimenez, D. Vazquez, J. Davies, and D. 301-333. In J. N. Strathern, E. W. Jones, and J. R. Broach Schindler. 1978. 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gomery, and B. D. Hall. 1979. Sequence of the gene for Ribosomal recessive suppressors cause a respiratory deficiency iso-1-cytochrome c in Saccharomyces cerevisiae. Cell in yeast Saccharomyces cerevisiae. Mol. Gen. Genet. 16:753-761. 185:319-323. 30. Surguchov, A. P., E. M. Pospelova, and V. N. Smirnov. 1981. 32. Webster, T. D., and R. C. Dickson. 1983. Direct selection of Synergistic action of genetic and phenotypic suppression of Saccharomyces cerevisiae resistant to the antibiotic G418 fol- nonsense mutations in yeast Saccharomyces cerevisiae. Mol. lowing transformation with a DNA vector carrying the kanamy- Gen. Genet. 183:197-198. cin-resistance gene of Tn9O3. Gene 26:243-252. 31. Ter-Avanesyan, M. D., J. Zimmermann, S. G. Inge-Vechtomov, 33. Zaret, K. S., and F. Sherman. 1982. DNA sequence required for A. B. Sudarikov, V. N. Smirnov, and A. P. Surguchov. 1982. efficient transcription termination in yeast. Cell 28:563-573.