JOURNAL OF BACTERIOLOGY, JUlY 1985, p. 8-14 Vol. 163, No. 10021-9193/85/070008-07$02.00/0Copyright © 1985, American Society for Microbiology

Characterization of Saccharomyces cerevisiae MutantsSupersensitive to Aminoglycoside Antibiotics

JOACHIM F. ERNST* AND RUSSELL K. CHANDepartment of Microbiology, 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 theaminoglycoside antibiotics G418, hygromycin B, destomycin A, and gentamicin X2. In addition, the mutantsare sensitive to apramycin, kanamycin B, lividomycin A, neamine, neomycin, paromomycin, and tobramy-cin-antibiotics which do not inhibit wild-type strains. Mapping studies suggest that supersensitivity is causedby mutations in at least three genes, denoted AGS), AGS2, andAGS3 (for aminoglycoside antibiotic sensitivity).Mutations in all three genes are required for highest antibiotic sensitivity; ags1 ags2 double mutants haveintermediate 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 Tn903phosphotransferase and that destomycin A is inactivated by the hygromycin B phosphotransferase. ags strainsare improved host strains for vectors carrying the phosphotransferase genes because a wide spectrum ofaminoglycoside antibiotics can be used to select for plasmid maintenance.

Several approaches have led to the discovery of dominantselectable transformation markers in the yeast Sac-charomyces cerevisiae. First, procaryotic antibiotic resis-tance genes have been expressed in yeast, and it has beendemonstrated that certain aminoglycoside antibiotics selectfor the growth of strains expressing the corresponding resis-tance gene. The kanamycin phosphotransferase (APH) en-coded by a gene of transposon Tn9O3, which inactivates theantibiotic G418 (8, 10, 32), and the gene encodinghygromycin B phosphotransferase (HPH) (7, 12) have beenused in this fashion. Second, certain proteins have beenoverproduced in yeast by inserting the encoding genes onhigh-copy-number vectors; yeast strains transformed withthese vectors are resistant to specific metabolic inhibitors ofthe overproduced enzymes. Overproduction of enzymes (15,22), copper chelatin (11), and a ribosomal protein (5) ren-dered yeast resistant to the corresponding specific inhibitors.Whereas the overproduction approach usually requires

the use of high-copy-number vectors, the gene encodingAPH renders yeast resistant to G418 even at low copynumbers (32). In addition, the genes encoding APH andHPH are expressed in S. cerevisiae, as well as in Escherichiacoli (7, 8, 32), allowing for antibiotic selection in bothorganisms with yeast shuttle vectors (2). However, therelatively high concentrations of G418 and hygromycin B (7,32) needed in selective growth media have hampered the useof phosphotransferase genes as dominant selectable markersin yeast. We describe here mutants of S. cerevisiae that areparticularly sensitive to G418 and hygromycin B. Thesestrains are also sensitive to a series of other aminoglycosideantibiotics, many of which are inactivated by the Tn9O3phosphotransferase. Thus, in our approach the modificationof the yeast host allows optimal use of the existing phospho-transferase genes.

MATERIALS AND METHODSStrains and growth conditions. The yeast strains used in

this study are listed in Table 1. Standard yeast genetic

* Corresponding author.

procedures were used for crosses and scoring of phenotypes(26). The Ags- phenotype was routinely scored on 1% yeastextract-2% peptone-2% glucose-2% Bacto-agar (Difco Lab-oratories, Detroit, Mich.) (YPD) agar (26) containing 20 jig ofG418 per ml or 20 ,ug of hygromycin B per ml; in addition,zones of inhibition surrounding antibiotic-containing filterdisks on yeast lawns were determined in some cases. Con-centration-dependent inhibition by antibiotics was deter-mined by inoculating 5 ml of liquid YPD containing antibiot-ics with 50 ,ul of a saturated yeast culture grown in minimalmedium (26); starter cultures of transformed strains weregrown selectively (without uracil) in minimal medium. Afterincubation at 30'C for 72 h on a rotating wheel, the opticaldensity at 600 nm was recorded. Sensitivity to chlorampheni-col (4 mg/ml), cycloheximide (0.5 jig/ml), or tetracycline (2mg/ml) was determined on YP agar (26) containing 4% glyc-erol, sensitivity to oligomycin (5 jig/ml) was determined onYP agar containing 3% glycerol, and sensitivity to dequali-nium chloride (5 ,ug/ml) was determined on YPD agar. Plat-ing efficiencies were determined on YPD agar for whole cellsand on YPD agar containing 1 M sorbitol for spheroplasts.The ability of ags mutants to suppress nonsense mutationswas assessed by crossing an ags strain with a strain carryingnonsense mutations. The resulting diploid was sporulated,and tetrads were dissected. The presence of ags spores thatexpressed the phenotype of the unsuppressed marker wastaken as evidence that ags did not suppress that mutation.

Plasmids. Standard procedures were used for the con-struction and amplification of plasmids (14). For construc-tion of pEX-2, the CYCI terminator region was subclonedfirst. The 270-base-pair HaeIII-to-HindIII fragment contain-ing the terminator (29, 33) was isolated and inserted betweenthe HincII and HindIII sites of bacteriophage M13mp8 (14).The EcoRI-to-HindIII terminator fragment was isolatedfrom double-stranded phage DNA and ligated with thefollowing two fragments to construct pEX-1: (i) the 4.85-kilobase HindIII-to-BamHI fragment of plasmid pAB107 (S.Baim and F. Sherman, unpublished results)-pAB107 con-tains the 0.85-kilobase EcoRI-to-HindIII ARSI fragment ofYRp7 (2) inserted between the EcoRI and HindIII sites of

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TABLE 1. Yeast strainsStrain Genotype Origin

BJ1991 MATaz pep4-3 prbl-1122 E. Jones,ura3-52 leu2 trpl Carnegie-

MellonUniversity

HR12S-llb MATa leu2-3 leu2-112 G. Sprague,trpl ura3-52 his3 his4 University of

OregonMB50OC-T1OC MATa his7 met2 tup7 gall L. F. Bisson (1)

agsRC1678 MATa ura3-52 trpl tup7 HR125-llb x

ags MB50OC-TlOCRC1705 MATa ura3-52 trpl leu2 BJ199i x RC1678

prbl-1122RC1707 MATa ura3-52 trpl tup7 Same tetrad as

pep4-3 prbl-1122 ags RC1705

YIp5 (2); (ii) the 1.1-kilobase BamHI-to-EcoRI fragment ofplasmid pAB63 carrying the cycl-13 promoter (Baim andSherman, unpublished results). The cycl-13 mutation is asingle nucleotide change altering the ATG translation startcodon to ATA (27). The sequence of the junction cycl-13promoter-M13 sequences-CYCI terminator is given in Fig.1A. pEX-2 was constructed by insertion of the 1.47-kilobaseHincII fragment carrying the origin of replication of theyeast 2,u circle (8) into the single PvuII site of pEX-1. Theconstruction of pLG89 from pEX-2 has been describedpreviously (7). pGH-1 and pEX-4 were constructed byinsertion of the 1.7-kilobase PvuII fragment of plasmidpAJ50 (10) carrying the APH-coding gene of Tn9O3 (16) intothe filled-in SalI sites of pEX-2 and pLG89, respectively(Fig. 1B). The Sall sites were restored in these construc-tions; in pGH-1, a 0.6-kilobase Sall fragment containingupstream sequences of the cycl-13 promoter and pBR322sequences was deleted during the construction. Transforma-tion of yeast strains with plasmids was performed by thespheroplast method (26) or by the salt method (9).

Reagents. G418 sulfate (Geneticin) was obtained fromGIBCO Europe Ltd., Paisley, United Kingdom, and fromSchering Corp., Bloomfield, N.J. Hygromycin B was ob-tained from Eli Lilly & Co., Indianapolis, Ind. Kanamycin Asulfate (95% kanamnycin A, 5% kanamycin B), neomycinsulfate (90 to 95% neomycin B), tobramycin, chlorampheni-col, cycloheximide, tetracycline, oligomycin (65%oligomycin A, 20% oligomycin B, 15% oligomycin C), ami-kacin, and dequalinium chloride were obtained from SigmaChemical Co., St. Louis, Mo. Paromomycin was obtainedfrom Farmitalia, Milano, Italy. Neamine (91%), netilmicin(58.4%), apramycin (86%), sisomicin, lividomycin A, genta-micin X2, kanamycin B, destomycin A, and ribostamycinwere gifts fronm J. Davies. The enzymes used in plasmidconstructions were from New England Biolabs, Beverly,Mass., and were used according to the recommendation ofthe manufacturer.

RESULTS

Mutants with enhanced sensitivity to amninoglycoside anti-biotics. Wild-type strains of S. cerevisiae are relativelyresistant to the aminoglycoside antibiotic G418 (8, 18).During routine strain constructions we discovered that strainMB500C-T1OC (1) and some of its outcrossed progeny weremore sensitive to G418 and other aminoglycoside antibiotics

than were other laboratory strains. This phenotype wasdenoted Ags-, for aminoglycoside antibiotic sensitivity.Comparisons of wild-type and Ags- strains for antibiotic

sensitivity demonstrated that Ags- strains are 10- to 20-foldmnore sensitive to G418 and hygromycin B than are wild-typestrains (Fig. 2). In complex growth medium, 50% inhibitionwas obtained at 1 to 2 ,ug/ml. Ags- strains were also moresensitive to gentamicin X2 and destomycin A-two antibiot-ics that inhibit growth of wild-type strains (Fig. 3, Fig. 4).To verify that the Ags- strain could be used as host for

yeast vectors carrying the known phosphotransferase genes(7, 10), we constructed plasmids that carry either the HPH-encoding gene (pLG89) or the Tn9O3 APH-encoding gene(pEX-4) or both (pGH41) (Fig. 1). All constructed plasmidscarry the yeast URA3 gene in addition to the antibioticresistance marker(s). An Ags+ ura3 strain and an Ags- ura3strain were transformed with plasmids pEX-2, pLG89, orpGH-1, selecting for uracil prototrophy. Quantitative aniti-biotic sensitivity tests were performed on the untransformedand transformed strains (Fig. 3, Fig. 4). As expected, anAgs+ transformant expressing APH (encoded on pEX-4) wasresistant to G418 at all antibiotic concentrations tested.Similarly, an Ags- strain expressing APH was more resis-tant to G418 than the untransformed Ags- host and the Ags-host carrying plasmid pLG89. However, at G418 concentra-tions greater than 20 ,ug/ml, inhibition by G418 was seeneven when the Ags- strain contained pEX-4. The presenceof plasmid pLG89 rendered Ags+ and Ags- strains moreresistant to hygromycin B (Fig. 3, Fig. 4). As describedpreviously (7), hygromycin B resistance was incomnplete at

A EcoRI SMO I BOmHITTAATAIJACTGAATTC CCGGGGATCCGTC CCCCTTTTCC

cyc/-I3 promoter ------- Ml3mp8----- *-_CYC/ terminator

BARSI

PEX-2 B B G89 I y R9.3kb 310.3kb Hy

2,p cyc/- 3 Boni

URA3 smR S

pEX-4 B pH- HygR11i kb A11.4kb Hy

KmR

FIG. 1. Plasmid constructions. (A) sequence of the promoter-terminator junction in pEX-2. The boxed nucleotides indicate theATG start codon of the CYCI gene, which is changed to ATA by thecycl-13 mutation (27). (B) construction of pEX-4 and pGH-1. Theconstruction of pLG89 has been described previously (7). Yeastsequences are drawn as heavy black lines, and pBR322 sequencesare drawn as thin lines. Antibiotic resistance genes are drawn asopen boxes. Arrows indicate the direction of transcription.

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10 ERNST AND CHAN

high antibiotic concentrations; in our experiments an Ags+pLG89 transformant was fully resistant up to 50 jig/ml andan Ags- pLG89 transformant was resistant up to 10 pugIml.We demonstrated that antibiotics gentamicin X2 and

destomycin A have properties similar to G418 andhygromycin B, respectively. Both antibiotics acted on wild-type strains; gentamicin X2 was inactivated by APH, anddestomycin 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-typestrains. Apramycin, kanamycin B, lividomycin A, neamine,neomycin, paromomycin, and tobramycin inhibited thegrowth of the Ags- strain (Table 2). Amikacin, gentamicinB, kanamycin A, netilmicin, ribostamycin, and sisomicinshowed no inhibitory effect on Ags+ or Ags- strains. Growthtests in liquid YPD medium (Fig.- 5) demonstrated thatneamine, kanamycin B, lividomnycin A, and paromomycinare inactivated by APH, since Ags- strains transformed withpEX-4 grew to higher densities as compared with theuntransformed strain. The difference in growth was small forneamine and for kanamycin B; it was virtually absent for

1.0

0.1

c

0

0otD

1.0

0.I

20 40

Concentration (,ug/ml )

FIG. 2. Inhibition of RC1705 (Ags+) (A) and RC1707 (Ags-) (O)by G418 (A) and hygromycin B (B). The assay was as described inthe text. OD, Optical density.

E * 4.

0

o 200 400 c loo 200 D0D

1.0

0.I

200 400 40 80

Concentration (Mug/mi)

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

neomycin. Therefore, it was of interest to determine whetherthese antibiotics could be used to select for yeast transform-ants expressing the aminoglycoside-inactivating enzyme. Totest this possibility, we grew an Ags- strain transformedwith plasmid pGH-1 (which carries the genes encoding APHand HPH) in the presence of antibiotics for approximately 48generations (Table 2). Generally, the concentration of anti-biotics was chosen such as to allow maximal growth of atrapsformant expressing the resistance gene, while inhibitingmaximally the untransformed Ags- host. Concentrations forneamine, kanamycin B, and neomycin were chosen to allowsufficient growth. After growth, single colonies were testedfor the maintenance of the URA3 gene also present on theyeast vector, The results show that all antibiotics that areinactivated in yeast as shown by the growth tests also willselect for plasmid maintenance. This result was also ob-tained with an Ags- strain transformed with pEX-4 or withpLG89, indicating that the presence of both the APH- andthe HPH-encoding genes on pGH-1 does not influence theselection for either resistance gene. The percentage of plas-mid maintenance varied, since the antibiotic concentrationduring the growth period was not optimized. Surprisingly,neomycin appears to select for plasmid maintenance, eventhough the selective growth advantage of a yeast strainexpressing APH is negligible. As expected, tobramycin andapramycin conferred no selective advantage for plasmidmaintenance, since neither antibiotic is inactivated by eitherAPH or HGH (4).

Genetic characterization of ags. To determine the geneticbasis for the Ags- phenotype we crossed strains BJ1991

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e 80~

0oDto 40 80o 20 400

0.I

40 80 4 8

Concentration (Mg/mi)

FIG. 4. Inhibition of untransformed and transformed RC1707(Ags-) by antibiotics. Panels are as in Fig. 3. Cells wereuntransformed (l) or transformed with plasmid pEX-4 (A), enco4-ing kanamycin phosphotransferase or with plasmid pLG89 (0),encoding hygromycin B phosphotransferase. The assay was asdescribed 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 thesegregation of antibiotic sensitivity was determined in theprogeny. Three phenotypes were found in the progeny: thetwo parental phenotypes, Ags+ and Ags-, and, in addition,

TABLE 2. Properties of antibiotics

Antibiotic Inhibition Inactivationb Selectionc

Ags+ Ags- pEX-4 pLG89 (CLognc) Ura+/total

Apramycin 130 - - 172 4/40Destomycin A 30 3 - + 5 40/40G418 30 2 + - 20 40/40Gentamicin X2 250 10 + - 50 39/40Hygromycin B 25 1 - + 10 40/40Kanamycin B 210 + - 350 15/40Lividomycin A 70 + - 280 38/40Neamine 360 + - 455 21/40Neomycin 60 - - 38 30/40Paromomycin 120 + - 120 36/40Tobramycin 140 - - 234 1/40

a Concentrations required for 50% inhibition (72 h) of RC1705 and RC1707.b Growth difference between RC1707 transformed with pEX-4 (expressing

kanamycin phosphotransferase) or with pLG89 (expressing hygromycin Bphosphotransferase) and untransformed RC1707.

RC1707 transformed with pGH-1 grown 48 generations in YPD plusantibiotics; 40 single colonies were tested for plasmid maintenance (Ura+).

a phenotype with intermediate sensitivity to aminoglycosideantibiotics. Mutants of this intermediate phenotype showedenhanced sensitivities to G418, hygromycin B, gentamicinX2, destomycin A, and paromomycin, with 50% growthinhibition at concentrations of 8, 8.5, 45, 8.5, and 80 ,uwg/ml,respectively. These enhanced sensitivities, as comparedwith the wild-type, were not associated with the additionalantibiotic sensitivities of the Ags- mutant (Table 2). Theintermediate 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 intermediatephenotype was resistant to 20 jig of G418 per ml, butsensiti've to 40 ,ug of G418 per ml.Analyses of the cross described above revealed that 25 of

30 tetrads showed 2:2 segregation for Ags+:Ags- when thecriterion for Ags- was sensitivity to 40 jig of G418 per ml; ofthe remaining tetrads four segregated 3:1 and one segregated4:0. The presence of 4:0 and 3:1 segregation suggested thatsensitivity is determined by more than one gene. If thecriterion for Ags- was sensitivity to 20 ,ug of G418 per ml,then 14 out of 30 tetrads showed 2:2 segregation forAgs+:Ags-; of the 16 tetrads that were not 2:2, 10 were 3:1and six were 4:0.The simplest explanation for our segregation results is that

the original Ags- mutant phenotype is determined by muta-tions in three genes. Two genes, denoted agsl and ags2, arerequired for sensitivity to 40 jig of G418 per ml (intermediatephenotype); at least one more mutation, denoted ags3, isrequired for the increased sensitivity of the Ags- strain.Segregation of genetic markers and sensitivity to 40 ,ug ofG418 per ml showed that at least one ags mutation iscentromere linked; further analysis revealed that this muta

_~~~~~~~~~~~~

E

(00 160 2100C260 D0

10

, . , , ,.

200 400 600 200 400

Concentration ( mg/mI)

FIG. 5. Inhibition of untransformed and transformed RC1707(Ags-) by (A) paromomycin, (B) lividomycin A, (C) neamine, or(D), kanamycin B. Synmbols are as in Fig. 4. The assay was asdescribed in the text. OD, Optical density.

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tion maps 8 centimorgans from LEU2 on chromosome III(Table 3). We arbitrarily define agsl as the mutation that ismost closely linked to LEU2. Two arguments suggest thatagsl is located distal to LEU2 on the left arm ofchromosomeIII. First, agsl shows a greater frequency of second-divisionsegregation than LEU2, as deternmined with the tightly linkedcentromere marker tup7. Second, the distance between agsl

and the MAT locus on the other side of the centromere isgreater than the distance between LEU2 and MAT. Finally,the high proportion (25 of 30) of 2:2 tetrads suggests thatags2, the second gene required for the intermediate mutantphenotype, is linked to agsl.

Reversion tests (Table 4) demonstrated that Ags- strainshave a plating efficiency of less than 2 x 10-7 on YPD platescontaining 25 and 40 ,ug of G418 per ml. Table 4 also showsthat spheroplasts are more sensitive to G418 than are intactcells; the significant difference in antibiotic sensitivity be-tween Ags+ and Ags- observed inl intact cells, however, isalso observed with spheroplasts, albeit at lower antibioticconcentrations. Thus, although the cell wall may act as a

barrier to aminoglycoside antibiotics, an altered cell wallappears not to be the reason for the Ags- phenotype.

Since sensitivity to paromomycin is a phenotype that hasbeen reported for other mutants (13, 20, 21, 30), we decidedto check the Ags- strains for some of the properties associ-ated with those previously described mutants. As deter-mined by replica plating, Ags- strains are not more resistantto oligomycin, cyclohexi'mide, chloramphenicol, or tetra-cycline thanl wild-type strains, nor are they more sensitive todequalinium chloride than wild-type strains. Although theoriginal MB50OC-TlOC strain does not grow on glycerolplates at 37°C, ags segregants can be found that do grow on

glycerol plates at 370C. Ags- strains are also not osmoticallysensitive, since they can grow on plates containing 1 Msorbitol. Crosses of Ags- strains to strains carrying ochremutations revealed that the Ags- phenotype does notcosegregate with suppression of ade2-1, leu2-1, trp548,tyrl-I, lysJ-L, hisS-2, ura4-1, can1-100, and trpS-2.

DISCUSSION

Any mutants of S. cerevisiae with enhanced aminoglyco-side antibiotic sensitivity are potentially improved hoststrains for vectors expressing appropriate resistance genes.In particular, the ags mutants we describe in this papersignificantly broaden the applicability of the twophosphotransferases, APH (10, 16) and HPH (7, 12). Weshow with Ags- host strains that the kanamycin phospho-transferase will inactivate neamine, kanamycin B,

TABLE 3. Mapping of agslaMarker pairs PD NPD TT cM %SDS

agslb and leu2 21 0 4 8agslb and tup7 12 9 4 16agslb and MAT 12 1 12 36Ieu2 and MAT 25 0 10 14.3leu2 and tup7 14 19 2 6

a Tetrad analysis of diploid formed by crossing RC1678 with BJ1991. PD;parental ditype; NPD, nonparental ditype; TT, tetratype; cM, distance incentimorgafis as calculated by the equation of Perkins (19); %SDS, percentageof second-divisiotn segregation.

b Ags- was scored as failure to grow after replication to YPD platescontaining 40 F.g of G418 per ml; only tetrads where Ags+ and Ags-segregated 2:2 are included.

TABLE 4. Plating efficienciesaPlating efficiencyb

G418 concn Whole cells Spheroplasts

Ags+ Ags- Ags+ Ags-

0 1.0 1.0 1.0 1.02 1.1 0.9 0.65 0.35 1.0 0.5 ND 7 x 1o-410 1.1 <4 x 10-5 0.2 <2 x 10-425 0.9 <2 x 10-7 0.2 <2 x 10-440 0.43 <2 x 10-7 ND ND100 2.7 x 10-4 <2 x 10-7 ND ND

a RC1705 (Ags+) and RC1707 (Ags-) were used. ND, Not determined.b Cells grown/cells plated.

neomycin, paromomycin, and lividomycin A-antibioticswhich do not inhibit wild-type strains. In addition, enhancedsensitivity of Ags- strains was observed for G418 andhygromycin B, antibiotics which have been used previouslyfor selection, but to which wild-type cells are relativelyresistant (7, 8, 18). Another new finding described here isthat the antibiotics gentamicin X2 and destomycin A can beused like G418 and hygromycin B, respectively.Rank et al. (20, 21, 23) previously described a mutation

designated pdrl, which causes collateral sensitivity to sev-eral agents, including neomycin and paromomycin, butwhich causes also a pleiotropic resistance to a number ofother compounds including chloramphenicol, cyclohexi-mide, or tetracycline. In addition, pdrl mutants were shownto be particularly sensitive to increased osmolarity, pH, andtemperature; also, pdrl caused partial respiratory deficiency(20, 21). None of the pdrl phenotypic traits that we testedwas found to be associated with the ags mutation describedin this paper. Ags- strains were not more resistant tooligomycin, chloramphenicol, cycloheximide, or tetra-cycline than were Ags+ strains and also showed no enhancedsensitivity to osmnotic pressure, pH, and temperature. Mostimportantly, Ags- strains were fully respiratory proficient.Furthermore, we have mapped one of the genes involved,agsl, 8 centimorgans distal to LEU2 on chromosome III,whereas pdrl has been mhapped to chromnosome VII.

Strains carrying certain omnipotent suppressors andantisuppressors have also been reported to be more sensitiveto neomycin and paromomycin (13, 30). Some of thesestrains show additional deficiencies, such as osmotic sensi-tivity, temperature sensitivity, and respiratory deficiency(31). Altered ribosomal proteins are associated with mutantscarrying omnipotent suppressors (25). Since phenotypicsuppression of nonsense and missense mutations can beshown with neomycin and paromomycin in wild-type strains(17, 28), it appears plausible that the increased sensitivity ofomnipotent suppressor strains to these antibiotics is due toenhanced mistranslation. However, the ags mutation did notsuppress trp548, ade2-1, or leu2-J, (all ochre mutations thatare suppressed by known omnipotent suppressors) andcaused none of the above-mentioned additional deficienciesof omnipotent suppressor mutations. Furthermore, noknown omnipotent suppressor gene maps in the vicinity ofagsl (8 centimorgans distal to LEU2 on chromosome III).SUP53, a gene encoding a leucine inserting UAG suppressor(25), is probably the closest known gene to agsl.The mechanism by which the ags mutations enhance

sensitivity to certain aminoglycoside antibiotics is notknown. Conceivably, mutations of the target site of anti-

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biotic action, i.e., the ribosome (3, 6, 25), or mutationsfacilitating access of the antibiotics to the target site (or both)may lead to the Ags- phenotype. However, enhanced sen-sitivity is limited to certain aminoglycoside antibiotics.Therefore, a general permeability increase of the yeast cellmembrane is probably not the cause of the Ags- phenotype.Our data also show that an alteration of the cell wall isprobably not involved in the Ags- phenotype.As we have shown, the multigenic composition of the

Ags- phenotype makes it difficult to analyze genetically. Abetter approach to identify all of the genetic components ofthe Ags- phenotype would be to clone the genes by comple-mentation. With the availability of the cloned genes, it mightalso be desirable to construct nonrevertable (null) ags muta-tions in vitro and to insert them into the genome bytransplacement (24). Toward this goal we have recentlycloned at least one of the ags genes (Ernst, unpublishedresults).Ags- mutants have obvious applications for research and

biotechnology. The amount of antibiotic required to selectfor a yeast strain carrying a vector with an inactivatingphosphotransferase gene is greatly reduced. In addition,easily available antibiotics such as paromomycin, kanamy-cin B, and neomycin can be used for selection in complexgrowth media. If yeast is used as host to produce mammalianproteins it may be desirable if an antibiotic not toxic formammalian cells is used during fermentation. Mammaliancells are not sensitive or only slightly sensitive to kanamycinB, neomycin, and lividomycin A (M. Hirschi, unpublishedresults). Furthermore, the strategy described in this paper,to first establish host strains with enhanced aminoglycosideantibiotic sensitivity that then can be used in conjunctionwith the existing phosphotransferases, may well be ap-plicable to species other than S. cerevisiae.

ACKNOWLEDGMENTS

The initial experiments for the construction of pEX-2 were per-formed in the laboratory of F. Sherman, University of Rochester,Rochester, N.Y. We thank D. Pietras for help with M13 construc-tions and M.-F. Planche and R. Guenin for skillful technical assis-tance. We acknowledge J. Davies for useful discussions and forgenerously supplying antibiotics for this study.

LITERATURE CITED1. Bisson, L. F., and J. Thorner. 1982. Mutations in the PHO80

gene confer permeability to 5'-mononucleotides in Sac-charomyces cerevisiae. Genetics 102:341-359.

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3. Cabanas, M., D. Vazquez, and J. Modolell. 1978. Dual interfer-ence of hygromycin B with translocation and with aminoacyl-tRNA recognition. Eur. J. Biochem. 87:21-27.

4. Davies, J. E. 1983. Resistance to aminoglycosides: mechanismsand frequency. Rev. Infect. Dis. 5:S261-S267.

5. Fried, H. M., and J. R. Warner. 1981. Cloning of yeast gene fortrichodermin resistance and ribosomal protein L3. Proc. Natl.Acad. Sci. U.S.A. 78:238-242.

6. Gonzales, A., A. Jimenez, D. Vazquez, J. Davies, and D.Schindler. 1978. Studies on the mode of action of hygromycin B,an inhibitor of translocation in eukaryotes. Biochim. Biophys.Acta 521:459-469.

7. Gritz, L., and J. Davies. 1983. Plasmid encoded hygromycin Bphosphotransferase gene and its expression in Escherichia coliand Saccharomyces cerevisiae. Gene 25:179-188.

8. Hollenberg, C. P. 1982. Cloning with 2 [. vectors and the

expression of foreign genes in Saccharomyces cerevisiae. Curr.Top. Microbiol. Immunol. 96:119-144.

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