MOLECULAR AND CELLULAR BIOLOGY, Aug. 1991, p. 4235-4243 Vol. 11, No. 80270-7306/91/084235-09$02.00/0Copyright (C 1991, American Society for Microbiology

Cloning and Characterization of a Gene Which Determines OsmoticStability in Saccharomyces cerevisiae

LUBOMIRA I. STATEVA,' STEPHEN G. OLIVER,2* LAURENCE J. TRUEMAN,2 AND PENCHO V. VENKOV'Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria,' and Department of Biochemistry

and Applied Molecuilar Biology, UMIST, Manchester, M60 IQD, United Kingdom2

Received 4 March 1991/Accepted 6 May 1991

The srbl-1 mutation of Saccharomyces cerevisiae is an ochre allele which renders the yeast dependent on anosmotic stabilizer for growth and gives the cells the ability to lyse on transfer to hypotonic conditions. A DNAfragment which complements both of these phenotypic effects has been cloned. This clone contains a functionalgene which is transcribed into a 2.3-kb polyadenylated mRNA molecule. Transformation of yeast strainscarrying defined suppressible alleles demonstrated that the cloned fragment does not contain a nonsensesuppressor. Integrative transformation and gene disruption experiments, when combined with classical geneticanalysis, confirmed that the cloned fragment contained the wild-type SRB1 gene. The integrated marker wasused to map SRB1 to chromosome XV by Southern hybridization and pulsed-field gel electrophoresis. Adisruption mutant created by the insertion of a TRP1 marker into SRBI displayed only the lysis abilityphenotype and was not dependent on an osmotic stabilizer for growth. Lysis ability was acquired by growth in(or transfer to) an osmotically stabilized environment, but only under conditions which permitted budding. Itis inferred that budding cells lyse with a higher probability and that weak points in the wall at the site ofbudding are involved in the process. The biotechnological potential of the cloned gene and the disruptionmutant is discussed.

The cell wall of the yeast Saccharomyces cerevisiaepresents a significant barrier to the recovery of high-molec-ular-weight compounds, such as DNA and RNA, and largerstructures, such as organelles, ribosomes, or viruslike par-ticles (28). A number of gentle lysis procedures, such asprotoplasting and glass bead breakage (28), have been de-veloped to solve this problem on the laboratory scale.However, these techniques are rarely practicable on anindustrial scale and, even for laboratory work, conditionallysis mutants of S. cerevisiae have always provided anattractive alternative (26, 30, 32). There have been fewreports on the isolation of such lysis mutants, and little isknown about how the genes defined by the mutations deter-mine the ability of the yeast cell wall and/or membrane towithstand stresses such as elevated temperature and osmoticshock.

Temperature-sensitive cly mutants which lyse after pro-longed incubation at the restrictive temperature of 36°C wereisolated by Hartwell (8) and Hartwell and McLaughlin (10),but nothing is known about their mechanism of lysis. Mu-tants lacking the outer chain of the N-linked mannoproteinsof the cell wall have been reported (1). These mnn9 mutantsexhibit a physically distorted cell wall, grow slowly, andgradually lyse during growth. A third class of yeast lysismutants is defined by the fragile mutant S. cerevisiaeVY1160 isolated by Venkov et al. (29). Cells of this mutantgrow normally in medium containing osmotic stabilizers(10% sorbitol or mannitol; 1.6% NaCl) but lyse spontane-ously on transfer to a hypotonic solution. This phenotypehas been found to be determined by a recessive nuclearmutation designated srbl (15). The mutation is pleiotropic inits effects and, in addition to the lysis phenotype, leads toincreased cell permeability to a wide range of compounds(nucleosides, antibiotics, DNA, and proteins), reduced

* Corresponding author.

spore viability (15), and elevated levels of protein excretion(31). The srbl-J mutation (previous designation, srbl; 15)has been found to produce defects in both the cell wall andthe membrane. The walls of mutant cells contain less man-nan than do those of the parent, and this mannan has anincreased proportion of short side chains (17). The glucanstructure of the walls of srbl-l mutants has also been foundto be altered, being less branched in nature than that of thewalls of the wild type (2a). The pleiotropic effects of themutation are not confined to the wall, however, sincealterations in the lipid composition of the plasma membranehave also been observed (22b).

It is not clear which of these effects is a direct conse-quence of the mutation and which is secondary in nature.Nevertheless, the range of effects suggests that the wild-typeSRB1 gene encodes a protein which plays a major role indetermining the structural integrity of the yeast cell. This,together with the practical utility of the mutant in thelaboratory and its potential for the commercial production ofyeast extracts (26, 30), means that it is important to charac-terize the SRBI gene and identify its product. In this paper,we report the cloning and characterization of SRBI, as wellas the construction by gene disruption of mutations whichshed further light on its physiological role.

MATERIALS AND METHODS

Strains and media. The S. cerevisiae and Escherichia colistrains used in this study are listed in Table 1.The media used for the growth of yeast cells (YEPD,

presporulation, sporulation, and selective dropout minimalmedia with appropriate combinations of amino acids orbases) were prepared as described by Sherman et al. (24),and those used for the growth of bacterial cells (LB andLB-ampicillin) were prepared as described by Maniatis et al.(18). When necessary, all media for yeast cells were solidi-fied by the addition of 2% agar and those for bacterial cells

4235

4236 STATEVA ET AL.

TABLE 1. Yeast and bacterial strains used in this work

Organism and strain Genotype or phenotype Reference orsource

S. cerevisiaeVY1160 MATa srbl tsl ts2 leu2 gal2 Mal- Met- Thr- Trp- rho 291SL MATa srbl leu2 tsl Our collectionaVY15 MATa srbl leu2 ura3 Our collectiona4STLU MATa srbl trpl leu2 ura3 Our collection'7SLU MATa srbl Ieu2 ura3 Our collection'S288C MATot Mal- gaI2 G. FinkYPH1 (=YNN214) MATa ura3-52 lys2-801 (amber) ade2-101 (ochre) 25YPH98 MATa ura3-52 lys2-801 (amber) ade2-101 (ochre) leu2AJ trplAl 25YPH274 MATo ura3-52 lys2-801 (amber) ade2-101 (ochre) trplAl his3A200 leu2A1 25

MATa ura3-52 Iys2-801 (amber) ade2-101 (ochre) trpJAJ his3A200 leu2AJYP148b MATa ura3-52 Iys2 ade his7 trpJAJ (TRPJ) (URA3) P. Hieter

E. coliHB101 hsdR hsdM recA13 supE44 lacZ24 leuB proA2 thi-J SmrDH1 F- recAl endAI gyrA96 thi-I hsdRI7 r- M+ supE44 7

a All srbl-l-containing strains are from our laboratory collection. They were obtained by crossing the original srbl-I donor mutant VY1160 (28) with differentwild-type strains and selecting for appropriate srbl-l-containing segregants among the meiotic progeny of the diploids.

b YP148 is a derivative of YP80 (MATcs ura3-52 Iys2 ade his7 trpl-1) in which chromosome VII is represented on two "chromosome fragments-thehigher-molecular-weight one being marked by URA3. Band 11 of an OFAGE separation of chromosomes from this strain contains only chromosome XV.

were solidified by the addition of 1.5% agar. For propagationof fragile yeast strains, all media were supplemented with10% sorbitol.

Yeast library and plasmids. The recombinant DNA poolcontaining ca. 5-kb fragments from a partial Sau3A digest ofyeast genomic DNA ligated into the BamHI site of theyeast-E. coli shuttle vector YEp13 (3) was generously pro-vided by K. D. Entian. The following parent plasmids wereused: YCp5O (16), YRp7 (27), pBR322::URA3 (a kind giftfrom G. Tchumper), YIp5 (23), pUC18, and pMA700 (a kindgift from M. Tuite). The S. cerevisiae TRPI gene wasisolated as a 1.4-kb EcoRI fragment from YRp7; URA3 wasisolated as a 1.1-kb Hindlll fragment from pBR322:: URA3;and HIS3 was isolated as a 1.8-kb BamHI fragment frompMA700. Plasmids YIpSUP61+ and pYleu2SUP6, contain-ing amber suppressor genes for tRNAser and tRNATYr,respectively, were a kind gift from J. Abelson.

Plasmid constructions for gene integration and disruptionby targeted transformation. The EcoRI site in YIp5 wasdestroyed; the resultant plasmid, YIp5*, was cut withBamHI and dephosphorylated. The SRBI gene was isolatedas a 4.0-kb BamHI fragment from the recombinantYEP13::SRBJ plasmid and inserted into Ylp5*. To target theintegration event to the chromosomal location of SRBI, welinearized plasmid YIp5*::SRBJ at the EcoRI site shown tobe internal to the SRBJ coding region.The plasmid for the gene disruption experiment was

constructed on the basis of pUC18. The EcoRI site in thepolylinker was eliminated. The resultant plasmid, pUC18*,was digested with PstI and dephosphorylated. The 1.5-kbPstl fragment originating from the SRBI sequences (Fig. 1)was purified and ligated to the pUC18* vector. The resultantplasmid, Lpl, was digested with EcoRI, dephosphorylated,and ligated to the 1.4-kb EcoRI fragment from YRp7 con-taining TRPJ. As a result, plasmid Lp2, in which the SRBJcoding region was interrupted by the insertion of the TRPIgene, was obtained. This plasmid was linearized beforetransformation at the HpaI site, generating ends homologousto the SRBI chromosomal sequence.

Transformation. S. cerevisiae strains containing the srbl-Jmutation were transformed by the method of Philipova (22a).

This method is similar to the whole-cell transformationmethod of Ito et al. (13) but does not involve the use of alkalications. Instead, mid-exponential-phase cells were treatedfor 30 min with DNA and incubated for 60 min with 35% PEG4000. Wild-type yeast strains were transformed by the sphero-plast method of Hinnen et al. (12), as modified by Burgers andPercival (4). E. coli strains were transformed by the calciumchloride method as described by Maniatis et al. (18).

Nucleic acid preparation. Yeast genomic DNA was pre-pared from spheroplasts (6). Bacterial plasmid DNA wasisolated on a large scale by the alkaline lysis method ofBirnboim and Doly (2) as described by Maniatis et al. (18).The DNA minipreparations of bacterial plasmid DNA wereobtained by a modification of the latter method involving 15min of incubation with 0.75% diethylpyrocarbonate at 65°Cbefore ethanol precipitation.

All DNA fragments used as radioactive probes or forsubcloning were gel purified either by extraction from low-melting-temperature agarose as described by Maniatis et al.(18) or by electroelution with a Biotrap (Schleicher &Schuell). Poly(A)+ RNA for Northern (RNA) analysis wasisolated from total RNA by oligo(dT)-cellulose chromatog-raphy (21).

Gel electrophoresis of DNA and RNA, transfer to mem-branes, and hybridization. Agarose gel electrophoresis ofDNA was carried out in 0.8% agarose gels in TEB buffer(18). The size standards used were HindlIl- or HindIll-EcoRI-digested lambda DNA (Boehringer). DNA in gels wastreated, transferred to Hybond membranes, and hybridizedto labeled probes by the protocol for Southern analysisrecommended by Amersham International plc. Poly(A)+RNA was denatured in formaldehyde-formamide at 50°C for15 min, subjected to electrophoresis in 1.5% agarose-form-aldehyde gels [5 to 10 ,ug of poly(A)+ mRNA per lane],transferred to Hybond in 20x SSC (lx SSC is 0.15 M NaClplus 0.015 M sodium citrate), and hybridized to labelledDNA probes as recommended in the Amersham protocol.Molecular weight standards were rRNAs from E. coli(Boehringer) separated on a parallel lane, cut from the rest ofthe gel, and stained with ethidium bromide. Filters wereexposed to X-ray films with intensifying screens at -70°C.

MOL. CELL. BIOL.

CLONING OF A YEAST OSMOTIC STABILITY DETERMINANT 4237

Plasmid

R S B P RE P Bg B E H R

YEpl3::SRBI ---/..-.

|( z 8kb4.Ikb

R

YCp5O::EcoRI-1

Vector comAl

YEpl3 +

R

YCp50 -

< 5.4kb-

R

YCp5O: EcoRI-r

( - 3. 4kb -*

B

-I4. Okb -

B

-I| 4 Okb

FIG. 1. Restriction map of the recombinant YEp13-SRBJ plasmid. The dotted lines represent plasmid vector DNA. The solid linesrepresent genomic DNA. Restriction sites: R, EcoRI; E, EcoRV; B, BamHI; Bg, BglII; P, PstI; S, Sall; H, HindIll. Abbreviations: r, right;1, left; compl, complementation (+, positive; -, negative).

Enzymatic reactions. Restriction enzymes, calf intestinalphosphatase, T4 DNA ligase, the Klenow fragment of DNApolymerase I, and pancreatic RNase A were from Pharmaciaor Boehringer. Restriction digestion and treatment with calfintestinal phosphatase were performed in accordance withthe instructions of the manufacturers. Overhanging ends ofEcoRI fragments were converted to blunt ends with theKlenow fragment of DNA polymerase I. Ligations with T4DNA ligase were performed at 14°C overnight in the buffersrecommended by the manufacturers. DNA probes were

radiolabelled with 32P by use of the random primed labellingkit from Amersham. DNA and unincorporated nucleotideswere separated by the spun-column procedure (18).

Isolation, electrophoresis, and hybridization of yeast chro-mosomes. Yeast chromosomes were prepared as in accor-dance with the instructions of the manufacturer (BioRad) forthe CHEF DR-II apparatus and separated in 1% agarose gelswith 0.5 TEB buffer (18) by the contour-clamped homoge-neous electric field (CHEF) method (5). DNA in gels was

HCI depurinated. All subsequent steps of denaturation,neutralization, transfer to Hybond membranes, prehybrid-ization, and hybridization were in accordance with theSouthern analysis protocol recommended by Amersham.

Genetic methods. Standard yeast genetic methods of mat-ing, sporulation, and tetrad analysis were followed (24). Theconcomitant loss of plasmid markers and the mitotic stabilityof transformed strains were assayed by growing cultures tothe stationary phase in YEPD at 30°C and plating thecultures for single colonies on YEPD plates. These plates

were incubated at 30°C, and the colonies were replica platedto selective media to determine the frequency of loss of aparticular marker. Sorbitol dependence was tested by com-paring growth on media with and without 10% sorbitol.

Lysis test. In the present study, we did not use the publishedprocedure for the radioactive determination of cell lysis (29).Instead, the following test was routinely used. Cells weregrown to the mid-exponential phase in an osmotically stabi-lized medium (YEPD or minimal), harvested by centrifuga-tion, and suspended in water to 1/10 of culture volume. Afterbeing vortexed briefly, the cell suspension was centrifugedand the A260 and A280 of the supernatant (lysate) weremeasured. Under these conditions most srbl-J lysis mutantsyielded between 4 and 6 A260 units from 10 ml of culturesuspended in 1 ml of water, whereas SRBI+ strains yieldedonly 1.0 to 1.5 A260 units under similar conditions.

Control experiments demonstrated that wild-type cellsexhibited 95% viability following this lysis test, whereas srblmutants were only 15 to 30% viable, depending on theparticular mutant used. These data confirm that absorbancedata do indeed monitor cell lysis and not merely the leakageof material from permeable cells.

RESULTS

Cloning of SRBI. A DNA fragment which complementedthe srbl-J mutation and permitted the growth of strain 1SLin the absence of sorbitol was isolated from a YEp13-basedyeast genomic library. The transformant obtained was tested

R

B

YCp50: BamHI

YCp50 -

YEpl3:: BamHI

B

YCp50 -

YEpl3 +

VOL. 11, 1991

4238 STATEVA ET AL.

for its lysis ability in comparison with that of the parentaluntransformed strain and was found incapable of lysis uponosmotic shock (data not shown). This transformant, contain-ing the putative SRBJ clone, was subjected to a mitoticstability test as described in Materials and Methods. A totalof 250 colonies grown under nonselective conditions weretested, and 70% of them were found to have simultaneouslylost their ability to grow without leucine and sorbitol.

Total yeast DNA was isolated from this SRBJ-containingtransformant and used to transform E. coli HB101. SeveralE. coli transformants were tested and found to contain a15.5-kb recombinant plasmid. This plasmid, designatedYEpl3::SRBJ, was used to transform other srbl-J mutants(7SLU, 4STLU, and VY15) and, in all cases, was able tocomplement both their growth dependence upon 10% sor-bitol and their ability to spontaneously lyse upon osmoticshock. For these reasons, the plasmid was used for furtheranalysis.

Restriction mapping and subcloning. The restriction map ofthe 4.8-kb insert of yeast genomic DNA in YEpl3::SRB1 isshown in Fig. 1. This figure also includes information aboutthe vectors and subfragments used for the subcloning exper-iments. Complementation of srbl-I with the different con-structs was tested with all the srbl-l hosts shown in Table 1.The data in Fig. 1 demonstrate that it was not possible tocomplement the srbl-J mutation with a fragment smallerthan the 4.0-kb BamHI fragment. Moreover, this fragmentwas capable of complementation only when present on amulticopy vector. Since neither of the EcoRI subclones wascapable of complementation, it appeared quite likely that theEcoRI site is internal to the gene.

Southern and Northern analyses. To further characterisethe putative SRBJ-containing DNA fragment, we usedSouthern analysis. Genomic DNA from S. cerevisiae S288Cwas isolated and digested with HindIll or EcoRI and hybrid-ized with the labelled BamHI fragment (Fig. 1). The HindIll-digested DNA contained only one band, and the EcoRI-digested DNA contained two bands (data not shown). Theseresults showed that the cloned fragment contains sequenceswhich are unique in the S. cerevisiae genome.The distribution of restriction sites relative to the func-

tional gene was further examined by Northern analysis.Different subfragments were used as radioactive probes forhybridization with poly(A)+ mRNA isolated from VY1160.The results of the Northern analysis are shown in Fig. 2.These data showed that the srbl-l-complementing fragmentcontains sequences corresponding to part or all of two geneswith poly(A)+ mRNA transcripts of 2.7 and 2.3 kb, respec-tively. Taken together with the results of the complementa-tion experiments, the results of the Northern analysis permitthe following conclusions. (i) The only srbl-l-complement-ing subclone, the BamHI fragment, hybridizes to a singlepoly(A)+ mRNA species of 2.3 kb, which must thereforerepresent the SRBI transcript. (ii) The PstI fragment isinternal to the gene. (iii) The SRBI gene straddles the EcoRIsite, which may therefore be used for gene disruptionexperiments.

Suppressor activity testing of the srbl-1-complementingfragment. The srbl-J mutation was shown independently tobe ochre suppressible (25a). Taking this result into account,we decided to check the cloned DNA fragment and theconstructed subclones (Fig. 1) for their ability to comple-ment well-characterized amber and/or ochre suppressiblealleles in S. cerevisiae YPH1 and YPH98 (25). As controls inthis experiment, we used vectors YEp13 and YCp5O, as wellas plasmids YIpSUP61+ and pYleu2SUP6.

r

t2.., 10-_.0

FIG. 2. Northern analysis of YEpl3::SRBI. Poly(A)+ mRNAwas isolated from VY1160. The DNA fragments used as radioactiveprobes were as follows: lane 1, EcoRI-1; lane 2, EcoRI-r; lane 3,BamHl; lane 4, PstI; lane 5, BglII-HindIlI. The molecular weightmarkers were standard E. coli rRNAs (Boehringer) separated byelectrophoresis on a separate lane, cut out of the gel, stained withethidium bromide, and photographed under UV light. The molecularsizes of the poly(A)+ mRNA transcripts are given in kilobases.

Transformants were selected by complementation withthe plasmid marker URA3 (for YCp5O and derivatives andfor YIpSUP61J) or LEU2 (for YEp13 and derivatives andpYleu2SUP6). One hundred transformants of each typewere screened for complementation of the suppressiblealleles, lys2 (amber) and ade2 (ochre). With the exception ofthe two controls, YIpSUP61+ and pYleu2SUP6, none of theother plasmid DNAs used was found to complement thesuppressible alleles in the test strains. For this reason, weconcluded that the cloned fragment probably contains thegene of interest rather than a suppressor. At this stage,however, we were not able to exclude the possibility that thecloned fragment contains some type of suppressor, otherthan a nonsense suppressor.The cloned SRBJ fragment directs integration at a unique

and homologous site. To integrate cloned DNA into the yeastgenome, we first constructed a plasmid based on YIp5 (seeMaterials and Methods). Integrative plasmid YIp5*::SRBIwas linearized by EcoRI digestion and used to transform S.cerevisiae 7SLU to Ura+. Transformants were tested formitotic stability, and most of them were found mitoticallystable, indicating that the plasmid had been integrated into ayeast chromosome. One of these transformants, IT-2, waschosen at random for further analysis. The cloned SRBIfragment was shown to direct integration at a unique andhomologous site by Southern blot hybridization analysis ofchromosomal DNA isolated from IT-2 and the parent strain,7SLU. The results are shown in Fig. 3a.URA3 hybridized to a single EcoRI or BamHI fragment

from genomic DNA of the parent strain, 7SLU. However, ithybridized to an intense, second band of 9.5 kb (as expectedfrom the diagram) in the EcoRI-digested DNA of IT-2 (lane3) and identified two fragments of similar mobilities (5.5 kb)in the BamHI-digested DNA of IT-2 (doublet in lane 4).SRBJ, used as a radioactive probe, identified two EcoRIfragments and one Sall fragment in the genomic DNA fromthe parent strain. However, SRBI hybridized to three EcoRIfragments (lane 7) and identified two Sall fragments ofsimilar mobilities (doublet in lane 8) in the genomic DNAfrom transformant IT-2. These results are consistent with the

MOL. CELL. BIOL.

CLONING OF A YEAST OSMOTIC STABILITY DETERMINANT

B Ra

B

. 4 Ir_

b-----I

7.S

xR

B!B .. .B

I--,

.FL..-i

B R B S B R

Vi 4.0kb - - 5. 5kb ---------- 40kb

9. 5kb

FIG. 3. Southern blot hybridization analysis of integrative transformant IT-2 in comparison with parent strain 7SLU. (a) DNA blothybridization analysis. Genomic DNA was isolated from IT-2 and 7SLU and digested with EcoRI, BamHI, or Sall. The blot was probed withURA3 (1.1-kb Hindlll fragment) and SRBJ (4.0-kb BamHI fragment). The results are presented as follows: lanes 1 to 4, probed with URA3;lanes 5 to 8, probed with SRBI. The DNA from parent strain 7SLU is shown in lanes 1 and 5 (after EcoRI digestion), 2 (after BamHIdigestion), and 6 (after Sall digestion). The DNA from transformant IT-2 is shown in lanes 3 and 7 (EcoRI digested), 4 (BamHI digested), and8 (Sall digested). The sizes of the observed fragments were determined with ethidium bromide-stained, UV-photographed, HindIll-digestedA DNA molecular weight markers. (b) Diagram of the recombination event. For clarity, only the BamHl (B), EcoRI (R), and Sall (S) sitesare indicated. R indicates the EcoRI site in pBR322 which was destroyed in the construction as described in Materials and Methods. The sizesof the fragments are given in kilobases. The striped box represents chromosomal SRBI sequences, the stippled box represents the URA3gene, the black box represents plasmid-borne sequences of the SRBI gene, the open box represents pBR322 sequences, and the broken linerepresents chromosomal sequences flanking SRBI. The recombination event was directed by linearization of YIp5*::SRBI at the EcoRI siteshown to be internal to the gene.

scheme presented in Fig. 3b and indicate that the integrationof plasmid YIp5*::SRBJ has taken place at a unique andhomologous site.

In an alternative experiment, chromosomes were isolatedfrom integrative transformant IT-2 and test strain YP148.The latter strain has been manipulated to provide goodresolution of the usual doublets observed with other strainsin OFAGE separations of yeast chromosomes. The chromo-somes were separated as described in Materials and Meth-ods and hybridized with a 1.1-kb HindlIl fragment contain-ing the URA3 gene and the 4.0-kb BamHI fragment as aprobe for the putative SRBJ gene. The results of thisexperiment (data not shown) indicated that the URA3 gene,as part of integrative plasmid YIp5*::SRBJ, has been inte-grated into the chromosome from which the cloned fragmentoriginated. This chromosome was tentatively identified as

number XV.Localization of the SRBI gene on chromosome XV by CHEF

blot hybridization. The preliminary assignment of the clonedSRBJ gene to chromosome XV was confirmed with a similar

experiment. Chromosomes were isolated from test strainYP148 and separated by the CHEF method until most of thelower-molecular-weight chromosomes had run off the end ofthe gel. Under these conditions, good separation of all thedoublets containing the higher-molecular-weight chromo-somes was achieved, chromosome VII being separated fromXV and chromosome XIII being separated from XVI.The previous chromosomal blot hybridization analysis

indicated that the integration of the URA3 gene targeted bythe SRBI homologous ends was not on chromosome VII.Therefore, in the present experiment, we used probes onlyfor the other neighboring chromosomes, XIII and XV. Theresults are presented in Fig. 4. Part of the ILV2 gene (kindlyprovided by M. Kielland-Brandt) was used as a probe forchromosome XIII, and the HIS3 gene was used as a probefor chromosome XV (20). The 1.5-kb PstI fragment (Fig. 1)was used as a radioactive probe for the localization of theSRBJ sequences. Both the HIS3- and the SRBI-containingsequences hybridized to the same chromosome, XV (20).SRBI is not an essential gene but its disruptants are sensitive

B

VOL. 11, 1991 4239

4240 STATEVA ET AL.

FIG. 4. CHEF blot hybridization mapping of the srbl-comple-

menting DNA fragment. Chromosomes were loaded and separated

on six lanes, blotted onto Hybond membranes, and divided into

three parts; each part was hybridized to a specific probe. Lanes 1 to

6, lanes 7 and 8, and lanes 9 to 11 were hybridized against ILV2,

HIS3, and SRBI, respectively. Lane 6, however, was in fact divided

into two parts; the left half was hybridized against ILV2, and the

right half was hybridized against HIS3.

to osmotic shock. Gene disruption by integrative transforma-

tion (22) was performed with plasmid Lp2, whose construc-

tion is described in Materials and Methods. To target the

integration event to the chromosomal location of the SRBJ

gene, we linearized the plasmid by Hpal digestion and used

it to transform diploid strain YPH274 (25) to Trp'. A large

number of transformants were tested for mitotic stability,

and most of them proved to be stable during long-term

cultivation under nonselective conditions. One of these

stable transformants, YPH274-4, was sporulated, and 40

tetrads were dissected. Fifteen full tetrads were obtained on

medium with sorbitol. All segregants were scored for Trp',Ade', Srb', and mating type. In all four-spored asci, TRPJ

was found to segregate 2:2, while one of the other markers,

ade2, segregated 4 Ade-:0 Ade'. Surprisingly, neither of the

Trp segregants showed dependence upon the addition of an

osmotic stabilizer to the medium, one of the phenotypic

effects of the srbl-J mutation.

The segregants from two of the tetrads IV and XXVII (4a,

4b, 4c, and 4d and 27a, 27b, 27c, and 27d, respectively),were grown to the mid-exponential phase (5 x 107 cells per

ml) in YEPD with 10% sorbitol and tested for lysis upon

osmotic shock as described in Materials and Methods. The

results (Table 2), which are the average of two tests,

indicated that the segregation of the TRPJ marker corre-

sponds to the segregation of the ability of the strains to lyse

upon osmotic shock. Therefore, disruption of the SRBI gene

TABLE 2. Lysis testinga of SRBI gene disruptants

Segregant A260 units TRPlb

4a 4.25 +4b 1.754c 1.624d 4.55 +

27a 1.2527b 1.5527c 5.55 +27d 6.15 +

a The standard lysis test is described in Materials and Methods.b +, presence of the marker and therefore disruption of the SRBI gene; -,

absence of the marker.

leads to lysis ability, but not to growth dependence upon theaddition of an osmotic stabilizer to the medium.To verify that the disruptant srbl::TRPJ was allelic to the

srbl-J ochre mutation, we crossed original mutants anddisruptants of opposite mating types and tested the diploidsfor sorbitol dependence and lysis ability. Six different dip-loids were obtained, and none was found to be dependent onthe addition of an osmotic stabilizer to the medium. How-ever, all of them were able to lyse upon osmotic shock,yielding results in the range characteristic for srbl-J mutantsand disruptants (data not shown). One of the diploids(4STLU x 4d) was sporulated, and all spores of two four-spored asci were shown to be capable of lysis upon osmoticshock. The disrupted segregants from tetrad IV (Table 2, 4aand 4d) were transformed with the srbl-l-complementingplasmids YEpl3::SRBl and YEp13-BamHI in an attempt torestore their wild-type phenotype. A similar complementa-tion test was carried out with YCp5O: :BamHI (Fig. 1), a low-or single-copy plasmid. Three transformants of each typewere subjected to the routine lysis test after growth in thepresence of 10% sorbitol. The results (Table 3) showed thatall the constructs tested, including the low-copy-numberplasmid YCp5O: :BamHI, were able to complement the lysisphenotype caused by the srbl::TRPI disruption. This resultis in contrast to the inability of YCp5O::BamHI to comple-ment the phenotype caused by the original srbl-J allele (Fig.1). Therefore, the cloned fragment is able to complement thesensitivity to osmotic shock determined by both srbl-J andsrbl::TRPI alleles of the SRBJ gene. As a result of theseexperiments, we concluded that the SRBI gene has been

TABLE 3. Complementation of the srbl::TRPI lysis phenotypewith different SRBI constructs

Disruptant Transforming A200 unit(s)aplasmid

4a None 2.62YEpl3::SRBI 0.56YEpl3::BamHI 0.62YCp5O::BamHI 0.71

4d None 2.81YEpl3::SRB1 0.48YEpl3::BamHI 0.78YCpSO::BamHI 0.68

a The results are the averages for three transformants of each type. Thelysis yields in this experiment were lower than in the previous experiment,since cell concentrations were reduced by growth on selective minimalmedium to prevent plasmid loss.

MOL. CELL. BIOL.

CLONING OF A YEAST OSMOTIC STABILITY DETERMINANT 4241

TABLE 4. Comparative lysis testing' after growthin different media

A260 units released upon lysis followinggrowth in medium:

SegregantWith 10% Without 10csorbitol sorbitol

4a 4.2 1.34b 1.7 1.74c 1.5 1.64d 5.9 1.8

27a 1.6 1.527b 1.7 1.727c 4.9 1.727d 5.5 1.9

aThe standard lysis test is described in Materials and Methods.

cloned. This gene codes for a product which determinesosmotic stability in S. cerevisiae.

Characterization of the lysis ability of srbl::TRPI mutants.Given the independence of the disruptants from the additionof an osmotic stabilizer to the medium, the segregants fromthe two tetrads (described in Table 2) were grown to themid-exponential phase on media with and without 10%sorbitol, and the strains were lysed. The results (Table 4)showed that, unlike the wild-type segregants (4b, 4c, 27a,and 27b), which are not sensitive to changes in the osmoticpressure, the disruptants (4a, 4d, 27c, and 27d) spontane-ously lysed after transfer from osmotically stabilized me-dium to water. However, the srbl::TRPJ mutants behavedexactly like the wild-type segregants when grown in non-osmotically stabilized medium.To determine the conditions necessary for the acquisition

of lysis ability, we used one of the disruptant segregants (4d)and a wild-type segregant from the same tetrad (4b) as acontrol. The two strains were cultivated in YEPD, andaliquots of the cultures were suspended at different initialdensities (7 x 106 and 2.5 x 107 cells per ml, respectively) inYEPD with 10% sorbitol and grown at 30°C until themid-exponential phase (5 x 107 to 6 x 107 cells per ml). Thiswas accomplished after 5 and 3 h of growth, respectively.

In an alternative experiment, the same strains were grownin YEPD until the mid-exponential phase, and 25-ml aliquotswere harvested from the culture and suspended in the same

volume of either 10% sorbitol or YEPD with 10% sorbitol,each containing cycloheximide. All cultures were shaken at30°C for 3 or 5 h. No growth of the strains was observedunder these conditions. As controls, both types of segre-gants were grown to the mid-exponential phase in YEPDwith and without 10% sorbitol.The results of the lysis test carried out with all the variants

and controls are presented in Table 5. They showed that agrowth period equivalent to approximately two generationtimes (5 h) in 10% sorbitol-containing YEPD madesrbl::TRPJ cells sensitive to osmotic shock. However, uponstarvation for the same period of time in the presence of onlythe osmotic stabilizer, the cells of the same mutant did notacquire the ability to lyse upon osmotic shock. Therefore, itis not the osmotic stabilizer per se, but growth in anosmotically buffered nutritional medium, which is essentialfor lysis ability. To check this hypothesis, we grew the cellsin an osmotically stabilized medium which contained aninhibitor of growth, cycloheximide. Like srbl-J mutants,srbl:: TRPI disruptants were inhibited by a lower concentra-

TABLE 5. Acquisition of lysis ability upon osmotic shock of thesrbl::TRPI disruptant under different conditions

A260 unit(s) released upon lysisaCondition(s) for segregant:

4d (srbl::TRPI) 4b (SRBI)

YEPD 1.7 1.0YEPD + 10% sorbitol 4.6 1.7YEPD + 10%, sorbitol (3 h) 2.7 1.1YEPD + 10%S sorbitol (5 h) 4.7 1.310% Sorbitol (3 h) 1.5 0.510% Sorbitol (5 h) 1.4 0.5YEPD + 10%o sorbitol + CH (3 h) 5.2 1.6YEPD + 10% sorbitol + CH (5 h) 7.2 2.6

" The standard lysis test is described in Materials and Methods. Cyclohex-imide (CH) was used at 30 Fig/ml for the disruptant and 50 pLg/ml for thewild-type segregant.

tion of the drug (30 ,ug/ml) than were wild-type segregants(50 ug/ml).The results in Table 5 showed that the inhibition of growth

in an osmotically buffered, cycloheximide-containing me-dium increased the sensitivity of the disruptant cells toosmotic shock. The yield of A260 units increased by morethan 50%, a result which can most likely be attributed to thearrest of most (or all) of the cells in a budded state.To test the hypothesis that budded cells were more

susceptible to lysis than unbudded ones, we assessed theproportions of budded cells before and after lysis using alight microscope. Although lysed and unlysed cells may bedistinguished by their phase brightness, a more unambiguousway of discriminating between the two classes of cells wasfound to be the destruction of lysed "ghosts" by sonication.Table 6 shows the proportions of unbudded cells, beforelysis and after lysis and sonication, for the wild type and forsrbl-J and srbl::TRPI mutant strains. It was found thatsonication (two min at full power with an MSE Soniprobe 50)had little effect on wild-type cells. Considerable cell break-age was obtained for both srbl-J and disruption mutants. Ineach case, there was a large increase in the proportion ofunbudded cells in the population surviving lysis and sonica-tion.

DISCUSSIONA 4.0-kb fragment of S. cerevisiae DNA which comple-

ments the srbl-J mutation, shown to determine the lysisability of the osmotically fragile mutant VY1160, has beenisolated. The cloned fragment, however, is capable of com-plementing the srbl-J mutation only when carried on amulticopy plasmid (Fig. 1), suggesting the cloning of asuppressor rather than the wild-type SRBI gene. Given thisresult and the fact that the srbl-I mutation is an ochremutation (9), we checked the cloned fragment for suppressor

TABLE 6. Proportions of unbudded cells before lysis andafter lysis and sonication

% of unbudded cellsStrain Genotype Before After lysis and

lysis sonication

4b SRBl 41 424d srbl:: TRPI 47 797SLU srbl-l 28 81

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4242 STATEVA ET AL.

activity. All the subclones shown in Fig. 1 were transformedinto two different strains carrying well-defined suppressiblealleles, but none of them showed any suppressor activity ofthe nonsense type. However, at this stage, our experimentaldata did not exclude the possibility that the cloned fragmentcontains another type of suppressor. Indeed, the fact theBamHI subclone only complemented srbl-J when presenton a multicopy vector meant that the possibility of anextragenic mass-action suppressor had to be considered.However, the possibility that some upstream activatingsequences were missing from the SRBJ cloned fragmentshould also be taken into account, because this would alsoresult in a lower level of expression of the cloned gene.The identity of cloned yeast genes is usually confirmed by

integrative transformation, since plasmid integration intoyeast chromosomes has been shown to occur by homologousrecombination (11, 22). We also took advantage of thisapproach to demonstrate that the SRBJ gene had indeedbeen cloned. For the linearization of the plasmid constructedfor integrative transformation and for the gene disruptionexperiment, we used the unique EcoRI site found in thecloned fragment. On the basis of the subcloning and North-ern analysis results, this site was shown to be internal to thegene.

Disruption of the SRBJ-containing sequences wasachieved by cloning the 1.4-kb EcoRI TRPI fragment intothe EcoRI site of SRBI. Diploid strain YPH274 (25) waschosen as a host for the transformation for two mainreasons. First, this strain carries the deletion mutationtrpl-J, which does not retain homology to the TRPI select-able marker gene (24) present in the Lp2 vector. Conse-quently, there is no possibility of integration at the trpl-ilocus. Second, the SRBJ gene might be essential for growth;therefore, construction of a null allele would be lethal. Infact, the genetic analysis of the transformant showed that theSRBI gene is not essential for growth. In 15 four-spored asci,lysis ability always segregated together with the TRPImarker. Quite surprisingly, none of the analyzed segregantscarrying the disruption was found to be dependent on thepresence of an osmotic stabilizer in the nutritional me-dium for growth. Nevertheless, the heterozygous diploidsrbl-JIsrbJ:: TRPI was very fragile and generated asci withall four spores capable of lysis upon osmotic shock. Theseresults showed that we had indeed cloned the SRBI gene.The phenotype of cells carrying the disruption allele,

srbl::TRPI, was different from that of those containing theoriginal ochre mutation, srbl-J. Both alleles determine os-motic fragility and confer higher sensitivity to antibiotics(cycloheximide was used at 30 ,ug/ml for the disruptant andat 50 ,ug/ml for the wild-type segregant). However, thesrbl ::TRPI disruptants differed from the srbl-J cells in thatthey did not require the presence of an osmotic stabilizer forgrowth and did not grow more slowly than the wild-typesegregants (data not shown). The reason for these differ-ences is unknown, but one plausible explanation is that theproduct of the SRBI gene is a protein which is required forprotection against osmotic shock, but not for normal vege-tative growth. The three phenotypes that we have charac-terized may then be represented in the following manner. Afully functional Srbl protein is incorporated into the cellsurface of wild-type cells which, as a result, can grow in theabsence of an osmotic stabilizer and do not lyse uponosmotic shock. The ochre mutation results in a fragment ofthe Srbl protein being synthesized and incorporated into thecell surface of the srbl-J mutant. The presence of thisfragment makes the cells dependent on an osmotic stabilizer

for growth, while the absence of the Srbl function leads tolysis upon osmotic shock. In the disruptant, the Srbl proteinis simply absent from the cell surface. In this case, the cellswill not be dependent upon an osmotic buffer for growth, butthe absence of the Srbl protein will render them sensitive toosmotic shock. A specific prediction of this hypothesis isthat single-copy vectors carrying SRBI should be capable ofcomplementing the srbl::TRPI disruption but not the origi-nal srbl-J mutation. This is because, in the latter case, highlevels of Srbl protein would be required to compete with theochre fragment for incorporation into the cell surface. Thisprediction was confirmed by demonstrating that the single-copy construct YCp5O: :BamHI was able to complementsrbl::TRPI but not srbl-J (Fig. 1 and Table 3).The efficiency of lysis by strains carrying the srbl-J

mutation is directly proportional to the concentration of anosmotic stabilizer in the growth medium. The characteristicsof the disruption mutant can be regarded as an extremesrbl-J phenotype. The disruptant can grow in the absence ofany osmotic buffer without lysis but, if an osmotic buffer isincluded in the growth medium, it will lyse upon transfer tohypotonic conditions. The manner in which the disruptantacquires lysis ability is therefore important to our under-standing of the mode of action of the SRBI gene. A series ofexperiments, the results of which are presented in Table 5,were undertaken to define the requirements for the acquisi-tion of lysis ability. It was found that, while actively growingcells were capable of lysis, cells which were starved in thepresence of an osmotic stabilizer did not lyse. In contrast,when growth was inhibited by the addition of the proteinsynthesis inhibitor cycloheximide to osmotically bufferedmedium, there was a significant increase in the lysis ability ofdisruptant cells. We propose that budded cells are mostprone to lysis. Support for this idea came from experimentswhich demonstrated that osmotic shock followed by sonica-tion preferentially destroyed budded cells in populations ofboth the srbl-J mutant and the disruption mutants. Starva-tion leads to the arrest of cells in the unbudded state (9) andso protects them from lysis, whereas cycloheximide haltscells at all stages of the division cycle and permits lysis tooccur with high efficiency. It is likely that it is at the zone ofbud formation, where the cell wall is weakened for theincorporation of new material (1, 9), that holes are formedupon osmotic shock and lysis results. These data fit well witha previously reported observation that the lysis ability ofsrbl-J cells is dependent on the growth phase of the culture,with the mid-exponential phase being optimal for lysis (26).We have previously demonstrated the utility of srbl-J

strains for the production of high-protein yeast extracts foruse in human or animal nutrition (26). Both the cloned SRBJgene and the disruption mutant generated by use of thatcloned gene could have significant applications in biotech-nology. The complementation of srbl-J or similar mutationsby the cloned gene enables the establishment of very stablehost-vector combinations. This is because a self-selectingsystem is set up in which cells that lose the plasmid lyse andare not only removed from the population but also releasetheir biomass for the use of the remaining, plasmid-contain-ing cells. Excellent stability characteristics have been dem-onstrated in pilot experiments with SRBJ-containing vectorsand srbl-J hosts (unpublished results). The disruption mu-tant may be exploited in the commercial production of largemultisubunit protein complexes by recombinant yeast cells.Hepatitis B virus surface antigens are already produced on alarge scale with yeast cells (14), and it has been proposedthat the yeast Ty viruslike particles may be used as carriers

MOL. CELL. BIOL.

CLONING OF A YEAST OSMOTIC STABILITY DETERMINANT 4243

for antigens to other viruses, including human immunodefi-ciency virus (19). The srbl::TRPI disruption mutant couldbe exploited in such processes. The mutant may be grown ona large scale in the absence of an osmotic stabilizer, and thecells may be concentrated into a volume suitable for down-stream processing and then rapidly converted into a lysis-competent form by transfer to osmotically buffered condi-tions. Subsequent lysis would enable the easy recovery oflarge antigen-bearing complexes for vaccine preparation.

ACKNOWLEDGMENTS

We acknowledge the FEBS and the British Council for short-termfellowships to L.I.S. Patent protection has been sought for thisapproach under British patent application no. 9016031.

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