Vol. 144, No. 3JOURNAL OF BACTERIOLOGY, Dec. 1980, p. 1113-11180021-9193/80/12-1113/06$02.00/0

Isolation and Characterization of Temperature-Sensitive makMutants of Saccharomyces cerevisiaePATRICIA GUERRY-KOPECKOt* AND REED B. WICKNER

Laboratory ofBiochemical Pharnacology, National Institute ofArthritis, Metabolism, and DigestiveDiseases, Bethesda, Maryland 20205

The K1 killer plasmid of Saccharomyces cerevisiae is a 1.5-megadalton lineardouble-stranded ribonucleic acid molecule. Using simplified screening and com-plementation procedures, we have isolated mutants in three chromosomal genesthat are temperature sensitive for killer plasmid maintenance or replication. Oneof these genes, mak28-1, was located on chromosome X. Two of the temperature-sensitive mutants rapidly lost the wild-type killer plasmid of A364A during sporegermination and outgrowth at nonpermissive temperatures, but during vegetativegrowth, they only lowered the plasmid copy number. These two mutants did notlose two other wild-type K1 killer plasmids, indicating a heterogeneity of the killerplasmids in laboratory yeast strains.

Some strains ofSaccharomyces cerevisiae (K1killers) secrete a protein toxin which kills othersensitive strains. The genes coding for toxin pro-duction and resistance reside on a 1.5-megadal-ton linear double-stranded (ds) RNA moleculetermed either M1 or P2 (1, 2, 13, 17). A seconddsRNA molecule (L or P1) with a molecularweight of 3.0 x 106 is found in almost all Sac-charomyces strains whether they are killers ornot and codes for the protein coat in which bothM and L molecules reside intracellularly (8, 10).Both species of dsRNA are nonessential to

their host and segregate as plasmids (i.e., in anon-Mendelian fashion). There are, however, atleast 28 nuclear genes that are required for rep-lication or maintenance of the M dsRNA orkiller plasmid. These are designated makl, mak3through mak27 (14, 19, 21-23), petl8 (11), andspe2 (4). In addition, a pair of nuclear genes,kexl and kex2, are required for expression of thekiller phenotype (19, 23), and mutations in anyof four nuclear genes, skil through ski4, resultin overproduction of the killer toxin and bypassof the requirement for certain mak genes (15,16).The involvement of mak genes in the main-

tenance of the killer plasmid is not understood,and except for spe2, which encodes adenosyl-methionine decarboxylase (3), no mak geneproducts have been identified. In an effort tofurther study and control the interactions ofmak genes and the killer plasmid, we have iso-lated a series of temperature-sensitive (Ts) makmutants. In this communication we describe

t Present address: Genex Laboratories, Rockville, MD20852.

three such mak(Ts) mutants, at least one ofwhich represents a new mak gene. Differentwild-type killer plasmids show different require-ments for these mak genes.

MATERIALS AND METHODSYeast strains. Strains used in this study are listed

in Table 1.Media. Media were as described previously (24).

Buffered YPAD contained 0.05 M Tris, pH 7.4.Assay of killing activity. Killing activity was

assayed as described by Toh-e et al. (15).Mutagenesis. An overnight culture of A364A

grown at 300C in YPAD was treated with 4.3% ethylmethane sulfonate for 100 min at 20°C. This treatmentresulted in 20% survival based on colony-forming abil-ity.

Analysis of dsRNA. dsRNA was isolated as de-scribed previously (24). Agarose gel electrophoresiswas done as described by Toh-e et al. (15).

Genetic analysis. Genetic methods are those de-scribed by Mortimer and Hawthorne (12). Cytoductionwas performed as described by Conde and Fink (5).Genetic mapping was done by using the supertriploidmethod (22).

RESULTSIsolation of mak(Ts) mutants. Sixty-six

colonies which showed no killing zone (K-) orweak killing (KW) of a lawn of sensitive strain5X47 were isolated from ethyl methane sulfo-nate-treated A364A cells grown at 300C. Thesecells were screened to eliminate the followingother genotypes that produce the K- phenotype:(i) K- R+ plasmid or kex mutations-4 of the 66strains remained resistant (R+) to killing by thetoxin of wild-type killer strain M52, indicatingthat the nonkiller (K-) phenotype resulted from

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1114 GUERRY-KOPECKO AND WICKNER

a plasmid mutation or a kex mutation; (ii) sup-pressive plasmid mutations-strains in whichthe killer plasmid had become suppressive wouldhave a K- R- phenotype and, when crossed witha wild-type killer strain, would produce nonkillerdiploids; three such strains were found; (iii) dip-loid-dependent plasmid mutants (20)-all of theremaining strains, when crossed with wild-typenonkiller AN33, resulted in K- diploids, indicat-ing that none was a diploid-dependent plasmidmutation.The potential mak mutants were then tested

for their ability to maintain a wild-type killerplasmid introduced by cytoduction (i.e., hetero-karyon formation) (5). This screening method isconsiderably easier than that used previously(19, 23). All K- strains were made p- by growthon YPAD plus ethidium bromide and crossedwith PG18 a karl-1 leul K+ R+ at 300C. Duringcytoduction any plasmids present in either strainare transferred to the other via cytoplasmic mix-ing, but because nuclear fusion is rare in crossesinvolving karl-I mutants, each haploid parentalstrain can be recovered. In the above cross,leucine prototrophs which had become respira-tory competent by virtue of receiving the mito-chondrial genome from PG18 were grown at300C and tested for killing activity at 200C. Suchscreening narrowed the number ofpotential makmutants to 20. These 20 strains were thencrossed with the killer strain M52. The resultingtetrads were dissected, and the spores were ger-minated at 300C (the nonpermissive tempera-

ture). Seventeen strains showed 2 K+:2 K- seg-regation of killing activity, as expected for typi-cal mak mutants. When spores from thesecrosses were germinated at 200C (the permissivetemperature), three strains showed 4 K+:0 K-segregation, indicating that these strains hadtemperature-sensitive mutations affecting killerplasmid maintenance (Table 2). These three mu-tants are called tsl 7, ts42, and ts6O.Analysis ofdsRNA. All previously described

mak mutants affect maintenance of the MdsRNA species, but not that of the L species.Similarly, analyses ofdsRNA preparations made

TABLE 2. Meiotic segregation ofmak(Ts) mutant8aDiploid Segregation of kIillingchromo-

somal geno- 200C 3"type

tsl7/+ (16) 4 K+:O K- (34) 2 K+:2 K-(1) 0 K+:4 K-(1) 3 K+:1 K-

ts42/+ (15) 4 K+:O K- (19) 2 K+:2 K-(1) 3 K+:1 K- (1) 0 K+:4 K-(1) 2 K+.2 K-

t860/+ (9) 4 K+:O K- (28) 2 K+:2 K-a The numbers in parentheses represent the number

of tetrads showing the indicated segregation. Themak+ K+ R+ strains used in these crosses were eitherA364A or M52. Strain M52 carries the same K, killerplasmid as strain A364A.

TABLE 1. Straiss useda

Genotype

a adel ade2 lys2 tyrl his7 gall ural [KI-k,]

a thrl arg [KIL-o]a/a hisl/+ trpl/+ ura3/+ [KIL-o]a leu2-1 met5 [KIL-k,]a lysl [KIL-k,]a thrl [KIL-k,]a karl-i leul [KIL-k,]a ural adel thrl [KIL-k,]a ural ade2 tsl7 [KILR-k]a ural thr4 tsl 7 [KILR-k]a thr4 his7 ural tyrl ts42 [KIL-k,]a thr4 his7 ural tyrl ts42 [KIL-k,]a his7 ural adel ts6O [KIL-k,]a thr4 his7 ural tyrl ts6O [KIL-k]a metl trp3 tsl 7 [KIL-o]a tyrl ts42 [KIL-o]a thr4 his7 ural [KIL-o]a/a cdcll/+ ura2/+ adel/+ trpl/+ lys2/+met5/+ leu2/+ tsl7/+ [KIL-k,]

Source or reference

Hartwell et al. (8)

S. HenryR. B. WicknerR. K. MortimerF, ShermanWickner and Leibowitz (23)This workThis workThis workThis workThis workThis workThis workThis workThis workThis workThis workThis work

Desina-tion

A364A

AN335X47S3718M52PG1839-9BPG137PG139PG141PG160PG145PG150PG174PG132PG131PG300

Killer pheno-type

Ki+ Ri+

K- R-K- R-Ki+ Rl+K,+ Ri+Ki+ Ri+Ki+ R,+K,+ R,+Ki+ R,+Ki+ Ri+Ki+ Ri+Ki+ RD+Ki+ RD+K,+ Ri+K- R-K- R-

K,- R,-

Ki+ Ri+

'Phenotypes of strains with respect to K, killing ability (KM) and resistance (RM) to killing are denoted Ki+ R,+and K- R-. In this work K+ means K,+. The genotype of the killer plasmid is denoted by [KILR-k] for wild-typeK, killers or by [KIL-o] for wild type sensitive (carrying no plasmid).

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mak(Ts) MUTANTS OF S. CEREVISIAE 1115

from the three mak(Ts) mutants showed that grown at the permissive temperature (Fig. 1,only the M species is affected by temperature lanes 5-8). When such strains were shifted toshifts (Fig. 1). The M dsRNA was lacking in the nonpermissive temperature and grown for 9dsRNA preparations from mak(Ts) strains to 10 generations, the M dsRNA band was faintwhich had been germinated at the nonpermis- but not substantially fainter than in strains ger-sive temperature (Fig. 1, lanes 1-4). In contrast, minated and grown at the permissive tempera-the killer plasmid was present in dsRNA prep- ture. When the mutant tsl 7, germinated at thearations from mak(Ts) strains germinated and permissive temperature, was grown for 20 gen-

FIG. 1. Agarose gel electrophoresis ofdsRNA from wild-type kiler and mak(Ts) strains. Cells were grownin 100 ml of YPAD broth at the indicated temperature for 9 to 10 generations, except for (C), lanes 12 and 13,where growth was for 20 generations. dsRNA was extracted as described previously (24). The positions of Land M dsRNA are indicated. For the mak(Ts) mutants, the initial germination temperature as well as thetemperature at which the cells were vegetatively grown for dsRNA are indicated. (A) (Lane 1) A364A, cellsgrown at 30°C; (2) tsl7, spore germinated at 30°C, and cells grown at 30°C; (3) ts42, spore germinated at30°C, and cells grown at 30°C; (4) ts6O, spore germinated at 30°C, and cells grown at 30°C. (B) (Lane 5)A364A, cells grown at 30°C; (6) tsl7, spore germinated at 20°C, and cells grown at 20°C; (7) ts42, sporegerminated at 20°C, and cells grown at 20°C; (8) ts6O, spore germinated at 20°C, and cells grown at 20°C;(9) ts17, spore germinated at 20°C, and cells grown at 30°C (10 generations); (10) ts42, spore germinated at20°C, and cells grown at 30°C; (11) ts60, spore germinated at 20°C, and cells grown at 30°C. (C) (Lane 12)tsl 7, spore germinated at 20°C, and cells grown at 20°C; (13) tsl 7, spore germinated at 20°C, and cells grownat 30°C (20 generations).

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1116 GUERRY-KOPECKO AND WICKNER

erations at the nonpermissive temperature, MdsRNA was completely gone.Kinetics of loss of killing activity.

mak(Ts) mutants carrying the killer plasmidwere shifted from 20 to 340C in buffered YPADbroth, and the proportion ofK+ colonies remain-ing after various periods of growth was deter-mined. Figure 2 shows that the tsl7 mutantunderwent a slow, but clear-cut loss of killingactivity, with 50% of the cells becoming nonkill-ers after approximately nine generations. Lessthan 1% of the cells showed killing after 20generations at 340C. A control mak+ strain, 39-9B, remained 100% K+ after over 25 generationsunder the same growth conditions.

In contrast, tM42 and ts6O strains carrying thekiller plasmid showed much slower loss of kill-ing; more than 80% of ts42 cells remained K+after 30 generations at 340C and 96% of ts6O cellswere K+ after 26 generations at 340C (see Fig.2). This result is consistent with the dsRNAanalysis (Fig. 1), which shows little, if any, de-crease of M dsRNA in ts42 or ts6O strains after9 to 10 generations at the nonpermissive tem-perature.Complementation tests. Complementation

testing was accomplished by crossing tsl7 K+strains (grown at 2000) with strains carryingeach of the known mak mutations and assayingkilling activity in the resultant diploids aftergrowth at the nonpermissive temperature. All27 mak genes complemented tsl 7, suggestingthat tsl 7 is a new mak gene.

100 A 0 mak'~~~~~~~~~~ts6O

ts42

Cr_\-J.\

I50

L-OI\

0

75 10 15 20 25 30

NUMBER OF CELL GENERATIONSFIG. 2. Kinetics of loss of killing activity. Strains

carrying the killer plasmid were shfited from 20 to34°C in buffered YPAD broth. At various times sam-ples were diluted and plated to YPAD at 20°C, andthe proportion of colonies remaining K+ was deter-mined.

As shown in Fig. 2 and discussed above, oncethe killer plasmid is established in a ts42 or ts60haploid, it is difficult to lose even after prolongedvegetative growth at the nonpermissive temper-ature. Similarly, homozygous K+ diploids of thets42 or the ts6O strain remained K+ even afterrepeated subculturing at 300C. This inability todemonstrate K- homozygous diploids of the ts42or the ts6O strain at 300C precluded testing forcomplementation as used for the tsl 7 mutant. Itshould be noted, however, that meiotic segrega-tion of such homozygous K+ diploids showed 0K+:4 K- segregation when the spores were ger-minated at 300C. Examination of meiotic segre-gants ofa cross between the ts42 and ts6O strainsindicates that they are, despite their phenotypicsimilarities, mutations in distinct genes (Table3). In principle, such allelism tests could be donewith the ts42 or the ts6O strain and any of themak mutants.Plasmid specificity of ts42 and ts6O

strains. Early in the analysis of these mak(Ts)mutants, it was observed that ts42 and ts6Ostrains did not always show the expected 2 K+:2K- meiotic segregation when crossed with killerstrain 18 or S37 at the nonpermissive tempera-ture (see Table 4). This effect could be due to a

TABLE 3. Complementation testing of ts42 and ts6Ostrains at the nonpermissive temperaturea

Diploid chromosomal Segregationgenotype

ts60/ts6l (21) 0 K+:4 K-

ts42/ts42 (10) 0 K+:4 K-

ts42/ts6O (4) 2 K+:2 K-(4) 1 K+:3 K-(1) 3 K+:1 K-(1) 0 K+:4 K-

a Crosses between a mak (Ts) K+ strain and amak(Ts) K- strain were performed at the permissivetemperature; spore germination was at the nonpermis-sive temperature. The numbers in parentheses repre-sent the number of tetrads showing the indicatedsegregation.

TABLE 4. Meiotic segregation ofmak(Ts) mutantscrossed with killer strains 18 and S37

No. of tetrads with segregation pat-Cross tern:

4K+:OK- 3K+:1K_ 2K+:2K-18 x tsl7K- 0 2 1318 x ts42K- 16 2 118 x ts60K- 7 2 1S37 x tsl7K- 0 0 12S37 x ts42K- 8 1 5S37 x ts6OK- 10 1 3

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mak(Ts) MUTANTS OF S. CEREVISIAE 1117

suppressor in S37 and 18 which is lacking inA364A, or to a difference in the plasmids in thestrains such that the killers from S37 and 18 arenot dependent (or not as dependent) on the geneproducts of ts42 and ts6O strains as is the killerplasmid from A364A. To distinguish betweenthese possibilities, the following experiment wasconducted. Strains S37 and 18 were cured oftheir killer plasmid by growth at 370C (18) andcrossed with ts42 and ts6O strains harboring thekiller plasmid from A364A. Killing in thesecrosses segregated mainly 2 K+:2 K-. Such ex-periments, as summarized for the ts6O strain inTable 5, indicate that there is a distinction inthe ability of these plasmids to be maintained inthe ts42 or ts6O background.Mapping of the tsl 7 mutant. The tsl 7 mu-

tant was found to be on chromosome X, usingthe supertriploid mapping method of Wickner(22). This position was verified by examiningmitotic recombinants of PG300: of 9 cdcll mi-totic recombinants, none had become tsl 7; how-ever, 4 of 21 ura2 mitotic recombinants werealso tsM7. There is no linkage of tsl7 to SUP7(PD:NPD:T = 0:8:13), nor is tsl7 centromerelinked. Thus, the tsl7 mutant is located betweenSUP7 and ura2 on chromosome X.

DISCUSSIONAt least 28 chromosomal genes have been

described which affect the maintenance of thekiller plasmid in S. cerevisiae. At least one ofthe three mak(Ts) mutants described here, tsl7,represents a new mak gene, mak28-1, based oncomplementation testing and mapping data.

All three ofthese mak(Ts) mutants are typicalof previously described mak genes in severalways. Analysis of dsRNA in the cells shows thatonly the maintenance of the M dsRNA is af-fected. The inability to maintain the killer plas-

TABLE 5. Segregation of different killer plasmids inthe ts60 strain at the nonpermissive temperaturea

No. of tetrads withSource of segregation pattern:

Cross killerplasmid 4 K+: 3 K+: 2 K+:

0K- 1K- 2K-

S37 K- x 145 ts6OK+ A364A 0 2 20S37 K+ x 145 ts60K- S37 10 1 318 K- x 150 ts6OK+ A364A 0 1 918 K+ x 150 ts60K- 18 3 4 3

a Strains S37 and 18 were cured of their plasmidsby growth at 37°C and crossed with strain ts6O har-boring the plasmid from A364A. The control crossesinvolve wild-type killer strains S37 and 18 crossed withthe corresponding strain of ts60 which has lost theA364A plasmid by virtue of growth at the nonpermis-sive temperature.

mid in the tsl7, ts42, and ts6O strains can bebypassed by the presence of any of the four ski(superkiller) genes (data not presented).Two of these mak mutants, ts42 and ts6O, are

unusual, however. In crosses with these mutants,the killer plasmid segregates meiotically 2 K+:2K- at 300C and 4 K+:0 K- at 200C. But if atetrad germinated at 200C is restreaked at 300C,nonkillers segregate from two of the four sporesonly very gradually. If a ts42 or ts6O K+ cell isgrown at nonpermissive temperatures in broth,the loss of killing is extremely slight. Thoughthe strains tend to remain killers at 300C oncethe plasmid has established itself in the strain,very often the intensity of the zone in the killingassay can be observed to decrease gradually asthe cells are grown at the nonpermissive tem-perature. There may be a decrease in the num-ber of copies of the plasmid in ts6O K+ or ts42K+ cells vegetatively growing at 300C. Whensuch cells are plated at 200C to assay for killingactivity, the constraints of the mutations arereleased, and any cell with even a single copy ofthe killer plasmid at the time of plating will stillappear K+. Thus, it seems that the primarydefect in plasmid maintenance in these mutantsis during sporulation or meiosis rather than dur-ing vegetative growth. It has not yet been deter-mined whether any of the previously describednonconditional mak mutants are of this class.The other unusual feature of ts42 and ts6O

strains is that they seem to be mak mutants foronly one particular K1 killer plasmid, that fromstrain A364A. The plamids from two other wild-type strains, 18 and S37, are maintained in thepresence of ts42 and ts6O strains, albeit not per-fectly, much more readily than is that fromA364A. Although the plasmids are superficiallyidentical, these data suggest that some funda-mental differences exist among wild-type K1killer plasmids. The killer plasmid in anotherlaboratory strain, P28-24C, was shown to beindependent of the mak4, mak7, makll, andmakl7 genes and conferred the superkiller phe-notype (16).

LITERATURE CITED1. Bevan, E. A., A. J. Herring, and D. J. Mitchell. 1973.

Preliminary characterization of two species of ds RNAin yeast and their relationship to the "killer" character.Nature (London) 245:81-86.

2. Bostian, K. A., J. E. Hopper, D. T. Rogers, and D. J.Tipper. 1980. Translational analysis of the ds RNAgenome killer-associated virus-like particle of Saccha-romyces cerevisiae: M ds RNA encodes toxin. Cell 19:403-414.

3. Cohn, M. S., C. W. Tabor, and H. Tabor. 1978. Isolationand characterization of Saccharomyces cerevisiae mu-tants deficient in S-adenosylmethionine decarboxylase,spermidine, and spermine. J. Bacteriol. 134:208-213.

4. Cohn, M. S., C. W. Tabor, H. Tabor, and R. B. Wick-

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5. Conde, J., and G. R. Fink. 1976. A mutant of Saccha-romyces cerevisiae defective for nuclear fusion. Proc.Natl. Acad. Sci. U.S.A. 73:3651-3655.

6. Fried, H. M., and G. R. Fink. 1978. Electron microscopicheteroduplex analysis of "killer" double-stranded RNAspecies from yeast. Proc. Natl. Acad. Sci. U.S.A. 75:4224-4228.

7. Harris, M. S. 1978. Virus-like particles and double-stranded RNA from killer and nonkiller strains of Sac-charomyces cerevisiae. Microbios 21:161-176.

8. Hartwell, L H., J. Culotti, and B. Reid. 1970. Geneticcontrol of the cell-division cycle in yeast. I. Detectionof mutants. Proc. Natl. Acad. Sci. U.S.A. 66:352-359.

9. Herring, A. J., and E. A. Bevan. 1974. Virus-like par-ticles associated with the double-stranded RNA speciesfound in killer and sensitive strains ofthe yeast Saccha-romyces cerevisiae. J. Gen. Virol. 22:387-394.

10. Hopper, J. E., K. Bostian, L B. Rowe, and D. J.Tipper. 1977. Translation of the L-species dsRNA ge-nome of the killer-associated virus-like particles of Sac-charomyces cerevisiae. J. Biol. Chem. 252:9010-9017.

11. Leibowitz, M. J., and R. B. Wickner. 1978. petl8: achromosomal gene required for cell growth and formaintenance of mitochondrial DNA and the killer plas-mid of yeast. Mol. Gen. Genet. 165:115-121.

12. Mortimer, R. K., and D. C. Hawthorne. 1975. Geneticmapping in yeast. Methods Cell Biol. 11:221-223.

13. Palfree, R., and H. Bussey. 1979. Yeast killer toxin:purification and characterization of the protein toxinfrom Saccharomyces cerevisiae. Eur. J. Biochem. 93:487493.

14. Somers, J. M., and E. A. Bevan. 1968. The inheritanceof the killer character in yeast. Genet. Res. 13:71-83.

15. Toh-e, A., P. Guerry, and R B. Wickner. 1978. Chro-mosomal superkiller mutants of Saccharomyces cere-visiae. J. Bacteriol. 136:1002-1007.

16. Toh-e, A., and R. B. Wickner. 1980. "Superkiller" mu-tations suppress chromosomal mutations affecting dou-ble-stranded RNA killer plasmid replication in Saccha-romyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 77:527-530.

17. Vodkin, M., F. Katterman, and G. R. Fink. 1974. Yeastkiller mutants with altered double-stranded ribonucleicacid. J. Bacteriol. 117:681-686.

18. Wickner, R. B. 1974. "Killer character" of Saccharomy-ces cerevisiae: curing by growth at elevated tempera-tures. J. Bacteriol. 117:1356-1357.

19. Wickner, R. B. 1974. Chromosomal and nonchromosomalmutations affecting the "killer character" of Saccharo-myces cerevisiae. Genetics 76:423432.

20. Wickner, R. B. 1976. Mutants of the killer plasmid ofSaccharomyces cerevisiae dependent on chromosomaldiploidy for expression and maintenance. Genetics 82:273-285.

21. Wickner, R. B. 1978. Twenty-six chromosomal genesneeded to maintain the killer double-stranded RNAplasmid ofSaccharomyces cerevisiae. Genetics 88:419-425.

22. Wickner, R. B. 1979. Mapping chromosomal genes ofSaccharomyces cerevisiae using an improved geneticmapping method. Genetics 92:803-821.

23. Wickner, R. B., and M. J. Leibowitz. 1976. Chromo-somal genes essential for replication of a double-stranded RNA plasmid of Saccharomyces cerevisiae:the killer character of yeast. J. Mol. Biol. 105:427-443.

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