JOURNAL OF BACTERIOLOGY, Dec. 1978, P. 1002-1007 0021-9193/78/0136-1002$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 136, No. 3 Printed in U.S.A.
Chromosomal Superkiller Mutants of Saccharomyces cerevisiae AKIO TOH-E,* PATRICIA GUERRY, AND REED B. WICKNER
Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Maryland 20014 Received for publication 21 September 1978
Yeast strains carrying a 1.5 x 106-dalton double-stranded RNA in virus-like particles secrete a protein toxin which is lethal to strains not carrying this species of double-stranded RNA. We find that recessive mutations in any of four chromosomal genes result in the superkiller phenotype, i.e. increased secretion of killer toxin activity by strains carrying the killer genome. These genes are designated skil through ski4 (for superkiller). ski3 and ski4 are located on chromosome XIV, and skil is on chromosome VII. A skil mutation results in a decreased rate of cell growth. The kexl and kex2 mutations are epistatic to each ski mutation.
Some strains of yeast (killers) kill other strains (sensitives) by secreting a protein toxin (3, 5, 17, 25). The killer phenomenon shows both Mendelian and non-Medelian inheritance (3, 17). A 1.5 x 106-dalton linear double-stranded (ds) RNA (M or P2) encapsulated in virus-like particles is believed to be the killer genome (1, 2, 11, 18, 19). Most laboratory strains have another kind of ds RNA (molecular weight, 3.0 x 106; L or P1 ds RNA [2, 19]) which codes for the coat protein (12) in which this L ds RNA is encapsulated (1, 4, 11). L may be unrelated to the killer phenomenon (for a review, see reference 21). At least 27 nuclear genes have been shown to be necessary for the replication or maintenance of the killer genome. They are designated petl8 (15; M. J. Leibowitz and R. B. Wickner, Mol. Gen. Genet., in press), makl through mak28 (17, 20, 22, 23; unpublished data), and spe2 (6). Two nuclear genes, kexl and kex2, are necessary for the expression of the killer phenotype (24). Because the killer toxin is thermolabile (25), wild-type killers show very little killing activity at 300C and are normally tested at 200C. We isolated mutants which showed an enhanced killer activity at 300C. These mutants, designated ski (super killer), each have a recessive mutation in a nuclear gene, whereas the superkiller reported by Vodkin et al. (19) resulted from a cytoplasmic mutation. In this communication, we report the identification of four ski genes. We have mapped skil, ski3, and ski4.
MATERIALS AND METHODS Yeast strains. Strains used in this study are listed in Table 1.
Media. YPAD, SD, YPG, presporulation medium, sporulation medium, complete minimal medium, and omission media were described previously (24). Agar (final concentration, 2%) was added to make solid media. Neutralized YPAD was prepared by adding 3 ml of 1 M potassium phosphate (pH 7.5) per 100 ml of YPAD (final pH, approximately 7.0). Assay of killing activity. Killing activity was assayed essentially as described by Somers and Bevan (17). Colonies grown on rich medium (YPAD) were replica plated onto a lawn of the sensitive strain 5 x 47 [KIL-o] on the same medium containing methylene blue and buffered at pH 4.7 with sodium citrate (MB medium) (24). Killing was indicated by a clear zone surrounding the killer strain, surrounded in turn by growth of the lawn. Isolation of ds RNA. The isolation scheme was essentially the same as that described previously (24). In brief, cells grown in modified YPAD (glucose concentration increased to 10%) to stationary phase were converted to spheroplasts by Zymolyase (13). After Pronase or proteinase K digestion, nucleic acids were extracted by phenol. The ds RNAs were separated from bulk RNA and DNA by CF-li cellulose column chromatography (9). To the ds RNA fraction eluted from the column was added 2 volumes of ethanol. The precipitate was dissolved in an appropriate buffer and subjected to further analysis. Mutagenesis. A364A cells grown for 1 day in YPAD at 30°C were treated with ethyl methane sulfonate by the method of Lindegren et al. (14). Ethyl methane sulfonate treatment was performed at 20°C for 45 min. (Survival was about 50% on the basis of colony-forming ability.) Agarose gel electrophoresis. A 1% agarose slab gel (13 by 11 by 0.3 cm) was prepared in 0.04 M tris (hydroxymethyl)aminomethane-acetate buffer (pH 7.6) containing 1 mM ethylenediaminetetraacetic acid. Electrophoresis was done under constant voltage (6.4 V/cm) for about 4 h. The current was about 40 mA. Electron microscopy. The ds RNA was prepared
1002
TABLE 1. Strainsa Designation
Killer pheno-
Genotype
type
A364A
K+ R+
a
adel ade2 tyrl Iys2 ural his7 gall
AN33
K- R-
a
[KIL-k] argl thrl [KIL-o]
5X47
K- R-
a his
tp
+
+
a
B55 AT95 AT94 95 80 AT17 AT18 AT19 AT20 AT21 AT22 AT23 AT25 AT26 AT27 AT28 AT202 AT257 1068
1003 the superkiller phenotype occurs in a chromosomal gene. Complementation tests among these mutants revealed four distinct genes, which are designated skil, ski2, ski3, and ski4. Diploids were constructed which were heterozygous for two ski mutations in different complementation groups. Meiotic analysis of these diploids showed no close linkage among the ski genes and confirmed the assignment of mutants to genes by the complementation tests. The superkiller phenotype of these mutants is shown in Fig. 1. The skil-1 mutant grows slowly on YPAD, but it shows neither temperature sensitivity nor respiratory deficiency. When a heterozygous was sporulated and tetrads were diploid, dissected, the superkiller phenotype always coincided with the slow growth character. These two phenotypes, however, could have resulted from two closely linked mutations. kex mutations are epistatic to ski mutations; mapping of skil, ski3, and ski4. Two nuclear genes, kexl and kex2, are required for toxin synthesis (24). The ski mutations, which give rise to overproduction of toxin activity, might be altered in some stage(s) of toxin synthesis. To study the phenotype of double mutants containing one ski and one kex mutation, each ski strain was crossed with a kexl or kex2 strain, and diploids were analyzed meiotically. Killing activity was scored at 30°C so that three phenotypes (kex, ski, and wild type) could be distinguished (Table 2). Two spore clones in every ascus were K-, and the rest were wild-type killers or superkillers or one wild-type killer and one superkiller. This segregation pattern indicates that double mutants, containing both ski and kex, show the K- phenotype of the kex mutation. Thus, superkiller strains continue to depend on the kex gene products for toxin production. Analysis of the crosses in Table 2 revealed that ski4 is very closely linked to kex2 (chromosome XIV); ski3 is loosely linked to kex2; and skil showed possible linkage to kexl (see below). Although ski4 and kex2 are closely linked, they are different genes because they complement; ski4 + + ke2 diploids are wild-type killers. Linkage could not be detected between ski3 and Iys9, rna2, or pet8 (genes linked in meiosis to the centromere of chromosome XIV [15]). Five Iys mitotic recombinants from a-+ SUPERKILLER MUTANTS OF YEAST
VOL. 136, 1978
+
ura3
[KIL-o]
K++ R+ K++ R+ K++ R+ K- R+ K- R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K+ R+
A364A ski2-1 [KIL-k] a his7 skil-) [KIL-k] a adel skil-) [KIL-k] a argl thrl kexl-l [KIL-k] a ade2 ural kex2-1 [KIL-k] a lys2 tyrl his7 ski2-1 [KIL-k] a ade2 thrl ski2-1 [KIL-k] a ade2 tyrl his7 ski3-1 [KRL-k] a ade2 argl ski3-1 [KIR-k] a thrl argl ski4-1 [KIL-k] a his7 adel (or ade2) ski4-1 [KIL-k] a adel (or ade2) his7 ski3-2 [KRL-k] a ade2 thrl argl tyrl ski2-2 [KIL-k] a adel (or ade2) his7 ski2-2 [KIL-k] a tyrl ski3-3 [KIL-k] a adel thrl argl ski3-3 [KIL-k] a lys2 his7 ski2-3 [KIL-k] a his7 skil-) [KIL-k] a hisl metl3 leul trp5 aro2 lys5 ade5 chy2 [KIL-k] a Phenotypes of strains with regard to their killing ability (K) and resistance (R) to killing are denoted K+ R+, K- R+, K+ R-, or K- R-. Superkillers are denoted K+. The genotype of the killer plasmid is noted by [KIL-k] (wild-type killer plasmid) and [KILr-o] (no killer plasmid).
for electron microscopy by the aqueous method of Davis et al. (8) and examined in a Phillips 300 electron microscope. A minimum of 100 ds RNA molecules from each strain were measured by using a Neumonics Digitizer. The measurements were calibrated by reference to an external standard of open circular simian virus 40 DNA. Genetic analysis. Genetic methods were as described previously (see Literature Cited in reference 15). Cytoduction was carried out as described by Conde and Fink (7). RESULTS
Isolation of ski mutants. Eight mutants which showed an increased killing zone (K++) at 300C were isolated from ethyl methane sulfonate-treated A364A cells. Each mutant killed only strains sensitive to the wild-type toxin. None of these mutants had acquired ability to kill wild-type killer strains. Also, each mutant showed greater killing ability than the wild type when tested at 200C. Each mutant was crossed with a wild-type nonkiller (AN33). Because all resulting diploids showed the same killing activity as that of wild type, the mutations causing the superkiller phenotype are recessive. When diploids from each cross were dissected, essentially all tetrads (7 to 13) showed 2 K++: 2 K+ segregation. Thus, each mutation responsible for
+il-,
diploid were isolated. None of these were superkillers, indicating that the fragment of chromosome XIV carrying ski3, ski4, kex2, and
1004
TOH-E, GUERRY, AND WICKNER
J. BACTERIOL. w
*..: ,''''.'..'.'.: 'S ..
w.
sk 3 ....... ~ ~ ~ ~ ~ ~ . ::.:...~ ~
.... ......
FIG. 1. Superkiller phenotype. A cell suspension of each strain was spotted onto MB medium previously spread with a lawn of a sensitive strain (5 x 47) and incubated at 30°C for 2 days. Top row, from left: wildtype kiler, wild-type nonkiller. Second row, from left: ski2-2, ski2-1, ski2-3. Third row, from left: ski3-3, ski32, ski3-1. Fourth row: ski4-1. Fifth row: skil-1.
TABLE 2. kex and ski mutations: linkage and epistasis No. with the following tetrad type:"
Cross kexl-l (95) x skil-I (AT94)
x ski2-1 (AT17)) x ski2-2 (AT26) x ski2-3 (AT15)
K+K:1 2 K+ 1:2 K(T) (PD)
2 K+:2 K-
(NPD)
6
4
1
12
27
6
petx is on the opposite side of the centromere from Iys9 (Fig. 2). The location of skil was defined further. Linkage data between genes on chromosome VII are listed in Table 3. The crossover patterns in individual tetrads were also analyzed to determine the arrangement of skil, aro2, and lys5 (Table 4). These results indicate that skil is located between kexl and lys5 (12.5 min from Iys5). The map positions of skil, ski3, and ski4 are shown in Fig. 2. ski mutants still depend on the killer
plasmid for killing ability. skil, ski2, ski3, and ski4 strains were grown at 37°C in rich a treatment shown to cure the killer medium, 1 5 x ski4-1 (AT22) 0 from wild-type strains. This treatment plasmid kex2-1 (80) induced a high frequency of nonkillers in each 2 6 3 x skil-I (AT95) strain, as expected if the ski mutants, like wildx Aki2-I (AT18) 6 21 4 type killers, depend on the killer plasmid for 4 x ski2-2 (AT25) killing ability. This was further tested in the case of ski2-2. The killer plasmid was reintroduced x ski3-1 (AT20) 8 48 34 x ski3-3 (AT28)J into a ski2-2 heat-cured K- R- p- strain by cytoduction (cytoplasmic mixing without nux ski4-1 (AT21) 2 0 97 clear fusion [7]) from a karl K+ R+ p+ strain. a PD, Parental ditype ascus; T, tetratype ascus; NPD, non- The cytoductants (judged by aquisition of p+) parental ditype ascus. Strains which are kex- ski+ [KIL-k] were superkillers. have the K- R+ phenotype (20). Each kex and ski mutation Analysis of ds RNA from ski mutants. In segregates 2+:2- in all crosses. The K- segregants must be kex-, since kex+ ski' and kex+ ski- segregants are always K+ wild-type strains, the weight ratio of M to L ds and K++, respectively. Tetrads that are 2 K++:2 K- must be 2 RNAs is about 1:10 (2, 19, 23), and the content kex+ ski-:2 kex- ski', all spores being of two types (ditype) in of M ds RNA is about 3 nmol of nucleotide per the parental configuration. Tetrads that are 2 K+:2 K- must g of cells (wet weight) (19, 23). These values are be 2 kex+ ski+:2 kex- ski-. The fact that kex- ski- segregants are K- (the kex phenotype) is expressed by saying kex is relatively constant from strain to strain. ds RNA was extracted from stationary cultures of wildepistatic to ski. x ski3-1 (AT19) x ski3-2 (AT23) J
7
24
9
SUPERKILLER MUTANTS OF YEAST
VOL. 136, 1978
1005
TABLE 3. Linkage of skil to Iys5 on chromosome viia
4lo4
Gene Gene
to07 41
skil PD NPD T Distances (cM)
\l-
lys5
aro2
metl3
cyh2
59 1 12 12.5
53 1 22 18.5
34 0 29 22
46 6 115 45
Iys5 PD NPD T Distances (cM)
I I I
kexl 32 41 Distances (cM) 'For each pair of genes, the number of parental ditype (PD; no crossovers), nonparental ditype (NPD; two crossovers), and tetratype (T; one crossover) asci observed is given. Except for the segregation of Akil relative to cyh2, the data come from a single cross: W205 (1068 x AT95). The distances shown for kexl for comparison are from Wickner and Leibowitz (24). Distances are calculated in centimorgans (cM) by the method of Perkins (16).
-o
t
0
(NY
4--q
4Nv
4,)e
66 0 8 5
TABLE 4. Gene order of skil, Iys5, and aro2a Crossovers needed for _
the following gene or-
0
cn
0
Type of tetrad
No. of asci observed
der:
skil- lys5- skilIys5-
,e. \\0Q -.-
e9, -e 10
x
-5
K.
Zb
ski)- aro2-
aro2 Iys5 1 1 0 1. aro2 and skil PD; Iys5 T with aro2 and skil 1 1 12 2 2. aro2 and Iys5 PD; skil T with aro2 and Iys5 2 7 1 1 3. Iys5 and skil PD; aro2 T with Iys5 and skil a The data (no. of asci observed) was obtained by examination of the same tetrads used in Table 3 to demonstrate linkage. Tetrads were selected in which two of the three markers were in the parental ditype (PD; nonrecombinant) configuration with each other, but each of these two was in the tetratype (T; single crossover) configuration with the third marker. If the third marker is the middle marker on the genetic map, this requires two crossovers (presumably a rare event in this short interval). If the third marker is on one end, one crossover will give this result. The type of tetrad which was not observed (type 1) must represent that segregation of markers requiring a double crossover. A type 1 tetrad requires a double crossover if the gene order is skil-lys5-aro2. Thus, the gene order is skil-lys5-aro2.
aro2 2
_
I
0
0
type and ski mutants as described above and equal amounts of ds RNA from each strain were analyzed by agarose slab gel electrophoresis. The ds RNA bands were visualized by the fluorescence emitted from the ds RNA-ethidium bromide complex under UV light (Fig. 3). As shown in Fig. 3, ski2, ski3, and ski4 strains have more M ds RNA relative to L ds RNA than does wild type. The skil strain, however, has roughly the same ratio of M ds RNA to L ds RNA as the
1006
J. BACTERIOL.
TOH-E, GUERRY, AND WICKNER
TABLE 5. ds RNA in ski mutantsa Total ds LdosRNA Mds N RNA Strain (no/g (RunAo (nmol/g (uo of s cells) of cel of cells)
M
FIG. 3. ds RNA of ski mutants on agarose gel electrophoresis. ds RNA purified by CF-lI column chromatography was precipitated by ethanol and dissolved in a small amount of electrophoresis buffer. A 50-gd amount of each sample (absorbance at 260 nm, approximately 1) was applied to lanes 1 through 8. Lane 1, strain AT154 wild type; lane 2, strain AT15 ski2-3; lane 3, strain AT9 ski2-1; lane 4, strain AT17 ski2-1; lane 5, strain AT26 ski2-2; lane 6, strain AT27 ski3-3; lane 7, strain AT21 ski4-1; lane 8, strain AT30 skil-l.
wild type. The molecular weights of M ds RNA in ski2, ski3, and ski4 were the same as wild type. The skil strain has two bands close to each other in the M ds RNA region. The population of L and M ds RNA in some of the ds RNA preparations was analyzed by measuring contour lengths of ds RNA molecules by electron microscopy. Table 5 shows the amounts of M and L ds RNAs in various strains. The data for the wild-type killer are close to the published values. The results in Table 5 suggest an absolute and relative increase in M ds RNA in ski mutants. However, considerable variability of the results was observed. Further study of this point by other methods will be necessary.
DISCUSSION Four ski genes (skil through ski4) were found by looking for mutants which gave rise to a larger killing zone than wild type at 300C. The killing ability of these mutants is greater even at
I ML
0.10 2.5 25.5 28 Wild type A364A 0.32 4.9 15.1 20 Wild type A364A 0.39 6.5 16.5 23 skil-1 AT95 0.55 10.6 19.4 30 skil-1 AT257 1.16 58 50 108 ski2-1 B55 1.22 11 9.5 20.5 ski2-3 AT202 0.36 4 11 15 ski3-3 AT27 0.60 10 16.8 26.8 ski3-3 AT27 1.97 16.6 8.4 25 ski4-1 AT21 0.66 6.3 9.5 15.8 ski4-1 AT21 a ds RNA was isolated as described in the text. A total of 90 to 100% of the isolated material was ds based on pancreatic ribonuclease sensitivity in high and low salt. The ratio of M to L molecules was determined by electron microscopy (see text). These data, the total ds RNA recovered from each strain, and the known molecular weights of L and M, were used to calculate the values shown in the table.
a lower temperature (20 and 2500) than that of the wild type. All ski mutations described here are recessive mutations occurring in nuclear genes. A mutant showing a similar superkiller phenotype has been described by Vodkin et al. (19), which was located in a cytoplasmic genome. ski mutants remain dependent upon the killer plasmid for their killing phenotype. Since kex mutations are epistatic to ski mutations, ski mutants are also dependent upon the kex gene products for toxin production. The skil gene may be essential for growth since our lone mutant allele results in decreased growth rate. Since killer toxin is rapidly degraded at 300C (25), the superkiller phenotype in one or more of these mutants could result from a mutation which makes the toxin heat resistant. If these strains overproduce a normal toxin, this might be due to changes in toxin processing or transport, translation of toxin message, transcription of toxin genes, or copy number of M ds RNA. Further studies will be directed toward distinguishing these possibilities. ACKNOWLEDGMENTS We are grateful to R. K. Mortimner, S. Fogel, J. Bassel, and R. Contopoulos for sending us many strains essential for this work.
LITERATURE CITED 1. Adler, J., H. A. Wood, and R. F. Bozarth. 1976. Viruslike particles from killer, neutral, and sensitive strains
of Saccharomyces cerevisiae. J. Virol.
17:472-476.
2. Bevan, E. A., A. J. Herring, and D. J. Mitchell. 1973. Preliminary characterization of two species of ds RNA in yeast and their relationship to the "killer" character.
Nature (London) 245:81-86.
3. Bevan, E. A., and J. M. Somers. 1968. Somatic segregation of the killer (k) and neutral (n) cytoplasmic genetic determinants in yeast. Genet. Res. 14:71-77.
4. Buck, K. W., P. Lhoas, and B. K. Street. 1973. Virus
VOL. 136, 1978
particles in yeast. Biochem. Soc. Trans. 1:1141-1142. 5. Bussey, H. 1972. Effects of yeast killer factor on sensitive cells. Nature (London) New Biol. 236:73-75. 6. Cohn, M. S., C. W. Tabor, H. Tabor, and R. B. Wickner. 1978. Spermidine or spermine requirement for killer double-stranded RNA plasmid replication in yeast. J. Biol Chem. 253:5225-5227. 7. Conde, J., and G. R. Fink. 1976. A mutant of Saccharomyces cerevisiae defective for nuclear fusion. Proc. Natl. Acad. Sci. U. S. A. 73:3651-3655. 8. Davis, R. W., M. Simon, and N. Davidson. 1971. Electron microscope hetero-duplex methods for mapping regions of base sequence homology in nucleic acids. Methods Enzymol. 21:413-427. 9. Franklin, R. M. 1966. Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc. Natl. Acad. Sci. U. S. A. 56:1504-1511. 10. Hartwell, L. H., J. Culotti, and B. Reid. 1970. Genetic control of the cell division cycle in yeast. I. Detection of mutants. Proc. Natl. Acad. Sci. U. S. A. 66:352-359. 11. Herring, A. J., and E. A. Bevan. 1974. Virus-like particles associated with the double-stranded RNA species found in killer and sensitive strains of the yeast Saccharomyces cerevisiae. J. Gen. Virol. 22:387-394. 12. Hopper, J. E., K. A. Bostian, L B. Rowe, and D. J. Tipper. 1977. Translation of the L-species ds RNA genome of the killer-associated virus-like particles of Saccharomyces cerevisiae. J. Biol. Chem. 262:90109017. 13. Kaneko, T., K. Kitamura, and Y. Yamamoto. 1973. Susceptibilities of yeasts to yeast cell wall lytic enzyme of Arthrobacter luteus. Agric. Biol. Chem. 37: 2295-2302. 14. Lindegren, G., Y. L. Hwang, Y. Oshima, and C. C. Lindegren. 1965. Genetical mutants induced by ethylmethanesulfonate in Saccharomyces. Can. J. Genet. Cytol. 7:491-499.
SUPERKILLER MUTANTS OF YEAST
1007
15. Mortimer, R. K., and D. C. Hawthorne. 1975. Genetic mapping in yeast, p. 221-223. In D. M. Prescott (ed.), Methods in cell biology, vol. 11. Academic Press Inc., New York. 16. Perkins, D. D. 1949. Biochemical mutants of the smut fungus Ustilago maydis. Genetics 34:607-626. 17. Somers, J. M., and E. A. Bevan. 1968. The inheritance of the killer character in yeast. Genet. Res. 13:71-83. 18. Sweeney, T. K., A. Tate, and G. R. Fink. 1976. A study of the transmission and structure of double stranded RNAs associated with the killer phenomenon in Saccharomyces cerevisiae. Genetics 84:27-42. 19. Vodkin, M., F. Katterman, and G. R. Fink. 1974. Yeast killer mutants with altered double-stranded ribonucleic acid. J. Bacteriol. 117:681-686. 20. Wickner, R. B. 1974. Chromosomal and nonchromosomal mutations affecting the "killer character" of Saccharomyces cerevisias. Genetics 76:423-432. 21. Wickner, R. B. 1976. Killer of Saccharomyces cerevisiae: a double-stranded RNA plasmid. Bacteriol. Rev. 40: 757-773. 22. Wickner, R. B. 1978. Twenty-six chromosomal genes needed to maintain the killer double-stranded RNA plasmid of Saccharomyces cerevisias. Genetics 88: 419-425. 23. Wickner, R. B., and M. J. Leibowitz. 1976. Chromosomal genes essential for replication of a doublestranded RNA plasmid of Saccharomyces cerevisiae: the killer character of yeast. J. Mol. Biol. 105:427-443. 24. Wickner, R. B., and M. J. Leibowitz. 1976. Two chromosomal genes required for killing expression in killer strains of Saccharomyces cerevisiae. Genetics 82: 429-442. 25. Woods, D. R., and E. A. Bevan. 1968. Studies on the nature of the killer factor produced by Saccharomyces cerevisiae. J. Gen. Microbiol. 51:115-126.
Vol. 136, No. 3 Printed in U.S.A.
Chromosomal Superkiller Mutants of Saccharomyces cerevisiae AKIO TOH-E,* PATRICIA GUERRY, AND REED B. WICKNER
Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Maryland 20014 Received for publication 21 September 1978
Yeast strains carrying a 1.5 x 106-dalton double-stranded RNA in virus-like particles secrete a protein toxin which is lethal to strains not carrying this species of double-stranded RNA. We find that recessive mutations in any of four chromosomal genes result in the superkiller phenotype, i.e. increased secretion of killer toxin activity by strains carrying the killer genome. These genes are designated skil through ski4 (for superkiller). ski3 and ski4 are located on chromosome XIV, and skil is on chromosome VII. A skil mutation results in a decreased rate of cell growth. The kexl and kex2 mutations are epistatic to each ski mutation.
Some strains of yeast (killers) kill other strains (sensitives) by secreting a protein toxin (3, 5, 17, 25). The killer phenomenon shows both Mendelian and non-Medelian inheritance (3, 17). A 1.5 x 106-dalton linear double-stranded (ds) RNA (M or P2) encapsulated in virus-like particles is believed to be the killer genome (1, 2, 11, 18, 19). Most laboratory strains have another kind of ds RNA (molecular weight, 3.0 x 106; L or P1 ds RNA [2, 19]) which codes for the coat protein (12) in which this L ds RNA is encapsulated (1, 4, 11). L may be unrelated to the killer phenomenon (for a review, see reference 21). At least 27 nuclear genes have been shown to be necessary for the replication or maintenance of the killer genome. They are designated petl8 (15; M. J. Leibowitz and R. B. Wickner, Mol. Gen. Genet., in press), makl through mak28 (17, 20, 22, 23; unpublished data), and spe2 (6). Two nuclear genes, kexl and kex2, are necessary for the expression of the killer phenotype (24). Because the killer toxin is thermolabile (25), wild-type killers show very little killing activity at 300C and are normally tested at 200C. We isolated mutants which showed an enhanced killer activity at 300C. These mutants, designated ski (super killer), each have a recessive mutation in a nuclear gene, whereas the superkiller reported by Vodkin et al. (19) resulted from a cytoplasmic mutation. In this communication, we report the identification of four ski genes. We have mapped skil, ski3, and ski4.
MATERIALS AND METHODS Yeast strains. Strains used in this study are listed in Table 1.
Media. YPAD, SD, YPG, presporulation medium, sporulation medium, complete minimal medium, and omission media were described previously (24). Agar (final concentration, 2%) was added to make solid media. Neutralized YPAD was prepared by adding 3 ml of 1 M potassium phosphate (pH 7.5) per 100 ml of YPAD (final pH, approximately 7.0). Assay of killing activity. Killing activity was assayed essentially as described by Somers and Bevan (17). Colonies grown on rich medium (YPAD) were replica plated onto a lawn of the sensitive strain 5 x 47 [KIL-o] on the same medium containing methylene blue and buffered at pH 4.7 with sodium citrate (MB medium) (24). Killing was indicated by a clear zone surrounding the killer strain, surrounded in turn by growth of the lawn. Isolation of ds RNA. The isolation scheme was essentially the same as that described previously (24). In brief, cells grown in modified YPAD (glucose concentration increased to 10%) to stationary phase were converted to spheroplasts by Zymolyase (13). After Pronase or proteinase K digestion, nucleic acids were extracted by phenol. The ds RNAs were separated from bulk RNA and DNA by CF-li cellulose column chromatography (9). To the ds RNA fraction eluted from the column was added 2 volumes of ethanol. The precipitate was dissolved in an appropriate buffer and subjected to further analysis. Mutagenesis. A364A cells grown for 1 day in YPAD at 30°C were treated with ethyl methane sulfonate by the method of Lindegren et al. (14). Ethyl methane sulfonate treatment was performed at 20°C for 45 min. (Survival was about 50% on the basis of colony-forming ability.) Agarose gel electrophoresis. A 1% agarose slab gel (13 by 11 by 0.3 cm) was prepared in 0.04 M tris (hydroxymethyl)aminomethane-acetate buffer (pH 7.6) containing 1 mM ethylenediaminetetraacetic acid. Electrophoresis was done under constant voltage (6.4 V/cm) for about 4 h. The current was about 40 mA. Electron microscopy. The ds RNA was prepared
1002
TABLE 1. Strainsa Designation
Killer pheno-
Genotype
type
A364A
K+ R+
a
adel ade2 tyrl Iys2 ural his7 gall
AN33
K- R-
a
[KIL-k] argl thrl [KIL-o]
5X47
K- R-
a his
tp
+
+
a
B55 AT95 AT94 95 80 AT17 AT18 AT19 AT20 AT21 AT22 AT23 AT25 AT26 AT27 AT28 AT202 AT257 1068
1003 the superkiller phenotype occurs in a chromosomal gene. Complementation tests among these mutants revealed four distinct genes, which are designated skil, ski2, ski3, and ski4. Diploids were constructed which were heterozygous for two ski mutations in different complementation groups. Meiotic analysis of these diploids showed no close linkage among the ski genes and confirmed the assignment of mutants to genes by the complementation tests. The superkiller phenotype of these mutants is shown in Fig. 1. The skil-1 mutant grows slowly on YPAD, but it shows neither temperature sensitivity nor respiratory deficiency. When a heterozygous was sporulated and tetrads were diploid, dissected, the superkiller phenotype always coincided with the slow growth character. These two phenotypes, however, could have resulted from two closely linked mutations. kex mutations are epistatic to ski mutations; mapping of skil, ski3, and ski4. Two nuclear genes, kexl and kex2, are required for toxin synthesis (24). The ski mutations, which give rise to overproduction of toxin activity, might be altered in some stage(s) of toxin synthesis. To study the phenotype of double mutants containing one ski and one kex mutation, each ski strain was crossed with a kexl or kex2 strain, and diploids were analyzed meiotically. Killing activity was scored at 30°C so that three phenotypes (kex, ski, and wild type) could be distinguished (Table 2). Two spore clones in every ascus were K-, and the rest were wild-type killers or superkillers or one wild-type killer and one superkiller. This segregation pattern indicates that double mutants, containing both ski and kex, show the K- phenotype of the kex mutation. Thus, superkiller strains continue to depend on the kex gene products for toxin production. Analysis of the crosses in Table 2 revealed that ski4 is very closely linked to kex2 (chromosome XIV); ski3 is loosely linked to kex2; and skil showed possible linkage to kexl (see below). Although ski4 and kex2 are closely linked, they are different genes because they complement; ski4 + + ke2 diploids are wild-type killers. Linkage could not be detected between ski3 and Iys9, rna2, or pet8 (genes linked in meiosis to the centromere of chromosome XIV [15]). Five Iys mitotic recombinants from a-+ SUPERKILLER MUTANTS OF YEAST
VOL. 136, 1978
+
ura3
[KIL-o]
K++ R+ K++ R+ K++ R+ K- R+ K- R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K++ R+ K+ R+
A364A ski2-1 [KIL-k] a his7 skil-) [KIL-k] a adel skil-) [KIL-k] a argl thrl kexl-l [KIL-k] a ade2 ural kex2-1 [KIL-k] a lys2 tyrl his7 ski2-1 [KIL-k] a ade2 thrl ski2-1 [KIL-k] a ade2 tyrl his7 ski3-1 [KRL-k] a ade2 argl ski3-1 [KIR-k] a thrl argl ski4-1 [KIL-k] a his7 adel (or ade2) ski4-1 [KIL-k] a adel (or ade2) his7 ski3-2 [KRL-k] a ade2 thrl argl tyrl ski2-2 [KIL-k] a adel (or ade2) his7 ski2-2 [KIL-k] a tyrl ski3-3 [KIL-k] a adel thrl argl ski3-3 [KIL-k] a lys2 his7 ski2-3 [KIL-k] a his7 skil-) [KIL-k] a hisl metl3 leul trp5 aro2 lys5 ade5 chy2 [KIL-k] a Phenotypes of strains with regard to their killing ability (K) and resistance (R) to killing are denoted K+ R+, K- R+, K+ R-, or K- R-. Superkillers are denoted K+. The genotype of the killer plasmid is noted by [KIL-k] (wild-type killer plasmid) and [KILr-o] (no killer plasmid).
for electron microscopy by the aqueous method of Davis et al. (8) and examined in a Phillips 300 electron microscope. A minimum of 100 ds RNA molecules from each strain were measured by using a Neumonics Digitizer. The measurements were calibrated by reference to an external standard of open circular simian virus 40 DNA. Genetic analysis. Genetic methods were as described previously (see Literature Cited in reference 15). Cytoduction was carried out as described by Conde and Fink (7). RESULTS
Isolation of ski mutants. Eight mutants which showed an increased killing zone (K++) at 300C were isolated from ethyl methane sulfonate-treated A364A cells. Each mutant killed only strains sensitive to the wild-type toxin. None of these mutants had acquired ability to kill wild-type killer strains. Also, each mutant showed greater killing ability than the wild type when tested at 200C. Each mutant was crossed with a wild-type nonkiller (AN33). Because all resulting diploids showed the same killing activity as that of wild type, the mutations causing the superkiller phenotype are recessive. When diploids from each cross were dissected, essentially all tetrads (7 to 13) showed 2 K++: 2 K+ segregation. Thus, each mutation responsible for
+il-,
diploid were isolated. None of these were superkillers, indicating that the fragment of chromosome XIV carrying ski3, ski4, kex2, and
1004
TOH-E, GUERRY, AND WICKNER
J. BACTERIOL. w
*..: ,''''.'..'.'.: 'S ..
w.
sk 3 ....... ~ ~ ~ ~ ~ ~ . ::.:...~ ~
.... ......
FIG. 1. Superkiller phenotype. A cell suspension of each strain was spotted onto MB medium previously spread with a lawn of a sensitive strain (5 x 47) and incubated at 30°C for 2 days. Top row, from left: wildtype kiler, wild-type nonkiller. Second row, from left: ski2-2, ski2-1, ski2-3. Third row, from left: ski3-3, ski32, ski3-1. Fourth row: ski4-1. Fifth row: skil-1.
TABLE 2. kex and ski mutations: linkage and epistasis No. with the following tetrad type:"
Cross kexl-l (95) x skil-I (AT94)
x ski2-1 (AT17)) x ski2-2 (AT26) x ski2-3 (AT15)
K+K:1 2 K+ 1:2 K(T) (PD)
2 K+:2 K-
(NPD)
6
4
1
12
27
6
petx is on the opposite side of the centromere from Iys9 (Fig. 2). The location of skil was defined further. Linkage data between genes on chromosome VII are listed in Table 3. The crossover patterns in individual tetrads were also analyzed to determine the arrangement of skil, aro2, and lys5 (Table 4). These results indicate that skil is located between kexl and lys5 (12.5 min from Iys5). The map positions of skil, ski3, and ski4 are shown in Fig. 2. ski mutants still depend on the killer
plasmid for killing ability. skil, ski2, ski3, and ski4 strains were grown at 37°C in rich a treatment shown to cure the killer medium, 1 5 x ski4-1 (AT22) 0 from wild-type strains. This treatment plasmid kex2-1 (80) induced a high frequency of nonkillers in each 2 6 3 x skil-I (AT95) strain, as expected if the ski mutants, like wildx Aki2-I (AT18) 6 21 4 type killers, depend on the killer plasmid for 4 x ski2-2 (AT25) killing ability. This was further tested in the case of ski2-2. The killer plasmid was reintroduced x ski3-1 (AT20) 8 48 34 x ski3-3 (AT28)J into a ski2-2 heat-cured K- R- p- strain by cytoduction (cytoplasmic mixing without nux ski4-1 (AT21) 2 0 97 clear fusion [7]) from a karl K+ R+ p+ strain. a PD, Parental ditype ascus; T, tetratype ascus; NPD, non- The cytoductants (judged by aquisition of p+) parental ditype ascus. Strains which are kex- ski+ [KIL-k] were superkillers. have the K- R+ phenotype (20). Each kex and ski mutation Analysis of ds RNA from ski mutants. In segregates 2+:2- in all crosses. The K- segregants must be kex-, since kex+ ski' and kex+ ski- segregants are always K+ wild-type strains, the weight ratio of M to L ds and K++, respectively. Tetrads that are 2 K++:2 K- must be 2 RNAs is about 1:10 (2, 19, 23), and the content kex+ ski-:2 kex- ski', all spores being of two types (ditype) in of M ds RNA is about 3 nmol of nucleotide per the parental configuration. Tetrads that are 2 K+:2 K- must g of cells (wet weight) (19, 23). These values are be 2 kex+ ski+:2 kex- ski-. The fact that kex- ski- segregants are K- (the kex phenotype) is expressed by saying kex is relatively constant from strain to strain. ds RNA was extracted from stationary cultures of wildepistatic to ski. x ski3-1 (AT19) x ski3-2 (AT23) J
7
24
9
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1005
TABLE 3. Linkage of skil to Iys5 on chromosome viia
4lo4
Gene Gene
to07 41
skil PD NPD T Distances (cM)
\l-
lys5
aro2
metl3
cyh2
59 1 12 12.5
53 1 22 18.5
34 0 29 22
46 6 115 45
Iys5 PD NPD T Distances (cM)
I I I
kexl 32 41 Distances (cM) 'For each pair of genes, the number of parental ditype (PD; no crossovers), nonparental ditype (NPD; two crossovers), and tetratype (T; one crossover) asci observed is given. Except for the segregation of Akil relative to cyh2, the data come from a single cross: W205 (1068 x AT95). The distances shown for kexl for comparison are from Wickner and Leibowitz (24). Distances are calculated in centimorgans (cM) by the method of Perkins (16).
-o
t
0
(NY
4--q
4Nv
4,)e
66 0 8 5
TABLE 4. Gene order of skil, Iys5, and aro2a Crossovers needed for _
the following gene or-
0
cn
0
Type of tetrad
No. of asci observed
der:
skil- lys5- skilIys5-
,e. \\0Q -.-
e9, -e 10
x
-5
K.
Zb
ski)- aro2-
aro2 Iys5 1 1 0 1. aro2 and skil PD; Iys5 T with aro2 and skil 1 1 12 2 2. aro2 and Iys5 PD; skil T with aro2 and Iys5 2 7 1 1 3. Iys5 and skil PD; aro2 T with Iys5 and skil a The data (no. of asci observed) was obtained by examination of the same tetrads used in Table 3 to demonstrate linkage. Tetrads were selected in which two of the three markers were in the parental ditype (PD; nonrecombinant) configuration with each other, but each of these two was in the tetratype (T; single crossover) configuration with the third marker. If the third marker is the middle marker on the genetic map, this requires two crossovers (presumably a rare event in this short interval). If the third marker is on one end, one crossover will give this result. The type of tetrad which was not observed (type 1) must represent that segregation of markers requiring a double crossover. A type 1 tetrad requires a double crossover if the gene order is skil-lys5-aro2. Thus, the gene order is skil-lys5-aro2.
aro2 2
_
I
0
0
type and ski mutants as described above and equal amounts of ds RNA from each strain were analyzed by agarose slab gel electrophoresis. The ds RNA bands were visualized by the fluorescence emitted from the ds RNA-ethidium bromide complex under UV light (Fig. 3). As shown in Fig. 3, ski2, ski3, and ski4 strains have more M ds RNA relative to L ds RNA than does wild type. The skil strain, however, has roughly the same ratio of M ds RNA to L ds RNA as the
1006
J. BACTERIOL.
TOH-E, GUERRY, AND WICKNER
TABLE 5. ds RNA in ski mutantsa Total ds LdosRNA Mds N RNA Strain (no/g (RunAo (nmol/g (uo of s cells) of cel of cells)
M
FIG. 3. ds RNA of ski mutants on agarose gel electrophoresis. ds RNA purified by CF-lI column chromatography was precipitated by ethanol and dissolved in a small amount of electrophoresis buffer. A 50-gd amount of each sample (absorbance at 260 nm, approximately 1) was applied to lanes 1 through 8. Lane 1, strain AT154 wild type; lane 2, strain AT15 ski2-3; lane 3, strain AT9 ski2-1; lane 4, strain AT17 ski2-1; lane 5, strain AT26 ski2-2; lane 6, strain AT27 ski3-3; lane 7, strain AT21 ski4-1; lane 8, strain AT30 skil-l.
wild type. The molecular weights of M ds RNA in ski2, ski3, and ski4 were the same as wild type. The skil strain has two bands close to each other in the M ds RNA region. The population of L and M ds RNA in some of the ds RNA preparations was analyzed by measuring contour lengths of ds RNA molecules by electron microscopy. Table 5 shows the amounts of M and L ds RNAs in various strains. The data for the wild-type killer are close to the published values. The results in Table 5 suggest an absolute and relative increase in M ds RNA in ski mutants. However, considerable variability of the results was observed. Further study of this point by other methods will be necessary.
DISCUSSION Four ski genes (skil through ski4) were found by looking for mutants which gave rise to a larger killing zone than wild type at 300C. The killing ability of these mutants is greater even at
I ML
0.10 2.5 25.5 28 Wild type A364A 0.32 4.9 15.1 20 Wild type A364A 0.39 6.5 16.5 23 skil-1 AT95 0.55 10.6 19.4 30 skil-1 AT257 1.16 58 50 108 ski2-1 B55 1.22 11 9.5 20.5 ski2-3 AT202 0.36 4 11 15 ski3-3 AT27 0.60 10 16.8 26.8 ski3-3 AT27 1.97 16.6 8.4 25 ski4-1 AT21 0.66 6.3 9.5 15.8 ski4-1 AT21 a ds RNA was isolated as described in the text. A total of 90 to 100% of the isolated material was ds based on pancreatic ribonuclease sensitivity in high and low salt. The ratio of M to L molecules was determined by electron microscopy (see text). These data, the total ds RNA recovered from each strain, and the known molecular weights of L and M, were used to calculate the values shown in the table.
a lower temperature (20 and 2500) than that of the wild type. All ski mutations described here are recessive mutations occurring in nuclear genes. A mutant showing a similar superkiller phenotype has been described by Vodkin et al. (19), which was located in a cytoplasmic genome. ski mutants remain dependent upon the killer plasmid for their killing phenotype. Since kex mutations are epistatic to ski mutations, ski mutants are also dependent upon the kex gene products for toxin production. The skil gene may be essential for growth since our lone mutant allele results in decreased growth rate. Since killer toxin is rapidly degraded at 300C (25), the superkiller phenotype in one or more of these mutants could result from a mutation which makes the toxin heat resistant. If these strains overproduce a normal toxin, this might be due to changes in toxin processing or transport, translation of toxin message, transcription of toxin genes, or copy number of M ds RNA. Further studies will be directed toward distinguishing these possibilities. ACKNOWLEDGMENTS We are grateful to R. K. Mortimner, S. Fogel, J. Bassel, and R. Contopoulos for sending us many strains essential for this work.
LITERATURE CITED 1. Adler, J., H. A. Wood, and R. F. Bozarth. 1976. Viruslike particles from killer, neutral, and sensitive strains
of Saccharomyces cerevisiae. J. Virol.
17:472-476.
2. Bevan, E. A., A. J. Herring, and D. J. Mitchell. 1973. Preliminary characterization of two species of ds RNA in yeast and their relationship to the "killer" character.
Nature (London) 245:81-86.
3. Bevan, E. A., and J. M. Somers. 1968. Somatic segregation of the killer (k) and neutral (n) cytoplasmic genetic determinants in yeast. Genet. Res. 14:71-77.
4. Buck, K. W., P. Lhoas, and B. K. Street. 1973. Virus
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particles in yeast. Biochem. Soc. Trans. 1:1141-1142. 5. Bussey, H. 1972. Effects of yeast killer factor on sensitive cells. Nature (London) New Biol. 236:73-75. 6. Cohn, M. S., C. W. Tabor, H. Tabor, and R. B. Wickner. 1978. Spermidine or spermine requirement for killer double-stranded RNA plasmid replication in yeast. J. Biol Chem. 253:5225-5227. 7. Conde, J., and G. R. Fink. 1976. A mutant of Saccharomyces cerevisiae defective for nuclear fusion. Proc. Natl. Acad. Sci. U. S. A. 73:3651-3655. 8. Davis, R. W., M. Simon, and N. Davidson. 1971. Electron microscope hetero-duplex methods for mapping regions of base sequence homology in nucleic acids. Methods Enzymol. 21:413-427. 9. Franklin, R. M. 1966. Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc. Natl. Acad. Sci. U. S. A. 56:1504-1511. 10. Hartwell, L. H., J. Culotti, and B. Reid. 1970. Genetic control of the cell division cycle in yeast. I. Detection of mutants. Proc. Natl. Acad. Sci. U. S. A. 66:352-359. 11. Herring, A. J., and E. A. Bevan. 1974. Virus-like particles associated with the double-stranded RNA species found in killer and sensitive strains of the yeast Saccharomyces cerevisiae. J. Gen. Virol. 22:387-394. 12. Hopper, J. E., K. A. Bostian, L B. Rowe, and D. J. Tipper. 1977. Translation of the L-species ds RNA genome of the killer-associated virus-like particles of Saccharomyces cerevisiae. J. Biol. Chem. 262:90109017. 13. Kaneko, T., K. Kitamura, and Y. Yamamoto. 1973. Susceptibilities of yeasts to yeast cell wall lytic enzyme of Arthrobacter luteus. Agric. Biol. Chem. 37: 2295-2302. 14. Lindegren, G., Y. L. Hwang, Y. Oshima, and C. C. Lindegren. 1965. Genetical mutants induced by ethylmethanesulfonate in Saccharomyces. Can. J. Genet. Cytol. 7:491-499.
SUPERKILLER MUTANTS OF YEAST
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15. Mortimer, R. K., and D. C. Hawthorne. 1975. Genetic mapping in yeast, p. 221-223. In D. M. Prescott (ed.), Methods in cell biology, vol. 11. Academic Press Inc., New York. 16. Perkins, D. D. 1949. Biochemical mutants of the smut fungus Ustilago maydis. Genetics 34:607-626. 17. Somers, J. M., and E. A. Bevan. 1968. The inheritance of the killer character in yeast. Genet. Res. 13:71-83. 18. Sweeney, T. K., A. Tate, and G. R. Fink. 1976. A study of the transmission and structure of double stranded RNAs associated with the killer phenomenon in Saccharomyces cerevisiae. Genetics 84:27-42. 19. Vodkin, M., F. Katterman, and G. R. Fink. 1974. Yeast killer mutants with altered double-stranded ribonucleic acid. J. Bacteriol. 117:681-686. 20. Wickner, R. B. 1974. Chromosomal and nonchromosomal mutations affecting the "killer character" of Saccharomyces cerevisias. Genetics 76:423-432. 21. Wickner, R. B. 1976. Killer of Saccharomyces cerevisiae: a double-stranded RNA plasmid. Bacteriol. Rev. 40: 757-773. 22. Wickner, R. B. 1978. Twenty-six chromosomal genes needed to maintain the killer double-stranded RNA plasmid of Saccharomyces cerevisias. Genetics 88: 419-425. 23. Wickner, R. B., and M. J. Leibowitz. 1976. Chromosomal genes essential for replication of a doublestranded RNA plasmid of Saccharomyces cerevisiae: the killer character of yeast. J. Mol. Biol. 105:427-443. 24. Wickner, R. B., and M. J. Leibowitz. 1976. Two chromosomal genes required for killing expression in killer strains of Saccharomyces cerevisiae. Genetics 82: 429-442. 25. Woods, D. R., and E. A. Bevan. 1968. Studies on the nature of the killer factor produced by Saccharomyces cerevisiae. J. Gen. Microbiol. 51:115-126.
