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Double-Stranded RNA Killer Plasmid Replication in Saccharomyces Cerevisiae (Ski Mutants/Mak Mutants) Akio TOH-E* and REED B

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Double-Stranded RNA Killer Plasmid Replication in Saccharomyces Cerevisiae (Ski Mutants/Mak Mutants) Akio TOH-E* and REED B Proc. Natl. Acad. Sci. USA Vol. 77, No. 1, pp. 527-530, January 1980 Genetics "Superkiller" mutations suppress chromosomal mutations affecting double-stranded RNA killer plasmid replication in Saccharomyces cerevisiae (ski mutants/mak mutants) AKIo TOH-E* AND REED B. WICKNERt Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205 Communicated by G. Gilbert Ashwell, October 17,1979 ABSTRACT Saccharomyces cerevisiae strains carrying a MATERIALS AND METHODS 1.5 X 106-dalton double-stranded RNA genome in virus-like particles (killer plasmid) secrete a protein toxin that kills strains Strains. Some of the strains of Saccharomyces cerevsiae used not carrying this plasmid. At least 28 chromosomal genes (mak in this study are listed in Table 1. Description of the phenotype genes) are required to maintain or replicate this plasmid. Re- and genotype of killer strains was presented previously (21). cessive mutations in any of four other chromosomal genes (ski Curing of the killer plasmid is done by growing killer strains for superkiller) result in enhanced toxin production. We report at an elevated temperature (37°C) (23). Mitochondrial DNA that many ski- mak- double mutants are able to maintain the killer plasmid, indicating that the SKIproducts have an effect was eliminated from strains by streaking to single colonies on on plasmid replication. The skil-) mutation suppresses (by- YPAD medium containing ethidium bromide at 30 ug/ml passes) all mak mutations tested except makl6-l. A variant killer (24). plasmid is described that confers the superkiller phenotype and, Media. YPAD, YPG, SD, presporulation medium, sporula- like chromosomal ski mutations, makes several mak genes tion medium, MB medium, and various omission media were dispensable for plasmid replication. described (25). Genetic Techniques. General techniques for yeast genetics Some strains of yeast kill other strains by secreting a protein (26) were followed. Cytoduction (cytoplasmic mixing) was toxin (1-4). The killer genome of Saccharomyces ceievisie is carried out with a karl (karyogamy) strain defective in nuclear a linear 1.5 X 106-dalton double-stranded (ds) RNA (M ds RNA fusion (27). On mating a karl strain with another strain, cyto- or P2) encapsulated in virus-like particles (5-11); this RNA plasmic mixing occurs, but the nuclei fail to fuse and separate codes for the killer toxin (12, 13). Most laboratory strains of yeast when the cell divides. Usually the recipient was p°, and the also have another species of virus-like particles containing a 3 donor of cytoplasm was p+. After mating donor and recipient X 106-dalton ds RNA (L or P1), which codes for the major coat in YPAD medium, donor nuclei were counter-selected by protein of the virus-like particles (14). plating the mating mixture on an appropriate selective plate. At least 28 nuclear genes are necessary to maintain the killer p+ strains having the genotype of recipient nuclei are cyto- plasmid (M ds RNA). These are maki and mak3-mak27 (refs. ductants. 5 and 15-17; unpublished results), petl8 (18, 19), and spe2 (20). RNA Extraction and Agarose Gel Electrophoresis. RNA Al mak mutants, including pet18 and spe2, lose M ds RNA but was extracted from intact cells (28). Electrophoresis was done retain L ds RNA. This result indicates that the mak genes are on agarose gels (1%) and photographs were taken after staining involved specifically in the replication or maintenance of M ds with ethidium bromide (21). RNA, although their exact functions are not known. Recessive mutations in any of four chromosomal genes (called RESULTS skil-ski4 for superkiller) result in increased production of toxin The recessive chromosomal ski mutations result in enhanced activity (21). Two lines of evidence suggest that ski genes may killer activity; thus, the SKI products might have a negative have some role in the replication of M ds RNA. (i) A particular regulatory function at some stage in the replication or expression mutant killer plasmid, called [KIL-sd], is unable to replicate in of the killer genome. The MAK products are each necessary SKI+ cells, but replicates normally in ski- strains (22) and (ii) for maintenance or replication of the killer genome (M ds ski2, ski3, and ski4 mutants seem to have more M ds RNA than RNA). Therefore, the phenotype of mak- ski- double mutants wild type (21). should give information about the functional relationship, if In this communication we show that ski mutations bypass the any, of MAK and SKI products. functions of some mak genes. These data strongly support the skil-1 Suppresses maklO-l. A skil-I strain (AT95) was idea that ski genes participate in the replication of M ds RNA. crossed with a maklO-l strain (M291). Asci containing more The bypass pattern suggests that there may be more than one than two killer clones appeared frequently (Table 2, cross pathway for the replication of M ds RNA. We also describe a superkiller plasmid variant [KIL-b], which, like the chromo- Abbreviations: [KIL-sdj, ski-dependent killer plasmid; [KIL-bi, su- somal ski - mutations, bypasses the requirement for many of perkiller bypass plasmid; ds, double-stranded; K+, killer phenotype; the mak genes. k-, nonkiller phenotype; R+, resistant to killer toxin; R-, sensitive to killer toxin; [KIL-o], absence of killer plasmid, [KIL-k], presence of wild-type killer plasmid. The publication costs of this article were defrayed in part by page * Present address: Department of Fermentation Technology, Faculty charge payment. This article must therefore be hereby marked "ad- of Engineering, Osaka University, Yamada-Kami Suita-Shi, Osaka, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Japan 565. this fact. t To whom reprint requests should be addressed. 527 Downloaded by guest on October 1, 2021 528 Genetics: Toh-e and Wickner Proc. Natl. Acad. Sci. USA 77 (1980) Table 1. Strains Table 3. Confirmation of genotype of spore clones from Killer cross W109 Strain phenotype Genotype Killer phenotype AT95 K++R+ a his7 skil-l AT257 K++R+ a his7skil-1 Spore Killer of diploid with: Assigned AT202 K++R+ a iys2 his7 ski2-3 Tetrad clone phenotype skil maklO genotype AT17 K++R+ a Iys2 tyrl his7ski2-1 W109-16 A K+ K++ K- skil maklO AT206 K++R+ a argl ski3-1 B K+ K+ K+ SKI MAK AT21 K++R+ a argl thrl ski4-1 C K+ K++ K- skil maklO 299 K-R- a iysl mak3-1 D K+ K+ K+ SKI MAK 737 K-R- a thrl lys2 mak4-1 W109-17 A 918 K+ K+ K+ SKI MAK K-R- a his6 adel arg4 mak6-1 B K+ K+ K+ SKI MAK 490 K-R- a Ieu2 met5 mak7-1 C K+ K++ K- skil maklO M291 K-R- a ura3 ilv3 canl maklO-l D K+ K++ K- skil maklO M292 K-R- a ura3 ilv3 cani maklO-l W489-4A K-R- a argl thrl Iys2 maklO-2 Superkiller strains (K++) give a clear killing zone at 300C and a AT34 K-R- aleu2ural cdc16makil wider zone at 20'C. Normal killers (K+) give little or no killing zone P28-24C K++R+ a [KIL-bi at 30'C and a smaller zone at 20'C. AT159 K+R+ a his4 karl-i [KIL-ki dissected. The SKI genes are not linked to any of the MAK AT171 K++R+ a his4 karl-i [KIL-b] genes in these crosses. Therefore, the criteria for the suppression 2403-20A K+-++R+ a his7 makl7skil of a mak mutation by a ski mutation was that the segregation 2404-7C K+-++R+ a his7 makl3 skil pattern with respect to the killer trait be 3K+:1K- on the av- 2405-1A K+-++R+ a his7 makl2 skil erage. The pattern of suppression is summarized in Table 4. The 1105 K-R- a adel mak16-1 ski) mutation suppresses all mak mutations tested so far except Several different specificities of killers ofSaccharomyces have been for mak16-1. Mutations in ski2, skiW, and ski4 do not suppress found, called K1, K2, etc. (3,4); they are distinguished by the strains mak)6, mak3, pet18, or maklO, but the rest of the mak muta- they can kill. All killer strains used in this study belong to the K1 tions tested can be suppressed by any of the ski mutations. specificity. K+-++ indicates strains whose killer phenotype was in- Translational suppressors are allele-specific and locus nonspe- termediate between K+ and K++. cific. The independently isolated mak)O mutations, mak)O-) W109), indicating that a suppressor of mak)O-1 was involved and mak)O-2, are both suppressed by the skil)- mutation in this cross. To test whether the suppressor of makIO-1 was (Table 2), and it is unlikely that so many mak mutations in skil-), we determined the genotype of spore clones of two asci different genes would be suppressible by the same translational from cross W109 by complementation with standard ski-) and suppressor (Table 4). Finally, translational (tRNA) suppressors mak)O-1 strains (Table 3). Of the 8 K+ clones, 16A, 16C, 17C, are generally dominant, whereas the suppressor activity of the and 17D were found to be both maklO-1 and skilc-I. When each ski- mutations is recessive (e.g., Table 3). spore clone of these two asci was crossed with a wild-type Suppressors of mak mutations may be isolated directly as [KIL-o] strain of appropriate mating type and meiotic spores killer sectors in mak spore clones from a mak-/ + [KIL-k] were dissected, K- clones frequently segregated from the diploid (29, 30).
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