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. 1986 May;6(5):1552–1561. doi: 10.1128/mcb.6.5.1552

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

R Esteban, R B Wickner
PMCID: PMC367681  PMID: 3537705

Abstract

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adler J., Wood H. A., Bozarth R. F. Virus-like particles from killer, neutral, and sensitive strains of Saccharomyces cerevisiae. J Virol. 1976 Feb;17(2):472–476. doi: 10.1128/jvi.17.2.472-476.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ball S. G., Tirtiaux C., Wickner R. B. Genetic Control of L-a and L-(Bc) Dsrna Copy Number in Killer Systems of SACCHAROMYCES CEREVISIAE. Genetics. 1984 Jun;107(2):199–217. doi: 10.1093/genetics/107.2.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bevan E. A., Herring A. J., Mitchell D. J. Preliminary characterization of two species of dsRNA in yeast and their relationship to the "killer" character. Nature. 1973 Sep 14;245(5420):81–86. doi: 10.1038/245081b0. [DOI] [PubMed] [Google Scholar]
  4. Brennan V. E., Bobek L. A., Bruenn J. A. Yeast deRNA viral transcriptase pause products: identification of the transcript strand. Nucleic Acids Res. 1981 Oct 10;9(19):5049–5059. doi: 10.1093/nar/9.19.5049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brennan V. E., Field L., Cizdziel P., Bruenn J. A. Sequences at the 3' ends of yeast viral dsRNAs: proposed transcriptase and replicase initiation sites. Nucleic Acids Res. 1981 Aug 25;9(16):4007–4021. doi: 10.1093/nar/9.16.4007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bruenn J. A. Virus-like particles of yeast. Annu Rev Microbiol. 1980;34:49–68. doi: 10.1146/annurev.mi.34.100180.000405. [DOI] [PubMed] [Google Scholar]
  7. Bruenn J., Bobek L., Brennan V., Held W. Yeast viral RNA polymerase is a transcriptase. Nucleic Acids Res. 1980 Jul 11;8(13):2985–2997. doi: 10.1093/nar/8.13.2985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buck K. W. Replication of double-stranded RNA in particles of Penicillium stoloniferum virus S. Nucleic Acids Res. 1975 Oct;2(10):1889–1902. doi: 10.1093/nar/2.10.1889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Buck K. W. Semi-conservative replication of double-stranded RNA by a virion-associated RNA polymerase. Biochem Biophys Res Commun. 1978 Oct 16;84(3):639–645. doi: 10.1016/0006-291x(78)90753-2. [DOI] [PubMed] [Google Scholar]
  10. Bussey H. Physiology of killer factor in yeast. Adv Microb Physiol. 1981;22:93–122. doi: 10.1016/s0065-2911(08)60326-4. [DOI] [PubMed] [Google Scholar]
  11. Cohn M. S., Tabor C. W., Tabor H., Wickner R. B. Spermidine or spermine requirement for killer double-stranded RNA plasmid replication in yeast. J Biol Chem. 1978 Aug 10;253(15):5225–5227. [PubMed] [Google Scholar]
  12. Conde J., Fink G. R. A mutant of Saccharomyces cerevisiae defective for nuclear fusion. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3651–3655. doi: 10.1073/pnas.73.10.3651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. FRAENKEL-CONRAT H., SINGER B., TSUGITA A. Purification of viral RNA by means of bentonite. Virology. 1961 May;14:54–58. doi: 10.1016/0042-6822(61)90131-3. [DOI] [PubMed] [Google Scholar]
  14. Fried H. M., Fink G. R. Electron microscopic heteroduplex analysis of "killer" double-stranded RNA species from yeast. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4224–4228. doi: 10.1073/pnas.75.9.4224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harris M. S. Virus-like particles and double stranded RNA from killer and non-killer strains of Saccharomyces cerevisiae. Microbios. 1978;21(85-86):161–176. [PubMed] [Google Scholar]
  16. Herring A. J., Bevan E. A. Virus-like particles associated with the double-stranded RNA species found in killer and sensitive strains of the yeast Saccharomyces cerevisiae. J Gen Virol. 1974 Mar;22(3):387–394. doi: 10.1099/0022-1317-22-3-387. [DOI] [PubMed] [Google Scholar]
  17. Hopper J. E., Bostian K. A., Rowe L. B., Tipper D. J. Translation of the L-species dsRNA genome of the killer-associated virus-like particles of Saccharomyces cerevisiae. J Biol Chem. 1977 Dec 25;252(24):9010–9017. [PubMed] [Google Scholar]
  18. Leibowitz M. J., Wickner R. B. Pet18: a chromosomal gene required for cell growth and for the maintenance of mitochondrial DNA and the killer plasmid of yeast. Mol Gen Genet. 1978 Oct 4;165(2):115–121. doi: 10.1007/BF00269899. [DOI] [PubMed] [Google Scholar]
  19. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  20. Newman A. M., Elliott S. G., McLaughlin C. S., Sutherland P. A., Warner R. C. Replication of double-stranded RNA of the virus-like particles in Saccharomyces cerevisiae. J Virol. 1981 Apr;38(1):263–271. doi: 10.1128/jvi.38.1.263-271.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Oliver S. G., McCREADY S. J., Holm C., Sutherland P. A., McLaughlin C. S., Cox B. S. Biochemical and physiological studies of the yeast virus-like particle. J Bacteriol. 1977 Jun;130(3):1303–1309. doi: 10.1128/jb.130.3.1303-1309.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ridley S. P., Sommer S. S., Wickner R. B. Superkiller mutations in Saccharomyces cerevisiae suppress exclusion of M2 double-stranded RNA by L-A-HN and confer cold sensitivity in the presence of M and L-A-HN. Mol Cell Biol. 1984 Apr;4(4):761–770. doi: 10.1128/mcb.4.4.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sclafani R. A., Fangman W. L. Conservative replication of double-stranded RNA in Saccharomyces cerevisiae by displacement of progeny single strands. Mol Cell Biol. 1984 Aug;4(8):1618–1626. doi: 10.1128/mcb.4.8.1618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Skipper N., Thomas D. Y., Lau P. C. Cloning and sequencing of the preprotoxin-coding region of the yeast M1 double-stranded RNA. EMBO J. 1984 Jan;3(1):107–111. doi: 10.1002/j.1460-2075.1984.tb01769.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sommer S. S., Wickner R. B. Co-curing of plasmids affecting killer double-stranded RNAs of Saccharomyces cerevisiae: [HOK], [NEX], and the abundance of L are related and further evidence that M1 requires L. J Bacteriol. 1982 May;150(2):545–551. doi: 10.1128/jb.150.2.545-551.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sommer S. S., Wickner R. B. Yeast L dsRNA consists of at least three distinct RNAs; evidence that the non-Mendelian genes [HOK], [NEX] and [EXL] are on one of these dsRNAs. Cell. 1982 Dec;31(2 Pt 1):429–441. doi: 10.1016/0092-8674(82)90136-2. [DOI] [PubMed] [Google Scholar]
  27. Thiele D. J., Leibowitz M. J. Structural and functional analysis of separated strands of killer double-stranded RNA of yeast. Nucleic Acids Res. 1982 Nov 11;10(21):6903–6918. doi: 10.1093/nar/10.21.6903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tipper D. J., Bostian K. A. Double-stranded ribonucleic acid killer systems in yeasts. Microbiol Rev. 1984 Jun;48(2):125–156. doi: 10.1128/mr.48.2.125-156.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Toh-E A., Guerry P., Wickner R. B. Chromosomal superkiller mutants of Saccharomyces cerevisiae. J Bacteriol. 1978 Dec;136(3):1002–1007. doi: 10.1128/jb.136.3.1002-1007.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Toh-E A., Wickner R. B. "Superkiller" mutations suppress chromosomal mutations affecting double-stranded RNA killer plasmid replication in saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 Jan;77(1):527–530. doi: 10.1073/pnas.77.1.527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Welsh D., Leibowitz M. J. Transcription of killer virion double-stranded RNA in vitro. Nucleic Acids Res. 1980 Jun 11;8(11):2365–2375. doi: 10.1093/nar/8.11.2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Welsh J. D., Leibowitz M. J. Localization of genes for the double-stranded RNA killer virus of yeast. Proc Natl Acad Sci U S A. 1982 Feb;79(3):786–789. doi: 10.1073/pnas.79.3.786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Welsh J. D., Leibowitz M. J., Wickner R. B. Virion DNA-independent RNA polymerase from Saccharomyces cerevisiae. Nucleic Acids Res. 1980 Jun 11;8(11):2349–2363. doi: 10.1093/nar/8.11.2349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wesolowski M., Wickner R. B. Two new double-stranded RNA molecules showing non-mendelian inheritance and heat inducibility in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Jan;4(1):181–187. doi: 10.1128/mcb.4.1.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wickner R. B. Double-stranded RNA replication in yeast: the killer system. Annu Rev Biochem. 1986;55:373–395. doi: 10.1146/annurev.bi.55.070186.002105. [DOI] [PubMed] [Google Scholar]
  36. Wickner R. B. Genetic control of replication of the double-stranded RNA segments of the killer systems in Saccharomyces cerevisiae. Arch Biochem Biophys. 1983 Apr 1;222(1):1–11. doi: 10.1016/0003-9861(83)90496-4. [DOI] [PubMed] [Google Scholar]
  37. Wickner R. B., Leibowitz M. J. Chromosomal genes essential for replication of a double-stranded RNA plasmid of Saccharomyces cerevisiae: the killer character of yeast. J Mol Biol. 1976 Aug 15;105(3):427–443. doi: 10.1016/0022-2836(76)90102-9. [DOI] [PubMed] [Google Scholar]
  38. Wickner R. B., Toh-e A. [HOK], a new yeast non-Mendelian trait, enables a replication-defective killer plasmid to be maintained. Genetics. 1982 Feb;100(2):159–174. doi: 10.1093/genetics/100.2.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wickner R. B. Twenty-six chromosomal genes needed to maintain the killer double-stranded RNA plasmid of Saccharomyces cerevisiae. Genetics. 1978 Mar;88(3):419–425. doi: 10.1093/genetics/88.3.419. [DOI] [PMC free article] [PubMed] [Google Scholar]

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