Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Jun;83(12):4433–4437. doi: 10.1073/pnas.83.12.4433

In vitro L-A double-stranded RNA synthesis in virus-like particles from Saccharomyces cerevisiae.

T Fujimura, R Esteban, R B Wickner
PMCID: PMC323747  PMID: 3520572

Abstract

Most strains of Saccharomyces cerevisiae harbor L-A double-stranded RNA (dsRNA), 4.5 kilobases long, contained in virus-like particles (VLPs). These L-A VLPs can be separated by CsCl density gradient centrifugation into a main peak of particles, containing full-length L-A dsRNA, which synthesizes only plus-strand single-stranded RNA (ssRNA), and a lighter fraction of VLPs, containing plus-strand ssRNA, which has L-A dsRNA-synthesizing activity. This dsRNA-synthesizing activity was present in particles from logarithmically growing cells but not from stationary-phase cells. The newly synthesized strand of dsRNA in the lightest particles was full-length minus strand. All or almost all of the new minus strand was synthesized in vitro, and the rate of chain elongation was approximately 100 nucleotides per minute. The lightest particles synthesized plus-strand ssRNA only after completion of dsRNA synthesis, indicating that the same particle contains dsRNA- and ssRNA-synthesizing enzyme(s). We also observed dsRNA-synthesizing activity in L-BC dsRNA-containing particles similar to that in L-A VLPs.

Full text

PDF
4434

Images in this article

4434Image
on p.4434
4434Image
on p.4434
4434Image
on p.4434
4435Image
on p.4435
4435Image
on p.4435
4435Image
on p.4435
4435Image
on p.4435
4435Image
on p.4435
4436Image
on p.4436
4436Image
on p.4436

Selected References

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

  1. 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]
  2. Bobek L. A., Bruenn J. A., Field L. J., Gross K. W. Cloning of cDNA to a yeast viral double-stranded RNA and comparison of three viral RNAs. Gene. 1982 Sep;19(2):225–230. doi: 10.1016/0378-1119(82)90010-5. [DOI] [PubMed] [Google Scholar]
  3. Bostian K. A., Sturgeon J. A., Tipper D. J. Encapsidation of yeast killer double-stranded ribonucleic acids: dependence of M on L. J Bacteriol. 1980 Jul;143(1):463–470. doi: 10.1128/jb.143.1.463-470.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Fujimura T., Wickner R. B. Thermolabile L-A virus-like particles from pet18 mutants of Saccharomyces cerevisiae. Mol Cell Biol. 1986 Feb;6(2):404–410. doi: 10.1128/mcb.6.2.404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hannig E. M., Leibowitz M. J., Wickner R. B. On the mechanism of exclusion of M2 double-stranded RNA by L-A-E double-stranded RNA in Saccharomyces cerevisiae. Yeast. 1985 Sep;1(1):57–65. doi: 10.1002/yea.320010107. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Herring A. J., Bevan E. A. Yeast virus-like particles possess a capsid-associated single-stranded RNA polymerase. Nature. 1977 Aug 4;268(5619):464–466. doi: 10.1038/268464a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Toh-e A., Sahashi Y. The PET18 locus of Saccharomyces cerevisiae: a complex locus containing multiple genes. Yeast. 1985 Dec;1(2):159–171. doi: 10.1002/yea.320010204. [DOI] [PubMed] [Google Scholar]
  21. Van Etten J. L., Burbank D. E., Cuppels D. A., Lane L. C., Vidaver A. K. Semiconservative synthesis of single-stranded RNA by bacteriophage phi 6 RNA polymerase. J Virol. 1980 Feb;33(2):769–773. doi: 10.1128/jvi.33.2.769-773.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. 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]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES