1887

Abstract

SUMMARY

Viroid-specific polymerase activity was detected in preparations rich in nuclei from infected with citrus exocortis viroid (CEV). The polymerase catalysed the synthesis of several RNAs, shown to be viroid-specific since they could not be observed in control experiments with healthy plants, and they contained CEV-specific sequences most of which were of the same polarity as the viroid RNA. The synthesis of the CEV-specific RNA species was greatly reduced in the presence of 1 μ--amanitin, suggesting the involvement of RNA polymerase II in this process. The structure of the viroid-specific RNA species was studied by chromatography on non-ionic cellulose, digestion with RNase under low and high ionic strength conditions, and analysis by polyacrylamide gel electrophoresis in non-denaturing and denaturing systems. The results showed that these RNAs synthesized contain unit and longer than unit length linear viroid strands forming multistranded complexes with single- and double-stranded regions. The RNAs therefore have the same structural properties as deduced for RNAs isolated from viroid-infected tissues which are the presumed replicative intermediates of the rolling circle mechanism proposed for viroid synthesis. A soluble fraction containing the polymerase-template complex responsible for the synthesis of the CEV-specific RNAs was isolated by treatment of the nuclei-rich preparation with heparin and DNase. This soluble fraction could be of interest in further studies to characterize the components of the polymerase-template complex involved in CEV replication.

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1989-10-01
2022-01-25
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References

  1. Ananiev E., Karagyozov L. 1984; Comparative effect of heparin, sarkosyl and aurintricarboxylic acid on RNA polymerase activity in isolated barley nuclei. Physiologie Végétale 22:555–563
    [Google Scholar]
  2. Branch A. D., Robertson H. D. 1984; A replication cycle for viroids and other small infectious RNAs. Science 223:450–455
    [Google Scholar]
  3. Branch A. D., Robertson H. D., Dickson E. 1981; Longer-than-unit-length viroid minus strands are present in RNA from infected plants. Proceedings of the National Academy of SciencesU.S.A. 786381–6385
    [Google Scholar]
  4. Branch A. D., Robertson H. D., Greer C., Gegenheimer P., Peebles C., Abelson J. 1982; Cell-free circularization of viroid progeny RNA by RNA ligase from wheat germ. Science 217:1147–1149
    [Google Scholar]
  5. Branch A. D., Benenfeld B. J., Robertson H. D. 1988; Evidence for a single rolling circle in the replication of potato spindle tuber viroid. Proceedings of the National Academy of SciencesU.S.A. 859128–9132
    [Google Scholar]
  6. Bruening G., Gould A. R., Murphy P. J., Symons R. H. 1982; Oligomers of avocado sunblotch viroid are found in infected avocado leaves. FEBS Letters 148:71–78
    [Google Scholar]
  7. Colpan M., Schumacher I., Bruggemann W., Sanger H. L., Riesner D. 1983; Large-scale purification of viroid RNA using Cs2SO4 gradient centrifugation and high-performance liquid chromatography. Analytical Biochemistry 131:257–265
    [Google Scholar]
  8. Flores R. 1986; Detection of citrus exocortis viroid in crude extracts by dot-blot hybridization: conditions for reducing spurious hybridization results and for enhancing the sensitivity of the technique. Journal of Virological Methods 13:161–169
    [Google Scholar]
  9. Flores R., Semancik J. S. 1982; Properties of a cell-free system for synthesis of citrus exocortis viroid. Proceedings of the National Academy of SciencesU.S.A. 796285–6288
    [Google Scholar]
  10. Franklin R. M. 1966; Purification and properties of the replicative intermediate of the RNA bacteriophage R 17. Proceedings of the National Academy of SciencesU.S.A. 551504–1511
    [Google Scholar]
  11. Grill L. K., Semancik J. S. 1978; RNA sequences complementary to citrus exocortis viroid in nucleic acid preparations from infected Gynura aurantiaca. Proceedings of the National Academy of SciencesU.S.A. 75896–900
    [Google Scholar]
  12. Hutchins C. J., Keese P., Visvader J. E., Rathjen P. D., Mcinnes J. L., Symons R. H. 1985; Comparison of multimeric plus and minus forms of viroid and virusoids. Plant Molecular Biology 4:293–304
    [Google Scholar]
  13. Hutchins C. J., Rathjen P. D., Forster A. C., Symons R. H. 1986; Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Research 14:3627–3640
    [Google Scholar]
  14. Igloi G. L. 1983; A silver stain for the detection of nanogram amounts of tRNA following two-dimensional electrophoresis. Analytical Biochemistry 134:184–188
    [Google Scholar]
  15. Ishikawa M., Meshi T., Ohno T., Okada Y., Sano T., Ueda I., Shikata E. 1984; A revised replication cycle for viroids: the role of longer than unit length RNA in viroid replication. Molecular and General Genetics 196:421–428
    [Google Scholar]
  16. Keese P., Symons R. H. 1987; The structure of viroids and virusoids. In Viroids and Viroid-like Pathogens. 1–47 Semancik J. S. Boca Raton: CRC Press;
    [Google Scholar]
  17. Kikuchi Y., Tyc K., Filipowicz W., Sänger H. L., Gross H. J. 1982; Circularization of linear viroid RNA via 2′-phosphomonoester, 3′, 5′-phosphodiester bonds by a novel type of RNA ligase from wheat germ and Chlamydomonas . Nucleic Acids Research 10:7521–7529
    [Google Scholar]
  18. Mcmaster G. K., Carmichael G. G. 1977; Analysis of single- and double-stranded nucleic acids on polyacrylarmde and agarose gels by using glyoxal and acridine orange. Proceedings of the National Academy of SciencesU.S.A. 744835–1838
    [Google Scholar]
  19. Marzluff W. F., Huang R. C. C. 1984; Transcription of RNA in isolated nuclei. In Transcription and Translation , a Practical Approach. 89–129 Hames B. D., Higgins S. J. Oxford & Washington D.C.: IRL Press;
    [Google Scholar]
  20. Miller W. A., Hall T. C. 1984; RN A-dependent RNA polymerase isolated from cowpea chlorotic mottle virus-infected cowpeas is specific for bromoviral RNA. Virology 132:53–60
    [Google Scholar]
  21. Morris T. J., Wright N. S. 1975; Detection on polyacrylamide gel of a diagnostic nucleic acid from tissue infected with potato spindle tuber viroid. American Potato Journal 52:57–63
    [Google Scholar]
  22. Mühlbach H.-P., Sanger H. L. 1979; Viroid replication is inhibited by alpha-amanitin. Nature, London 278:185–188
    [Google Scholar]
  23. Mühlbach H.-P., Faustmann O., Sanger H. L. 1983; Conditions for optimal growth of a PSTV-infected potato cell suspension and detection of viroid-complementary longer-than-unit-length RNA in these cells. Plant Molecular Biology 2:239–247
    [Google Scholar]
  24. Owens R. A., Diener T. O. 1982; RNA intermediates in potato spindle tuber viroid replication. Proceedings of the National Academy of SciencesU.S.A. 79113–117
    [Google Scholar]
  25. Rackwitz H. R., Rohde W., Sanger H. L. 1981; DNA-dependent RNA polymerase II of plant origin transcribes viroid RNA into full-length copies. Nature, London 291:297–301
    [Google Scholar]
  26. Robertson H. D., Branch A. D. 1987; The viroid replication process. In Viroids and Viroid-like Pathogens. 49–69 Semancik J. S. Boca Raton: CRC Press;
    [Google Scholar]
  27. Rohozinski J., Francki R. I.B., Chu P. W. G. 1986; The in vitro synthesis of velvet tobacco mottle virus-specific double-stranded RNA by a soluble fraction in extracts from infected Nicotiana clevelandii leaves. Virology 155:27–38
    [Google Scholar]
  28. Sänger H. L., Ramm K., Domdey H., Gross H. I., Henco K., Riesner D. 1979; Conversion of circular viroid molecules to linear strands. FEBS Letters 99:117–122
    [Google Scholar]
  29. Semancik J. S., Harper K. L. 1984; Optimal conditions for cell-free synthesis of citrus exocortis viroid and the question of specificity of RNA polymerase activity. Proceedings of the National Academy of SciencesU.S.A. 814429–4433
    [Google Scholar]
  30. Spiesmacher E., Mühlbach H.-P., Tabler M., Sänger H. L. 1985; Synthesis of (+) and (–) RNA molecules of potato spindle tuber viroid (PSTV) in isolated nuclei and its impairment by transcription inhibitors. Bioscience Reports 5:251–265
    [Google Scholar]
  31. Tabler M., Schnölzer M., Sänger H. L. 1985; Molecular cloning of potato spindle tuber viroid (PSTV) cDNA synthesized by enzymatic elongation of PSTV-specific DNA primers: a general strategy for viroid cloning. Bioscience Reports 5:143–158
    [Google Scholar]
  32. Tsagris M., Tabler M., Mühlbach H.-P., Sänger H. L. 1987; Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells. EMBO Journal 6:2173–2183
    [Google Scholar]
  33. Young N. D., Zaitlin M. 1986; An analysis of tobacco mosaic virus replicative structures synthesized in vitro. Plant Molecular Biology 6:455–465
    [Google Scholar]
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