1887

Abstract

Full-length cDNA to RNA 1, RNA 2 and RNA 3 of cucumber mosaic virus strain Q (CMV-Q) was amplified using the polymerase chain reaction (PCR). The first-strand primer contained a HI site and sequences complementary to the 3′ terminus of the RNA, The second-strand primers contained a HI site, a T7 promoter and sequences corresponding to the 5′ terminus of each RNA. After cleavage with HI, the PCR products were cloned into the HI site of the vector pEMBL9(+). Five clones of each RNA were selected and RNA transcripts were synthesized from each clone using T7 RNA polymerase. The constructs were designed to allow transcription to initiate precisely at the 5′ terminus of each RNA. All the transcripts were found to be infectious when inoculated onto cv. Samsun plants in sets of three, corresponding to RNA 1, RNA 2 and RNA 3. Of the transcript sets, four induced symptoms indistinguishable from symptoms induced by CMV-Q RNAs. However a fifth transcript set induced much more severe symptoms. Plasmids were also constructed to allow synthesis of transcripts with one or two additional G residues at the 5′ terminus of each RNA. Although the yields of such transcripts synthesized with T7 RNA polymerase were higher, their infectivity was lower than that of those with no additional residues at their 5′ termini.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-71-11-2503
1990-11-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/71/11/JV0710112503.html?itemId=/content/journal/jgv/10.1099/0022-1317-71-11-2503&mimeType=html&fmt=ahah

References

  1. Ahlquist P., Janda M. 1984; cDNA cloning and in vitro transcription of the complete brome mosaic virus genome. Molecular and Cellular Biology 4:2876–2882
    [Google Scholar]
  2. Ahlquist P., French R., Janda M., Loesch-Fries L. S. 1984; Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proceedings of the National Academy of Sciences, U.S.A 81:7066–7070
    [Google Scholar]
  3. Ahlquist P., French R., Bujarski J. J. 1987; Molecular studies of brome mosaic virus using infectious transcripts from cloned cDNA. Advances in Virus Research 32:215–242
    [Google Scholar]
  4. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7:1513–1523
    [Google Scholar]
  5. Chen E. Y., Seeburg P. H. 1985; Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4:165–170
    [Google Scholar]
  6. Dasmahapatra B., Dashupta R., Saunders K., Selling B., Gallagher T., Kaesberg P. 1986; Infectious RNA derived by transcription from cloned cDNA copies of the genomic RNA of an insect virus. Proceedings of the National Academy of Sciences, U.S.A 83:63–66
    [Google Scholar]
  7. Dawson W. O., Beck D. L., Knorr D. A., Grantham G. L. 1986; cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts. Proceedings of the National Academy of Sciences, U.S.A 83:1832–1836
    [Google Scholar]
  8. Dente L., Sollazzo M., Baldari C., Cesarini G., Cortese R. 1985; The pEMBL family of single-stranded vectors. In DNA Cloning: A Practical Approach 1 pp. 101–107 Glover D. M. Edited by Oxford: IRL Press;
    [Google Scholar]
  9. Domier L. L., Franklin K. M., Hunt A. G., Rhoads R. E., Shaw J. G. 1989; Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus. Proceedings of the National Academy of Sciences, U.S.A 86:3509–3513
    [Google Scholar]
  10. Dunn J. J., Studier F. W. 1983; Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. Journal of Molecular Biology 166:477–535
    [Google Scholar]
  11. Eggen R., Verver J., Wellink J., De Jong A., Goldbach R., Van Kammen A. 1989a; Improvements of the infectivity of in vitro transcripts from cloned cowpea mosaic virus cDNA: impact of terminal nucleotide sequences. Virology 173:447–455
    [Google Scholar]
  12. Eggen R., Verver J., Wellink J., Pleij K., Van Kammen A., Goldbach R. 1989b; Analysis of sequences involved in cowpea mosaic virus RNA replication using site-specific mutants. Virology 173:456–464
    [Google Scholar]
  13. Gopinathan K. P., Weymouth L. A., Kunkel T. A., Loeb L. A. 1979; Mutagenesis in vitro by DNA polymerase from an RNA tumour virus. Nature; London: 278:857–859
    [Google Scholar]
  14. Gould A. R., Symons R. H. 1982; Cucumber mosaic virus RNA 3. Determination of the nucleotide sequence provides the amino acid sequences of protein 3A and viral coat protein. European Journal of Biochemistry 126:217–226
    [Google Scholar]
  15. Habili N., Francki R. I. B. 1974; Comparative studies on tomato aspermy and cucumber mosaic virus. I. Physical and chemical properties. Virology 57:392–401
    [Google Scholar]
  16. Heaton L. A., Carrington J. C., Morris T. J. 1989; Turnip crinkle virus infection from RNA synthesized in vitro . Virology 170:214–218
    [Google Scholar]
  17. Janda M., French R., Ahlquist P. 1987; High efficiency T7 polymerase synthesis of infectious RNA from cloned brome mosaic virus cDNA and effects of 5′ extensions on transcript infectivity. Virology 158:259–262
    [Google Scholar]
  18. Kaplan G., Lubinski J., Dasgupta A., Racaniello V. R. 1985; In vitro synthesis of infectious poliovirus RNA. Proceedings of the National Academy of Sciences, U.S.A 82:8424–8428
    [Google Scholar]
  19. Keohavong P., Thilly W. G. 1989; Fidelity of DNA polymerases in DNA amplification. Proceedings of the National Academy of Sciences, U.S.A 86:9253–9257
    [Google Scholar]
  20. Kunkel T. A., Roberts J. D., Zakour R. A. 1987; Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods in Enzymology 154:367–382
    [Google Scholar]
  21. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
    [Google Scholar]
  22. Lot H., Marchoux G., Marrou J., Kaper J. M., West C. K., Van Vloten-Doting L., Hull R. 1974; Evidence for three functional RNA species in several strains of cucumber mosaic virus. Journal of General Virology 22:81–93
    [Google Scholar]
  23. Mead D. A., Szczesna-Skorupa E., Kemper B. 1986; Singlestranded DNA ‘blue’ T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Engineering 1:67–74
    [Google Scholar]
  24. Meshi T., Ishakawa M., Motoyoshi F., Semba K., Okada Y. 1986; In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus. Proceedings of the National Academy of Sciences, U.S.A 83:5043–5047
    [Google Scholar]
  25. Osman T. A. M., Buck K. W. 1989; Properties of three spontaneous mutants of red clover necrotic mosaic virus. Journal of General Virology 70:491–497
    [Google Scholar]
  26. Osman T. A. M., Dodd S. M., Buck K. W. 1986; RNA 2 of red clover necrotic mosaic virus determines lesion morphology and systemic invasion in cowpea. Journal of General Virology 67:203–207
    [Google Scholar]
  27. Peden K. W. C., Symons R. H. 1973; Cucumber mosaic virus contains a functionally divided genome. Virology 53:487–492
    [Google Scholar]
  28. Petty I. T. D., Hunter B. G., Wei N., Jackson A. O. 1989; Infectious barley stripe mosaic virus RNA transcribed in vitro from full-length genomic cDNA clones. Virology 171:342–349
    [Google Scholar]
  29. Quillet L., Guilley H., Jonard G., Richards K. 1989; In vitro synthesis of biologically active beet necrotic yellow vein virus RNA. Virology 172:293–301
    [Google Scholar]
  30. Rezaian M. A., Williams R. H. V., Gordon K. J. H., Gould A. R., Symons R. H. 1984; Nucleotide sequence of cucumber mosaic virus RNA 2 reveals a translation product significantly homologous to corresponding proteins of other viruses. European Journal of Biochemistry 143:277–284
    [Google Scholar]
  31. Rezaian M. A., Williams R. H. V., Symons R. H. 1985; Nucleotide sequence of cucumber mosaic virus RNA 1. Presence of a sequence complementary to part of the viral satellite RNA and homologies with other viral RNAs. European Journal of Biochemistry 150:331–339
    [Google Scholar]
  32. Rice C. M., Levis R., Strauss J. H., Huang H. V. 1987; Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker and in vitro mutagenesis to generate defined mutants. Journal of Virology 61:3809–3819
    [Google Scholar]
  33. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. 1988; Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
    [Google Scholar]
  34. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  35. Shaklee P. N., Miglietta J. J., Palmenberg A. C., Kaesberg P. 1988; Infectious positive- and negative-strand transcript RNAs from bacteriophage Qβ cDNA clones. Virology 163:209–213
    [Google Scholar]
  36. Steinhauer D. A., Holland J. J. 1987; Rapid evolution of RNA viruses. Annual Review of Microbiology 41:409–433
    [Google Scholar]
  37. Van Der Werf S., Bradley J., Wimmer E., Studier F. W., Dunn J. J. 1986; Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Proceedings of the National Academy of Sciences, U.S.A 83:2330–2334
    [Google Scholar]
  38. Vos P., Jaegle M., Wellink J., Verver J., Eggen R. A., Goldbach R. 1988; Infectious RNA transcripts from full-length DNA copies of the genomic RNAs of cowpea mosaic virus. Virology 166:33–41
    [Google Scholar]
  39. Weiland J. J., Dreher T. W. 1989; Infectious TYMV RNA from cloned cDNA: effects in vitro and in vivo of point substitutions in the initiation codons of two extensively overlapping ORFs. Nucleic Acids Research 17:4675–1687
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-71-11-2503
Loading
/content/journal/jgv/10.1099/0022-1317-71-11-2503
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error