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. 1984 Dec 20;3(13):3049–3053. doi: 10.1002/j.1460-2075.1984.tb02256.x

Study of the genetic organisation of a plant viral RNA genome by in vitro expression of a full-length DNA copy.

P Vos, J Verver, P van Wezenbeek, A van Kammen, R Goldbach
PMCID: PMC557815  PMID: 6549293

Abstract

The genetic approach for elucidating functions encoded by RNA plant viruses has been hampered by the lack of methods to select desired mutants following random mutagenesis. An alternative might be to copy RNA genomes into DNA and use methods for site-directed mutagenesis to modify specific regions of the DNA copy. Transcription of the DNA copy will subsequently produce viral RNA with desired mutations. We have constructed a full-length DNA copy of the smaller of the two cowpea mosaic virus (CPMV) RNAs, referred to as M RNA. The DNA copy was positioned downstream from the promoter of bacteriophage SP6 and using SP6 RNA polymerase, this copy and two derivatives of it containing a specific deletion and insertion, respectively, have been transcribed into RNA molecules which are efficiently translated in rabbit reticulocyte lysates. The results obtained show that the subsequent in vitro transcription and translation of DNA copies may be a powerful tool to unravel the genetic properties of viral RNA genomes.

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Selected References

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

  1. Daubert S. D., Bruening G., Najarian R. C. Protein bound to the genome RNAs of cowpea mosaic virus. Eur J Biochem. 1978 Dec 1;92(1):45–51. doi: 10.1111/j.1432-1033.1978.tb12721.x. [DOI] [PubMed] [Google Scholar]
  2. Davies J. W., Verver J. W., Goldbach R. W., Van Kammen A. Efficient reverse transcription of cowpea mosaic virus RNAs. Nucleic Acids Res. 1978 Dec;5(12):4643–4661. doi: 10.1093/nar/5.12.4643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Franssen H., Goldbach R., Broekhuijsen M., Moerman M., van Kammen A. Expression of Middle-Component RNA of Cowpea Mosaic Virus: In Vitro Generation of a Precursor to Both Capsid Proteins by a Bottom-Component RNA-Encoded Protease from Infected Cells. J Virol. 1982 Jan;41(1):8–17. doi: 10.1128/jvi.41.1.8-17.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Franssen H., Moerman M., Rezelman G., Goldbach R. Evidence That the 32,000-Dalton Protein Encoded by Bottom-Component RNA of Cowpea Mosaic Virus is a Proteolytic Processing Enzyme. J Virol. 1984 Apr;50(1):183–190. doi: 10.1128/jvi.50.1.183-190.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Godson G. N., Vapnek D. A simple method of preparing large amounts of phiX174 RF 1 supercoiled DNA. Biochim Biophys Acta. 1973 Apr 11;299(4):516–520. doi: 10.1016/0005-2787(73)90223-2. [DOI] [PubMed] [Google Scholar]
  6. Goldbach R. W., Schilthuis J. G., Rezelman G. Comparison of in vivo and in vitro translation of cowpea mosaic virus RNAs. Biochem Biophys Res Commun. 1981 Mar 16;99(1):89–94. doi: 10.1016/0006-291x(81)91716-2. [DOI] [PubMed] [Google Scholar]
  7. Goldbach R., Krijt J. Cowpea Mosaic Virus-Encoded Protease Does Not Recognize Primary Translation Products of M RNAs from Other Comoviruses. J Virol. 1982 Sep;43(3):1151–1154. doi: 10.1128/jvi.43.3.1151-1154.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goldbach R., Rezelman G. Orientation of the cleavage map of the 200-kilodalton polypeptide encoded by the bottom-component RNA of cowpea mosaic virus. J Virol. 1983 May;46(2):614–619. doi: 10.1128/jvi.46.2.614-619.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Green M. R., Maniatis T., Melton D. A. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell. 1983 Mar;32(3):681–694. doi: 10.1016/0092-8674(83)90054-5. [DOI] [PubMed] [Google Scholar]
  10. Huez G., Cleuter Y., Bruck C., Van Vloten-Doting L., Goldbach R., Verduin B. Translational stability of plant viral RNAs microinjected into living cells. Influence of a 3'-poly(A) segment. Eur J Biochem. 1983 Jan 17;130(1):205–209. doi: 10.1111/j.1432-1033.1983.tb07137.x. [DOI] [PubMed] [Google Scholar]
  11. Kassavetis G. A., Butler E. T., Roulland D., Chamberlin M. J. Bacteriophage SP6-specific RNA polymerase. II. Mapping of SP6 DNA and selective in vitro transcription. J Biol Chem. 1982 May 25;257(10):5779–5788. [PubMed] [Google Scholar]
  12. Klein R. D., Selsing E., Wells R. D. A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis. Plasmid. 1980 Jan;3(1):88–91. doi: 10.1016/s0147-619x(80)90037-2. [DOI] [PubMed] [Google Scholar]
  13. Klootwijk J., Klein I., Zabel P., van Kammen A. Cowpea mosaic virus RNAs have neither m7GpppN ... nor mono-, di- or triphosphates at their 5' ends. Cell. 1977 May;11(1):73–82. doi: 10.1016/0092-8674(77)90318-x. [DOI] [PubMed] [Google Scholar]
  14. Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
  15. Kozak M. Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Res. 1981 Oct 24;9(20):5233–5252. doi: 10.1093/nar/9.20.5233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lomonossoff G. P., Shanks M. The nucleotide sequence of cowpea mosaic virus B RNA. EMBO J. 1983;2(12):2253–2258. doi: 10.1002/j.1460-2075.1983.tb01731.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pelham H. R. Synthesis and proteolytic processing of cowpea mosaic virus proteins in reticulocyte lysates. Virology. 1979 Jul 30;96(2):463–477. doi: 10.1016/0042-6822(79)90104-1. [DOI] [PubMed] [Google Scholar]
  20. Rezelman G., Goldbach R., Van Kammen A. Expression of bottom component RNA of cowpea mosaic virus in cowpea protoplasts. J Virol. 1980 Nov;36(2):366–373. doi: 10.1128/jvi.36.2.366-373.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stanley J., Rottier P., Davies J. W., Zabel P., Van Kammen A. A protein linked to the 5' termini of both RNA components of the cowpea mosaic virus genome. Nucleic Acids Res. 1978 Dec;5(12):4505–4522. doi: 10.1093/nar/5.12.4505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Wieslander L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem. 1979 Oct 1;98(2):305–309. doi: 10.1016/0003-2697(79)90145-3. [DOI] [PubMed] [Google Scholar]
  26. Zabel P., Moerman M., van Straaten F., Goldbach R., van Kammen A. Antibodies Against the Genome-Linked Protein VPg of Cowpea Mosaic Virus Recognize a 60,000-Dalton Precursor Polypeptide. J Virol. 1982 Mar;41(3):1083–1088. doi: 10.1128/jvi.41.3.1083-1088.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. van Wezenbeek P., Verver J., Harmsen J., Vos P., van Kammen A. Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus. EMBO J. 1983;2(6):941–946. doi: 10.1002/j.1460-2075.1983.tb01525.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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