Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 May 25;16(10):4569–4581. doi: 10.1093/nar/16.10.4569

Complementary oligodeoxynucleotide mediated inhibition of tobacco mosaic virus RNA translation in vitro.

C Crum 1, J D Johnson 1, A Nelson 1, D Roth 1
PMCID: PMC336649  PMID: 3380688

Abstract

Two different "antisense" oligodeoxynucleotides and their RNA analogues, each complementary to non-overlapping sequences of 51 bases near the 5' end of TMV RNA, inhibit in vitro translation of the genomic RNA in a rabbit reticulocyte lysate. Inhibition is dependent upon complementarity, concentration, and hybridization of the oligomers with TMV RNA. Inhibition is observed at molar ratios of TMV RNA to antisense oligomers as low as 1:1.5. A plateau of inhibition at which 10-25% of the control signal remains is achieved by molar ratios of TMV RNA:antisense DNA or RNA greater than or equal to 1:15. The extent of inhibition is not increased by the simultaneous presence of both complementary fragments. Oligodeoxynucleotides and their RNA analogues identical to the same regions of TMV RNA have no direct effect on translation, however, they can block inhibition by the antisense fragments. Translation of BMV RNA is not affected by any of the oligodeoxynucleotides. Polyacrylamide gel electrophoresis shows translation of TMV p126 is selectively inhibited. We conclude that the observed inhibition of translation is due to direct interference with ribosome function.

Full text

PDF
4569

Images in this article

4577Image
on p.4577

Selected References

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

  1. Ahlquist P., Strauss E. G., Rice C. M., Strauss J. H., Haseloff J., Zimmern D. Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses. J Virol. 1985 Feb;53(2):536–542. doi: 10.1128/jvi.53.2.536-542.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beachy R. N., Zaitlin M., Bruening G., Israel H. W. A genetic map for the cowpea strain on TMV. Virology. 1976 Sep;73(2):498–507. doi: 10.1016/0042-6822(76)90411-6. [DOI] [PubMed] [Google Scholar]
  3. Blake K. R., Murakami A., Miller P. S. Inhibition of rabbit globin mRNA translation by sequence-specific oligodeoxyribonucleotides. Biochemistry. 1985 Oct 22;24(22):6132–6138. doi: 10.1021/bi00343a015. [DOI] [PubMed] [Google Scholar]
  4. Britten R. J., Graham D. E., Neufeld B. R. Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 1974;29:363–418. doi: 10.1016/0076-6879(74)29033-5. [DOI] [PubMed] [Google Scholar]
  5. Chang L. J., Stoltzfus C. M. Inhibition of Rous sarcoma virus replication by antisense RNA. J Virol. 1987 Mar;61(3):921–924. doi: 10.1128/jvi.61.3.921-924.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coleman J., Hirashima A., Inokuchi Y., Green P. J., Inouye M. A novel immune system against bacteriophage infection using complementary RNA (micRNA). Nature. 1985 Jun 13;315(6020):601–603. doi: 10.1038/315601a0. [DOI] [PubMed] [Google Scholar]
  7. Ecker J. R., Davis R. W. Inhibition of gene expression in plant cells by expression of antisense RNA. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5372–5376. doi: 10.1073/pnas.83.15.5372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Evans R. K., Haley B. E., Roth D. A. Photoaffinity labeling of a viral induced protein from tobacco. Characterization of nucleotide-binding properties. J Biol Chem. 1985 Jun 25;260(12):7800–7804. [PubMed] [Google Scholar]
  9. Filipowicz W., Haenni A. L. Binding of ribosomes to 5'-terminal leader sequences of eukaryotic messenger RNAs. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3111–3115. doi: 10.1073/pnas.76.7.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goelet P., Karn J. Tobacco mosaic virus induces the synthesis of a family of 3' coterminal messenger RNAs and their complements. J Mol Biol. 1982 Jan 25;154(3):541–550. doi: 10.1016/s0022-2836(82)80013-2. [DOI] [PubMed] [Google Scholar]
  11. Goelet P., Lomonossoff G. P., Butler P. J., Akam M. E., Gait M. J., Karn J. Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5818–5822. doi: 10.1073/pnas.79.19.5818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Green P. J., Pines O., Inouye M. The role of antisense RNA in gene regulation. Annu Rev Biochem. 1986;55:569–597. doi: 10.1146/annurev.bi.55.070186.003033. [DOI] [PubMed] [Google Scholar]
  13. Guilley H., Jonard G., Kukla B., Richards K. E. Sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA. Nucleic Acids Res. 1979 Apr;6(4):1287–1308. doi: 10.1093/nar/6.4.1287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gupta K. C. Antisense oligodeoxynucleotides provide insight into mechanism of translation initiation of two Sendai virus mRNAs. J Biol Chem. 1987 Jun 5;262(16):7492–7496. [PubMed] [Google Scholar]
  15. Izant J. G., Weintraub H. Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell. 1984 Apr;36(4):1007–1015. doi: 10.1016/0092-8674(84)90050-3. [DOI] [PubMed] [Google Scholar]
  16. Kawasaki E. S. Quantitative hybridization-arrest of mRNA in Xenopus oocytes using single-stranded complementary DNA or oligonucleotide probes. Nucleic Acids Res. 1985 Jul 11;13(13):4991–5004. doi: 10.1093/nar/13.13.4991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lawson T. G., Ray B. K., Dodds J. T., Grifo J. A., Abramson R. D., Merrick W. C., Betsch D. F., Weith H. L., Thach R. E. Influence of 5' proximal secondary structure on the translational efficiency of eukaryotic mRNAs and on their interaction with initiation factors. J Biol Chem. 1986 Oct 25;261(30):13979–13989. [PubMed] [Google Scholar]
  18. Liebhaber S. A., Cash F. E., Shakin S. H. Translationally associated helix-destabilizing activity in rabbit reticulocyte lysate. J Biol Chem. 1984 Dec 25;259(24):15597–15602. [PubMed] [Google Scholar]
  19. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  20. McGraw R. A., 3rd Dideoxy DNA sequencing with end-labeled oligonucleotide primers. Anal Biochem. 1984 Dec;143(2):298–303. doi: 10.1016/0003-2697(84)90666-3. [DOI] [PubMed] [Google Scholar]
  21. Melton D. A. Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc Natl Acad Sci U S A. 1985 Jan;82(1):144–148. doi: 10.1073/pnas.82.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Minshull J., Hunt T. The use of single-stranded DNA and RNase H to promote quantitative 'hybrid arrest of translation' of mRNA/DNA hybrids in reticulocyte lysate cell-free translations. Nucleic Acids Res. 1986 Aug 26;14(16):6433–6451. doi: 10.1093/nar/14.16.6433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Owens J. R., Haley B. E. A study of adenosine 3'-5' cyclic monophosphate binding sites of human erythrocyte membranes using 8-azidoadenosine 3'-5' cyclic monophosphate, a photoaffinity probe. J Supramol Struct. 1976;5(1):91–102. doi: 10.1002/jss.400050110. [DOI] [PubMed] [Google Scholar]
  24. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  25. Pestka S., Daugherty B. L., Jung V., Hotta K., Pestka R. K. Anti-mRNA: specific inhibition of translation of single mRNA molecules. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7525–7528. doi: 10.1073/pnas.81.23.7525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Scalla R., Romaine P., Asselin A., Rigaud J., Zaitlin M. An in vivo study of a nonstructural polypeptide synthesize upon TMV infection and its identification with a polypeptide synthesized in vitro from TMV RNA. Virology. 1978 Nov;91(1):182–193. doi: 10.1016/0042-6822(78)90366-5. [DOI] [PubMed] [Google Scholar]
  27. Shih D. S., Park I. W., Evans C. L., Jaynes J. M., Palmenberg A. C. Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA. J Virol. 1987 Jun;61(6):2033–2037. doi: 10.1128/jvi.61.6.2033-2037.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Siegel A., Hari V., Kolacz K. The effect of tobacco mosaic virus infection on host and virus-specific protein synthesis in protoplasts. Virology. 1978 Apr;85(2):494–503. doi: 10.1016/0042-6822(78)90456-7. [DOI] [PubMed] [Google Scholar]
  29. Urdea M. S., Ku L., Horn T., Gee Y. G., Warner B. D. Base modification and cloning efficiency of oligodeoxyribonucleotides synthesized by the phosphoramidite method: methyl versus cyanoethyl phosphorus protection. Nucleic Acids Symp Ser. 1985;(16):257–260. [PubMed] [Google Scholar]
  30. Zamecnik P. C., Stephenson M. L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A. 1978 Jan;75(1):280–284. doi: 10.1073/pnas.75.1.280. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES