Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1986 Apr 25;14(8):3215–3228. doi: 10.1093/nar/14.8.3215

The stiffness of dsRNA: hydrodynamic studies on fluorescence-labelled RNA segments of bovine rotavirus.

R Kapahnke, W Rappold, U Desselberger, D Riesner
PMCID: PMC339748  PMID: 3010231

Abstract

The sedimentation coefficients of dsRNA segments of bovine rotavirus were determined in the analytical ultracentrifuge. The eleven segments were separated by preparative gel electrophoresis, and isolated by elution from gel pieces. The RNA was labelled by the intercalating fluorescent dye ethidium bromide at a ratio bound dye per base pair between 0.003 to 0.018. The analytical ultracentrifuge was equipped with a fluorescence recording optics. Sedimentation coefficients could be determined with amounts of RNA as little as 8 ng. All sedimentation coefficients were extrapolated to zero-concentration, zero-dye binding, and zero-impurities from the preparative gel electrophoresis. The hydrodynamic model of flexible cylinders was applied for the interpretation of the sedimentation coefficients. All dsRNA segments of rotavirus (663-3409 base pairs) and the dsRNA5 of cucumber mosaic virus (335 base pairs) fit the model of a "worm-like" or flexible cylinder with a persistence length of 1125 A and a hydrated diameter of 30 A. The results are compared with data from the literature on the persistence lengths of the B- and Z-forms of dsDNA and of viroids.

Full text

PDF
3215

Images in this article

Selected References

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

  1. Baybutt H. N., McCrae M. A. The molecular biology of rotaviruses. VII. Detailed structural analysis of gene 10 of bovine rotavirus. Virus Res. 1984 Oct;1(7):533–541. doi: 10.1016/0168-1702(84)90011-x. [DOI] [PubMed] [Google Scholar]
  2. Bodkin D. K., Knudson D. L. Sequence relatedness of Palyam virus genes to cognates of the Palyam serogroup viruses by RNA-RNA blot hybridization. Virology. 1985 May;143(1):55–62. doi: 10.1016/0042-6822(85)90096-0. [DOI] [PubMed] [Google Scholar]
  3. Both G. W., Siegman L. J., Bellamy A. R., Ikegami N., Shatkin A. J., Furuichi Y. Comparative sequence analysis of rotavirus genomic segment 6--the gene specifying viral subgroups 1 and 2. J Virol. 1984 Jul;51(1):97–101. doi: 10.1128/jvi.51.1.97-101.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buck K. W., Ackermann H. W., Bozarth R. F., Bruenn J. A., Koltin Y., Rawlinson C. J., Ushiyama R., Wood H. A. Six groups of double-stranded RNA mycoviruses. Intervirology. 1984;22(1):17–23. doi: 10.1159/000149529. [DOI] [PubMed] [Google Scholar]
  5. Cohen J., Lefevre F., Estes M. K., Bremont M. Cloning of bovine rotavirus (RF strain): nucleotide sequence of the gene coding for the major capsid protein. Virology. 1984 Oct 15;138(1):178–182. doi: 10.1016/0042-6822(84)90159-4. [DOI] [PubMed] [Google Scholar]
  6. Diaz-Ruiz J. R., Kaper J. M. Cucumber mosaic virus-associated RNA 5. VI. Characterization and denaturation-renaturation behavior of the double-stranded form. Biochim Biophys Acta. 1979 Sep 27;564(2):275–288. doi: 10.1016/0005-2787(79)90225-9. [DOI] [PubMed] [Google Scholar]
  7. Dobos P., Hill B. J., Hallett R., Kells D. T., Becht H., Teninges D. Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes. J Virol. 1979 Nov;32(2):593–605. doi: 10.1128/jvi.32.2.593-605.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dyall-Smith M. L., Elleman T. C., Hoyne P. A., Holmes I. H., Azad A. A. Cloning and sequence of UK bovine rotavirus gene segment 7: marked sequence homology with simian rotavirus gene segment 8. Nucleic Acids Res. 1983 May 25;11(10):3351–3362. doi: 10.1093/nar/11.10.3351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elleman T. C., Hoyne P. A., Dyall-Smith M. L., Holmes I. H., Azad A. A. Nucleotide sequence of the gene encoding the serotype-specific glycoprotein of UK bovine rotavirus. Nucleic Acids Res. 1983 Jul 25;11(14):4689–4701. doi: 10.1093/nar/11.14.4689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Etten J. V., Lane L., Gonzalez C., Partridge J., Vidaver A. Comparative properties of bacteriophage phi6 and phi6 nucleocapsid. J Virol. 1976 May;18(2):652–658. doi: 10.1128/jvi.18.2.652-658.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Follett E. A., Desselberger U. Cocirculation of different rotavirus strains in a local outbreak of infantile gastroenteritis: monitoring by rapid and sensitive nucleic acid analysis. J Med Virol. 1983;11(1):39–52. doi: 10.1002/jmv.1890110106. [DOI] [PubMed] [Google Scholar]
  12. Garcia de la Torre J. G., Bloomfield V. A. Hydrodynamic properties of complex, rigid, biological macromolecules: theory and applications. Q Rev Biophys. 1981 Feb;14(1):81–139. doi: 10.1017/s0033583500002080. [DOI] [PubMed] [Google Scholar]
  13. Gaugain B., Barbet J., Capelle N., Roques B. P., Le Pecq J. B. DNA Bifunctional intercalators. 2. Fluorescence properties and DNA binding interaction of an ethidium homodimer and an acridine ethidium heterodimer. Biochemistry. 1978 Nov 28;17(24):5078–5088. doi: 10.1021/bi00617a002. [DOI] [PubMed] [Google Scholar]
  14. Goodman T. C., Nagel L., Rappold W., Klotz G., Riesner D. Viroid replication: equilibrium association constant and comparative activity measurements for the viroid-polymerase interaction. Nucleic Acids Res. 1984 Aug 10;12(15):6231–6246. doi: 10.1093/nar/12.15.6231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Imai M., Richardson M. A., Ikegami N., Shatkin A. J., Furuichi Y. Molecular cloning of double-stranded RNA virus genomes. Proc Natl Acad Sci U S A. 1983 Jan;80(2):373–377. doi: 10.1073/pnas.80.2.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kam Z., Borochov N., Eisenberg H. Dependence of laser light scattering of DNA on NaCl concentration. Biopolymers. 1981 Dec;20(12):2671–2690. doi: 10.1002/bip.1981.360201213. [DOI] [PubMed] [Google Scholar]
  17. Kovacic R. T., van Holde K. E. Sedimentation of homogeneous double-strand DNA molecules. Biochemistry. 1977 Apr 5;16(7):1490–1498. doi: 10.1021/bi00626a038. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Müller W., Crothers D. M. Studies of the binding of actinomycin and related compounds to DNA. J Mol Biol. 1968 Jul 28;35(2):251–290. doi: 10.1016/s0022-2836(68)80024-5. [DOI] [PubMed] [Google Scholar]
  20. Nelson J. W., Martin F. H., Tinoco I., Jr DNA and RNA oligomer thermodynamics: the effect of mismatched bases on double-helix stability. Biopolymers. 1981 Dec;20(12):2509–2531. doi: 10.1002/bip.1981.360201204. [DOI] [PubMed] [Google Scholar]
  21. Riesner D., Kaper J. M., Randles J. W. Stiffness of viroids and viroid-like RNA in solution. Nucleic Acids Res. 1982 Sep 25;10(18):5587–5598. doi: 10.1093/nar/10.18.5587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rixon F., Taylor P., Desselberger U. Rotavirus RNA segments sized by electron microscopy. J Gen Virol. 1984 Jan;65(Pt 1):233–239. doi: 10.1099/0022-1317-65-1-233. [DOI] [PubMed] [Google Scholar]
  23. Rosenberg J. M., Seeman N. C., Day R. O., Rich A. RNA double-helical fragments at atomic resolution. II. The crystal structure of sodium guanylyl-3',5'-cytidine nonahydrate. J Mol Biol. 1976 Jun 14;104(1):145–167. doi: 10.1016/0022-2836(76)90006-1. [DOI] [PubMed] [Google Scholar]
  24. Seeman N. C., Rosenberg J. M., Suddath F. L., Kim J. J., Rich A. RNA double-helical fragments at atomic resolution. I. The crystal and molecular structure of sodium adenylyl-3',5'-uridine hexahydrate. J Mol Biol. 1976 Jun 14;104(1):109–144. doi: 10.1016/0022-2836(76)90005-x. [DOI] [PubMed] [Google Scholar]
  25. Spiesmacher E., Mühlbach H. P., Tabler M., Sänger H. L. Synthesis of (+) and (-) RNA molecules of potato spindle tuber viroid (PSTV) in isolated nuclei and its impairment by transcription inhibitors. Biosci Rep. 1985 Mar;5(3):251–265. doi: 10.1007/BF01119595. [DOI] [PubMed] [Google Scholar]
  26. Taylor P., Rixon F., Desselberger U. Rise per base pair in helices of double-stranded rotavirus RNA determined by electron microscopy. Virus Res. 1985 Mar;2(2):175–182. doi: 10.1016/0168-1702(85)90247-3. [DOI] [PubMed] [Google Scholar]
  27. Thomas T. J., Bloomfield V. A. Chain flexibility and hydrodynamics of the B and Z forms of poly(dG-dC).poly(dG-dC). Nucleic Acids Res. 1983 Mar 25;11(6):1919–1930. doi: 10.1093/nar/11.6.1919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ward C. W., Elleman T. C., Azad A. A., Dyall-Smith M. L. Nucleotide sequence of gene segment 9 encoding a nonstructural protein of UK bovine rotavirus. Virology. 1984 Apr 15;134(1):249–253. doi: 10.1016/0042-6822(84)90292-7. [DOI] [PubMed] [Google Scholar]
  29. Wickner R. B. The killer double-stranded RNA plasmids of yeast. Plasmid. 1979 Jul;2(3):303–322. doi: 10.1016/0147-619x(79)90015-5. [DOI] [PubMed] [Google Scholar]
  30. Wu H. M., Dattagupta N., Crothers D. M. Solution structural studies of the A and Z forms of DNA. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6808–6811. doi: 10.1073/pnas.78.11.6808. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES