Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1985 Jan 25;13(2):355–367. doi: 10.1093/nar/13.2.355

Complexes of viroids with histones and other proteins.

P Wolff, R Gilz, J Schumacher, D Riesner
PMCID: PMC341000  PMID: 3889835

Abstract

Complexes of potato spindle tuber viroid (PSTV) with nuclear proteins have been studied by in vitro reconstitution of the complexes and by isolation and characterization of in vivo complexes under non-dissociating conditions. For in vitro reconstitution, nuclear proteins were separated by SDS-gel-electrophoresis, renatured and blotted onto nitrocellulose filters, and incubated with viroid. The viroid-protein complexes were crosslinked covalently, and the viroid containing protein bands were detected by northern hybridization with a radioactive cDNA probe. The histones, a 41,000 dalton protein and to a small extent a 31,000 dalton protein were found in complexes with viroids. Raising the strength to 0.4 M NaCl destroys the complexes with the 41,000 dalton proteins but not those with the histones. From nucleoli, which are known to obtain the majority of viroids under non-dissociating conditions (Schumacher et al., (1983) EMBO J. 2, 1549-1555), a nucleosomal fraction was prepared. Viroids were found predominantly in this nucleosomal fraction. They are bound in a complex of 12-15 svedberg units.

Full text

PDF
361

Images in this article

Selected References

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

  1. Alberts B. M., Amodio F. J., Jenkins M., Gutmann E. D., Ferris F. L. Studies with DNA-cellulose chromatography. I. DNA-binding proteins from Escherichia coli. Cold Spring Harb Symp Quant Biol. 1968;33:289–305. doi: 10.1101/sqb.1968.033.01.033. [DOI] [PubMed] [Google Scholar]
  2. Bloom K. S., Anderson J. N. Fractionation and characterization of chromosomal proteins by the hydroxyapatite dissociation method. J Biol Chem. 1978 Jun 25;253(12):4446–4450. [PubMed] [Google Scholar]
  3. Bowen B., Steinberg J., Laemmli U. K., Weintraub H. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 1980 Jan 11;8(1):1–20. doi: 10.1093/nar/8.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chaconas G., van de Sande J. H. 5'-32P labeling of RNA and DNA restriction fragments. Methods Enzymol. 1980;65(1):75–85. doi: 10.1016/s0076-6879(80)65012-5. [DOI] [PubMed] [Google Scholar]
  5. Colpan M., Schumacher J., Brüggemann W., Sänger H. L., Riesner D. Large-scale purification of viroid RNA using Cs2SO4 gradient centrifugation and high-performance liquid chromatography. Anal Biochem. 1983 May;131(1):257–265. doi: 10.1016/0003-2697(83)90164-1. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Gross H. J., Riesner D. Viroids: a class of subviral pathogens. Angew Chem Int Ed Engl. 1980;19(4):231–243. doi: 10.1002/anie.198002313. [DOI] [PubMed] [Google Scholar]
  8. Hillen W., Klein R. D., Wells R. D. Preparation of milligram amounts of 21 deoxyribonucleic acid restriction fragments. Biochemistry. 1981 Jun 23;20(13):3748–3756. doi: 10.1021/bi00516a013. [DOI] [PubMed] [Google Scholar]
  9. Jendrisak J. J., Burgess R. R. A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II. Biochemistry. 1975 Oct 21;14(21):4639–4645. doi: 10.1021/bi00692a012. [DOI] [PubMed] [Google Scholar]
  10. Jendrisak J. J., Burgess R. R. Studies of the subunit structure of wheat germ ribonucleic acid polymerase II. Biochemistry. 1977 May 3;16(9):1959–1964. doi: 10.1021/bi00628a032. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  13. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  14. Palen T. E., Cech T. R. Chromatin structure at the replication origins and transcription-initiation regions of the ribosomal RNA genes of Tetrahymena. Cell. 1984 Apr;36(4):933–942. doi: 10.1016/0092-8674(84)90043-6. [DOI] [PubMed] [Google Scholar]
  15. Rackwitz H. R., Rohde W., Sänger H. L. DNA-dependent RNA polymerase II of plant origin transcribes viroid RNA into full-length copies. Nature. 1981 May 28;291(5813):297–301. doi: 10.1038/291297a0. [DOI] [PubMed] [Google Scholar]
  16. Renz M. Polynucleotide-histone H1 complexes as probes for blot hybridization. EMBO J. 1983;2(6):817–822. doi: 10.1002/j.1460-2075.1983.tb01508.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Riesner D., Henco K., Rokohl U., Klotz G., Kleinschmidt A. K., Domdey H., Jank P., Gross H. J., Sänger H. L. Structure and structure formation of viroids. J Mol Biol. 1979 Sep 5;133(1):85–115. doi: 10.1016/0022-2836(79)90252-3. [DOI] [PubMed] [Google Scholar]
  18. Schumacher J., Randles J. W., Riesner D. A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids. Anal Biochem. 1983 Dec;135(2):288–295. doi: 10.1016/0003-2697(83)90685-1. [DOI] [PubMed] [Google Scholar]
  19. Schumacher J., Sänger H. L., Riesner D. Subcellular localization of viroids in highly purified nuclei from tomato leaf tissue. EMBO J. 1983;2(9):1549–1555. doi: 10.1002/j.1460-2075.1983.tb01622.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. van Wezenbeek P., Vos P., van Boom J., van Kammen A. Molecular cloning and characterization of a complete DNA copy of potato spindle tuber viroid RNA. Nucleic Acids Res. 1982 Dec 20;10(24):7947–7957. doi: 10.1093/nar/10.24.7947. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES