Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1984 Aug 1;99(2):672–679. doi: 10.1083/jcb.99.2.672

Drug-induced dispersal of transcribed rRNA genes and transcriptional products: immunolocalization and silver staining of different nucleolar components in rat cells treated with 5,6-dichloro-beta-D- ribofuranosylbenzimidazole

PMCID: PMC2113249  PMID: 6204996

Abstract

Upon incubation of cultured rat cells with the adenosine analogue 5,6- dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), nucleoli reversibly dissociate into their substructures, disperse throughout the nuclear interior, and form nucleolar "necklaces". We have used this experimental system, which does not inhibit transcription of the rRNA genes, to study by immunocytochemistry the distribution of active rRNA genes and their transcriptional products during nucleolar dispersal and recovery to normal morphology. Antibodies to RNA polymerase I allow detection of template-engaged polymerase, and monoclonal antibodies to a ribosomal protein (S1) of the small ribosomal subunit permit localization of nucleolar preribosomal particles. The results show that, under the action of DRB transcribed rRNA, genes spread throughout the nucleoplasm and finally appear in the form of several rows, each containing several (up to 30) granules positive for RNA polymerase I and argyrophilic proteins. Nucleolar material containing preribosomal particles also appears in granular structures spread over the nucleoplasm but its distribution is distinct from that of rRNA gene- containing granules. We conclude that, although transcriptional units and preribosomal particles are both redistributed in response to DRB, these entities retain their individuality as functionally defined subunits. We further propose that each RNA polymerase-positive granular unit represents a single transcription unit and that each continuous array of granules ("string of nucleolar beads") reflects the linear distribution of rRNA genes along a nucleolar organizer region. Based on the total number of polymerase I-positive granules we estimate that a minimum of 60 rRNA genes are active during interphase of DRB-treated rat cells.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Angelier N., Hernandez-Verdun D., Bouteille M. Visualization of Ag-NOR proteins on nucleolar transcriptional units in molecular spreads. Chromosoma. 1982;86(5):661–672. doi: 10.1007/BF00285609. [DOI] [PubMed] [Google Scholar]
  2. Arrighi F. E., Lau Y. F., Spallone A. Nucleolar activity in differentiated cells after stimulation. Cytogenet Cell Genet. 1980;26(2-4):244–250. doi: 10.1159/000131445. [DOI] [PubMed] [Google Scholar]
  3. Bernhard W. Drug-induced changes in the interphase nucleus. Adv Cytopharmacol. 1971 May;1:49–67. [PubMed] [Google Scholar]
  4. Bouteille M., Bouvier D., Seve A. P. Heterogeneity and territorial organization of the nuclear matrix and related structures. Int Rev Cytol. 1983;83:135–182. doi: 10.1016/s0074-7696(08)61687-3. [DOI] [PubMed] [Google Scholar]
  5. Busch H., Daskal Y., Gyorkey F., Smetana K. Silver staining of nucleolar granules in tumor cells. Cancer Res. 1979 Mar;39(3):857–863. [PubMed] [Google Scholar]
  6. De Capoa A., Pelliccia F., Marlekaj P., Ciofi-Luzzatto A. R., Buongiorno Nardelli M. Silver positivity of the NORs during embryonic development of Xenopus laevis. Exp Cell Res. 1983 Sep;147(2):472–478. doi: 10.1016/0014-4827(83)90231-8. [DOI] [PubMed] [Google Scholar]
  7. Derenzini M., Hernandez-Verdun D., Pession A., Novello F. Structural organization of chromatin in nucleolar organizer regions of nucleoli with a nucleolonema-like and compact ribonucleoprotein distribution. J Ultrastruct Res. 1983 Aug;84(2):161–172. doi: 10.1016/s0022-5320(83)90127-2. [DOI] [PubMed] [Google Scholar]
  8. Derenzini M., Pession A., Betts-Eusebi C. M., Novello F. Relationship between the extended, non-nucleosomal intranucleolar chromatin in situ and ribosomal RNA synthesis. Exp Cell Res. 1983 Apr 15;145(1):127–143. doi: 10.1016/s0014-4827(83)80015-9. [DOI] [PubMed] [Google Scholar]
  9. Egyházi E. Inhibition of Balbiani ring RNA synthesis at the initiation level. Proc Natl Acad Sci U S A. 1975 Mar;72(3):947–950. doi: 10.1073/pnas.72.3.947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Egyházi E., Pigon A., Rydlander L. 5,6-Dichlororibofuranosylbenzimidazole inhibits the rate of transcription initiation in intact Chironomus cells. Eur J Biochem. 1982 Mar 1;122(3):445–451. doi: 10.1111/j.1432-1033.1982.tb06458.x. [DOI] [PubMed] [Google Scholar]
  11. Fakan S., Puvion E. The ultrastructural visualization of nucleolar and extranucleolar RNA synthesis and distribution. Int Rev Cytol. 1980;65:255–299. doi: 10.1016/s0074-7696(08)61962-2. [DOI] [PubMed] [Google Scholar]
  12. Franke W. W., Denk H., Kalt R., Schmid E. Biochemical and immunological identification of cytokeratin proteins present in hepatocytes of mammalian liver tissue. Exp Cell Res. 1981 Feb;131(2):299–318. doi: 10.1016/0014-4827(81)90234-2. [DOI] [PubMed] [Google Scholar]
  13. Franke W. W., Schmid E., Vandekerckhove J., Weber K. Permanently proliferating rat vascular smooth muscle cell with maintained expression of smooth muscle characteristics, including actin of the vascular smooth muscle type. J Cell Biol. 1980 Dec;87(3 Pt 1):594–600. doi: 10.1083/jcb.87.3.594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Granick D. Nucleolar necklaces in chick embryo fibroblast cells. I. Formation of necklaces by dichlororibobenzimidazole and other adenosine analogues that decrease RNA synthesis and degrade preribosomes. J Cell Biol. 1975 May;65(2):398–417. doi: 10.1083/jcb.65.2.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Granick D. Nucleolar necklaces in chick embryo fibroblast cells. II. Microscope observations of the effect of adenosine analogues on nucleolar necklace formation. J Cell Biol. 1975 May;65(2):418–427. doi: 10.1083/jcb.65.2.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hancock R., Boulikas T. Functional organization in the nucleus. Int Rev Cytol. 1982;79:165–214. doi: 10.1016/s0074-7696(08)61674-5. [DOI] [PubMed] [Google Scholar]
  17. Hernandez-Verdun D., Derenzini M., Bouteille M. The morphological relationship in electron microscopy between NOR-silver proteins and intranucleolar chromatin. Chromosoma. 1982;85(4):461–473. doi: 10.1007/BF00327343. [DOI] [PubMed] [Google Scholar]
  18. Hernandez-Verdun D., Hubert J., Bourgeois C. A., Bouteille M. Ultrastructural localization of Ag-NOR stained proteins in the nucleolus during the cell cycle and in other nucleolar structures. Chromosoma. 1980;79(3):349–362. doi: 10.1007/BF00327325. [DOI] [PubMed] [Google Scholar]
  19. Hofgärtner F. J., Krone W., Jain K. Correlated inhibition of ribosomal RNA synthesis and silver staining by actinomycin D. Hum Genet. 1979 Apr 5;47(3):329–333. doi: 10.1007/BF00321025. [DOI] [PubMed] [Google Scholar]
  20. Hubbell H. R., Lau Y. F., Brown R. L., Hsu T. C. Cell cycle analysis and drug inhibition studies of silver staining in synchronous HeLa cells. Exp Cell Res. 1980 Sep;129(1):139–147. doi: 10.1016/0014-4827(80)90339-0. [DOI] [PubMed] [Google Scholar]
  21. Martini O. H., Gould H. J. Molecular weight distribution of ribosomal proteins from several vertebrate species. Mol Gen Genet. 1976 Dec 31;142(4):317–331. doi: 10.1007/BF00271255. [DOI] [PubMed] [Google Scholar]
  22. Puvion-Dutilleul F., Bernadac A., Puvion E., Bernhard W. Visualization of two different types of nuclear transcriptional complexes in rat liver cells. J Ultrastruct Res. 1977 Jan;58(1):108–117. doi: 10.1016/s0022-5320(77)80012-9. [DOI] [PubMed] [Google Scholar]
  23. Puvion-Dutilleul F., Nicoloso M., Bachellerie J. P. Altered structure of ribosomal RNA transcription units in hamster cells after DRB treatment. Exp Cell Res. 1983 Jun;146(1):43–52. doi: 10.1016/0014-4827(83)90322-1. [DOI] [PubMed] [Google Scholar]
  24. Puvion E., Puvion-Dutilleul F., Leduc E. H. The formation of nucleolar perichromatin granules. J Ultrastruct Res. 1981 Aug;76(2):181–191. doi: 10.1016/s0022-5320(81)80016-0. [DOI] [PubMed] [Google Scholar]
  25. Rose K. M., Stetler D. A., Jacob S. T. Protein kinase activity of RNA polymerase I purified from a rat hepatoma: probable function of Mr 42,000 and 24,600 polypeptides. Proc Natl Acad Sci U S A. 1981 May;78(5):2833–2837. doi: 10.1073/pnas.78.5.2833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Scheer U., Lanfranchi G., Rose K. M., Franke W. W., Ringertz N. R. Migration of rat RNA polymerase I into chick erythrocyte nuclei undergoing reactivation in chick-rat heterokaryons. J Cell Biol. 1983 Nov;97(5 Pt 1):1641–1643. doi: 10.1083/jcb.97.5.1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Scheer U., Rose K. M. Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1431–1435. doi: 10.1073/pnas.81.5.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schmid M., Müller H., Stasch S., Engel W. Silver staining of nucleolus organizer regions during human spermatogenesis. Hum Genet. 1983;64(4):363–370. doi: 10.1007/BF00292368. [DOI] [PubMed] [Google Scholar]
  29. Schwarzacher H. G., Mikelsaar A. V., Schnedl W. The nature of the Ag-staining of nucleolus organizer regions. Electron- and light-microscopic studies on human cells in interphase, mitosis, and meiosis. Cytogenet Cell Genet. 1978;20(1-6):24–39. doi: 10.1159/000130837. [DOI] [PubMed] [Google Scholar]
  30. Sehgal P. B., Darnell J. E., Jr, Tamm I. The inhibition by DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) of hnRNA and mRNA production in HeLa cells. Cell. 1976 Nov;9(3):473–480. doi: 10.1016/0092-8674(76)90092-1. [DOI] [PubMed] [Google Scholar]
  31. Stumph W. E., Wu J. R., Bonner J. Determination of the size of rat ribosomal deoxyribonucleic acid repeating units by electron microscopy. Biochemistry. 1979 Jun 26;18(13):2864–2871. doi: 10.1021/bi00580a030. [DOI] [PubMed] [Google Scholar]
  32. Tamm I., Hand R., Caliguiri L. A. Action of dichlorobenzimidazole riboside on RNA synthesis in L-929 and HeLa cells. J Cell Biol. 1976 May;69(2):229–240. doi: 10.1083/jcb.69.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tamm I., Kikuchi T. Early termination of heterogeneous nuclear RNA transcripts in mammalian cells: accentuation by 5,6-dichloro 1-beta-D-ribofuranosylbenzimidazole. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5750–5754. doi: 10.1073/pnas.76.11.5750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Trendelenburg M. F., Scheer U., Franke W. W. Structural organization of the transcription of ribosomal DNA in oocytes of the house cricket. Nat New Biol. 1973 Oct 10;245(145):167–170. doi: 10.1038/newbio245167a0. [DOI] [PubMed] [Google Scholar]
  35. Zandomeni R., Bunick D., Ackerman S., Mittleman B., Weinmann R. Mechanism of action of DRB. III. Effect on specific in vitro initiation of transcription. J Mol Biol. 1983 Jul 5;167(3):561–574. doi: 10.1016/s0022-2836(83)80098-9. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES