Skip to main content
SpringerLink
Log in

Immunolocalization of DNA at nucleolar structural components in onion cells

  • Published:
Chromosoma Aims and scope Submit manuscript

Abstract

Intranucleolar DNA, including ribosomal DNA (rDNA), was localized in situ in proliferating onion cells under the electron microscope using an anti-DNA monoclonal antibody and a postembedding indirect immunogold procedure. In the interphase nucleolus of this species, characterized by a very high amount of rRNA genes, we found DNA concentrated mostly in fibrillar centres (FCs) and in the region of the dense fibrillar component (DFC) immediately surrounding them. Clusters of gold particles were frequently seen covering both of these structural components of the nucleolus at the same time. Moreover, the same technique, applied to transcriptionally arrested quiescent onion cells, showed the nucleolar DFC devoid of DNA. Also, in mitotic cells at telophase, the prenucleolar material, which has the same morphological and cytochemical features as the DFC, does not contain DNA. These data suggest the existence of at least two subcomponents of the DFC in the onion cell nucleolus, one associated with pre-rRNA synthesis, and the other, with further processing of transcripts, already released from the rDNA template. We conclude that the first subcomponent forms part of the “transition between FC and DFC”, which is the in situ structural counterpart of pre-rRNA synthesis. This transition is morphologicaly sizeable in onion cells, because of their high number of rRNA genes and the large size of the DFC mass; however, it would be barely detectable in situ in other cell systems, where the whole DFC comprises only a thin layer and the amount of rDNA is considerably reduced.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

FC(s) :

fibrillar centre(s)

DFC :

dense fibrillar component

rDNA :

ribosomal DNA

rRNA :

ribosomal RNA

NOR :

nucleolar organizer region

PBS :

phosphate buffered saline

References

  • Alberts B, Bray D, Lewis J, Rall M, Robert K, Watson JD (1983) Molecular biology of the cell. Garland Publ, New York, p 1146

    Google Scholar 

  • Arroua ML, Hartung M, Devictor M, Berge-Lefranc JL, Stahl A (1982) Localization of ribosomal genes by in situ hybridization in the fibrillar centre of the nucleolus in the human spermatocyte. Biol Cell 44:337–340

    Google Scholar 

  • Benavente R, Reimer G, Rose KM, Hugle-Dorr B, Scheer U (1988) Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells. Chromosoma 97:115–123

    Google Scholar 

  • Brill SJ, Di Nardo S, Voelkel-Meiman K, Sternglanz R (1987) Need for DNA topoisomerase activity as a swivel for DNA replication and for transcription of ribosomal RNA. Nature 326:414–416

    Google Scholar 

  • Busch H, Lischwe MA, Michalik J, Chan PK, Busch RK (1982) Nucleolar proteins of special interest: silver-staining proteins B23 and C23 and antigens of human tumour nucleoli. In: Jordan EG, Cullis CA (eds) The nucleolus. Cambridge University Press, pp 43–71

  • Caizergues-Ferrer M, Belenguer P, Lapeyre B, Amalric F, Wallace MO, Olson MOJ (1987) Phosphorylation of nucleolin by a nucleolar type NII protein kinase. Biochemistry 26:7876–7883

    Google Scholar 

  • Deltour R, Mosen H (1987) Proposals for the macromolecular organization of the higher plant nucleolonema. Biol Cell 60:75–86

    Google Scholar 

  • Derenzini M, Hernandez-Verdun D, Bouteille M (1982) Visualization in situ of extended DNA filaments in nucleolar chromatin of rat hepatocytes. Exp Cell Res 141:463–469

    Google Scholar 

  • Devictor M, Hartung M, Stahl A (1987) Distribution of rDNA and of its transcription sites in the nucleolus of the human Sertoli cell. Chromosomes Today 9:295–300

    Google Scholar 

  • Esponda P, Gimenez-Martin G (1974) Cytochemical aspects of the nucleolar organizer in Allium cepa microspores. Chromosoma 45:203–213

    Google Scholar 

  • Fakan S, Puvion E (1980) The ultrastructural visualization of nucleolar and extranucleolar RNA synthesis and distribution. Int Rev Cytol 65:255–295

    Google Scholar 

  • Flavell RB, Martini G (1982) The genetic control of nucleolus formation with special reference to common breadwheat. In: Jordan EG, Cullis CA (eds) The nucleolus. Cambridge University Press, pp 113–128

  • Gard LC, Di Angelo S, Jacob ST (1987) Role of DNA topoisomerase in the transcription of supercoiled rRNA gene. Proc Natl Acad Sci USA 84:3185–3188

    Google Scholar 

  • Goessens G (1984) Nucleolar structure. Int Rev Cytol 87:107–158

    Google Scholar 

  • Goessens G, Lepoint A (1979) The nucleolus organizing regions (NOR's): recent data and hypotheses. Biol Cell 35:211–220

    Google Scholar 

  • Hadjiolov AA (1985) The nucleolus and ribosome biogenesis. Cell Biol Monogr, vol 12. Springer, Berlin Heidelberg New York, p 168

    Google Scholar 

  • Hernandez-Verdun D, Hubert J, Bourgeois CA, Bouteille M (1980) Ultrastructural localization of AgNOR stained proteins in the nucleolus during the cell cycle and in other nucleolar structures. Chromosoma 79:349–362

    Google Scholar 

  • Jordan EG (1984) Nucleolar nomenclature. J Cell Sci 67:217–220

    Google Scholar 

  • Jordan EG (1987a) At the heart of the nucleolus. Nature 329:489–490

    Google Scholar 

  • Jordan EG (1987b) Nucleolar organizers in plants. Chromosomes Today 9:272–283

    Google Scholar 

  • Karagyozov LK, Stoyanova BB, Hadjiolov AA (1980) Effects of cycloheximide on the in vivo and in vitro synthesis of ribosomal RNA in rat liver. Biochim Biophys Acta 607:295–303

    Google Scholar 

  • Knalmann VM, Burger EC (1983) Quantitative aspects of in situ hybridization on rRNA gene sites in Vicia species and Allium cepa. Mikroskopie 40:87–94

    Google Scholar 

  • Lapeyre B, Bourbon H, Amalric F (1987) Nucleolin, the major nucleolar protein of growing eukaryotic cells: an unusual protein structure revealed by the nucleotide sequence. Proc Natl Acad Sci USA 84:1472–1476

    Google Scholar 

  • Long EO, Dawid IB (1980) Repeated genes in eukaryotes. Annu Rev Biochem 49:727–764

    Google Scholar 

  • Medina FJ, Risueño MC, Moreno Diaz de la Espina S (1983) 3-D reconstruction and morphometry of fibrillar centres in plant cells in relation to nucleolar activity. Biol Cell 48:31–38

    Google Scholar 

  • Medina FJ, Solanilla EL, Sánchez-Pina MA, Fernández-Gómez ME, Risueño MC (1986a) Cytological approach to the nucleolar functions detected by silver staining. Chromosoma 94:259–266

    Google Scholar 

  • Medina FJ, Risueño MC, Moreno Díaz de la Espina S, Sánchez-Pina MA, Fernández-Gómez ME (1986b) Organization, distribution and transcriptional activity of rDNA in plant cells. In: Fakan S, Hernandez-Verdun D (eds) The nucleolus and the nucleolar organizer regions. Biol Cell 56:189–206

  • Miller OL, Beatty BR (1969) Visualization of nucleolar genes. Science 164:955–957

    Google Scholar 

  • Mirre C, Stahl A (1981) Ultrastructural organization, sites of transcription and distribution of fibrillar centres in the nucleolus of the mouse oocyte. J Cell Sci 48:105–126

    Google Scholar 

  • Motte P, Deltour R, Mosen H, Bronchart R (1988) Three-dimensional electron microscopy of the nucleolus and nucleolus-asociated chromatin (NAC) during early germination of Zea mays. Biol Cell 62:65–81

    Google Scholar 

  • Newman GR, Jasani B, Williams ED (1983) A simple postembedding system for the rapid demonstration of tissue antigens under the electron microscope. Histochem J 15:543–555

    Google Scholar 

  • Olson MOJ, Rivers ZM, Thompson BA, Kao WY, Case ST (1983) Interaction of nucleolar phosphoprotein C23 with cloned segments of rat ribosomal deoxyribonucleic acid. Biochemistry 22:3345–3351

    Google Scholar 

  • Pebusque MJ, Seite R (1981) Electron microscopic studies of silverstained proteins in nucleolar organizer regions: location in nucleoli of rat sympathetic neurons during light and dark periods. J Cell Sci 51:85–94

    Google Scholar 

  • Pebusque MJ, Dupuy-Coin AM, Cataldo C, Seite R, Bouteille M, Moens PB (1981) Three-dimensional electron microscopy of the nucleolar organizer regions (NORs) in sympathetic neurons. Biol Cell 41:59–62

    Google Scholar 

  • Ploton D, Bendayan M, Adnet JJ (1983) Ultrastructural localization of Ag-NOR proteins and nucleic acids in reticulated nucleoli. Biol Cell 49:29–34

    Google Scholar 

  • Raska I, Reimer G, Jarnik M, Kostrouch Z, Raska K (1989) Does the synthesis of ribosomal RNA take place within nucleolar fibrillar centers or dense fibrillar components? Biol Cell 65:79–82

    Google Scholar 

  • Risueño MC, Medina FJ (1986) The nucleolar structure in plant cells. Cell Biology Reviews (RBC) vol 7. Editorial Service University Basque Country-Springer International, Leioa Spain, p 154

  • Risueño MC, Medina FJ, Moreno Díaz de la Espina S (1982) Nucleolar fibrillar centres in plant meristematic cells: ultrastructure, cytochemistry and autoradiography. J Cell Sci 58:313–329

    Google Scholar 

  • Scheer U, Raska I (1987) Immunocytochemical localization of RNA polymerase I in the fibrillar centers of nucleoli. Chromosomes Today 9:284–294

    Google Scholar 

  • Scheer U, Rose KM (1984) Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc Natl Acad Sci USA 81:1431–1435

    Google Scholar 

  • Scheer U, Zentgraf H (1982) Morphology of nucleolar chromatin in electron microscopic spread preparations. In: Busch H (ed) The cell nucleus, vol 11. Academic Press, New York, pp 143–176

    Google Scholar 

  • Scheer U, Messner K, Hazan R, Raska I, Hansmann P, Falk H, Spiees E, Franke WW (1987) High sensitivity immunolocalization of double and single-stranded DNA by a monoclonal antibody. Eur J Cell Biol 43:358–371

    Google Scholar 

  • Sommerville J (1986) Nucleolar structure and ribosome biogenesis. Trends Biochem Sci 11:438–442

    Google Scholar 

  • Stahl A (1982) The nucleolus and nucleolar chromosomes. In: Jordan EG, Cullis CA (eds) The nucleolus. Cambridge University Press, pp 1–24

  • Stahl A, Luciani JM, Vagner-Capodano AM (eds) (1987) Nucleolus organizer. Chromosomes Today 9:242–310

  • Thiry M (1988) Study of RNA distribution in the nucleolar components of Ehrlich cell using RNase-gold method. Histochemistry 89:231–236

    Google Scholar 

  • Thiry M, Lepoint A, Goessens G (1985) Re-evaluation of the site of transcription in Ehrlich tumour cell nucleoli. Biol Cell 54:57–64

    Google Scholar 

  • Thiry M, Scheer U, Goessens G (1988) Localization of DNA within Ehrlich tumour cell nucleoli by immunoelectron microscopy. Biol Cell 63:27–34

    Google Scholar 

  • Tröster H, Spring H, Meissner B, Schultz P, Oudet O, Trendelenburg MF (1985) Structural organization of an active, chromosomal nucleolar organizer region (NOR) identified by light microscopy, and subsequent TEM and STEM electron microscopy. Chromosoma 91:151–163

    Google Scholar 

  • Vazquez-Nin GH, Chavez B, Tomas-Martin C (1973) A preferential staining method for chromatin in electron microscopy. J Microscopie 16:243–246

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Centro de Investigaciones Biológicas (C.S.I.C.), Velazquez 144, E-28006, Madrid, Spain

    Marta Martin, Susana Moreno Diaz de la Espina & Francisco Javier Medina

Authors
  1. Marta Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Susana Moreno Diaz de la Espina
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Francisco Javier Medina
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Martin, M., de la Espina, S.M.D. & Medina, F.J. Immunolocalization of DNA at nucleolar structural components in onion cells. Chromosoma 98, 368–377 (1989). https://doi.org/10.1007/BF00292390

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00292390

Keywords

Search

Navigation