Skip to main content
SpringerLink
Log in

N-band proteins of nucleolar organizers: chromosomal mapping, subnucleolar localization and rDNA binding

  • Published:
Chromosoma Aims and scope Submit manuscript

Abstract

The ribosomal DNA(rDNA)-containing chromatin in eukaryotes forms a unique architecture called the “secondary constriction” or “nucleolus organiser region (NOR)” on mitotic chromosomes. To gain more insight into non-histone chromosomal proteins (NHCP), termed “N-band proteins”, that are specifically associated with the NOR in a wide variety of eukaryotes, we attempted to: (1) identify the NHCP responsible for N-band staining; (2) determine their stoichiometry; (3) map them on metaphase chromosomes; (4) determine their subnucleolar localization and (5) examine their possible ability to bind rDNA. Based on several criteria, including chromosomal localization, solubility, association with chromatin, and intranuclear localization, two of the nucleolus-rich NHCP, termed component B of mol.wt 55,000 and component C of mol.wt. 41,000, were tentatively identified as N-band proteins. Immunological studies using a polyclonal, monospecific antibody raised against component C show that this protein is in fact associated with the chromosomal telomeres where NORs are located. In nucleoli, N-band proteins appear to be compartmentalized into a structure that probably corresponds to fibrillar components. Both components B and C are among several NHCPs that showed, under in vitro conditions, a strong affinity for rDNA cloned in lambda phage but not for calf thymus genomic DNA or phage vector DNA. The antibody against component C effectively suppressed in vitro transcription by RNA polymerase I in nucleoli and nucleolar chromatin. Component C appears to exist in the nucleus at 3.75–5.13×103 copies per rDNA unit or 0.09–0.13 copy per nucleotide. These findings support the hypothesis that the NOR is a chromosomal site, architecturally not only unique but also different from other chromatin regions in that constituent DNA, i.e., rDNA, is organized in a specific manner by interacting with specific NHCP, i.e., N-band proteins.

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

References

  • Alfageme CR, Rudkin GT, Cohen H (1976) Locations of chromosomal proteins in polytene chromosomes Proc Natl Acad Sci USA 73:2038–2042

    Google Scholar 

  • Alberts B, Herrick G (1971) DNA-cellulose chromatography. Methods Enzymol 21:198–217

    Google Scholar 

  • Basler J, Berezney RD, Pietras D, Matsui S, Sandberg AA, Hastie ND (1981) Hybridization of nuclear matrix associated DNA fragments. Biochemistry 20:6921–6929

    Google Scholar 

  • Black M, Ansley HR (1964) Histone staining with ammoniacal silver. Science 143:693–695

    Google Scholar 

  • Bowen B, Steinberg J, Laemmli UK, Weintraub H (1980) The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res 1:1–20

    Google Scholar 

  • Buys CHCH, Osinga J (1984) Selective staining of the same set of nucleolar phosphoproteins by silver and Giemsa. A combined biochemical and cytochemical study on staining of NORs. Chromosoma 89:387–396

    Google Scholar 

  • DeCapoa A, Pelliccia F, Marlekaj P, Ciofiluzzatto AR, Nardelli MB (1983) Silver positivity of the NORs during embryonic development of Xenopus laevis. Exp Cell Res 147:472–478

    Google Scholar 

  • Denhardt DT (1966) A membrane-filter technique for the detection of complimentary DNA. Biochem Biophys Res Commun 23:641–646

    Google Scholar 

  • Earnshaw WC, Laemmli UK (1984) Silver staining of the chromosome scaffold. Chromosoma 89:186–192

    Google Scholar 

  • Engel W, Zentes MT, Schmid M (1977) Activation of mouse ribosomal RNA genes at the 2-cell stage. Hum Genet 38:57–63

    Google Scholar 

  • Engelke DR, Ng SY, Shastry BS, Roeder RGH (1980) Specific interaction of a purified transcription factor with an internal control region of 5S RNA genes. Cell 19:717–728

    Google Scholar 

  • Fletcher JM (1979) Light microscope analysis of meiotic prophase chromosomes by silver staining. Chromosoma 72:241–248

    Google Scholar 

  • Fuke M, Dennis KJ, Busch H (1981) Characterization of cloned rat ribosomal DNA fragments. Mol Gen Genet 182:25–30

    Google Scholar 

  • Funaki K, Matsui S, Sasaki M (1975) Location of nucleolus organizers in animal and plant chromosomes by means of an improved N-banding technique. Chromosoma 49:357–370

    Google Scholar 

  • Goldberg MI, Perriard JC, Rutter WJ (1977) Purification of rat liver and mouse ascites DNA-dependent RNA polymerase I. Biochemistry 16:1655–1665

    Google Scholar 

  • Goodpasture C, Bloom SE (1975) Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma 53:37–50

    Google Scholar 

  • Goyanes CJ, Matsui S, Sandberg AA (1980) Chromatin organization of Chinese hamster chromosomes studied by histone-DNA crosslinking and trypsin digestion. Chromosoma 78:123–135

    Google Scholar 

  • Heitz E (1934) Über α- und β-heterochromatin sowie konstantz und bau der chromosomeren bei Drosophila. Biol Zbl 54:588–609

    Google Scholar 

  • Howell WM, Denton TE, Diamont JR (1975) Differential staining of the satellite regions of human acrocentric chromosomes. Experientia 31:260–262

    Google Scholar 

  • Hsu TC, Brinkley BR, Arrighi FE (1967) The structure and behavior of the nucleolus organizer in mammalian cells. Chromosoma 23:137–153

    Google Scholar 

  • Jack RS, Gehring WJ, Brack C (1981) Protein component from Drosophila larval nuclei showing sequence specificity for a short region near a major heat-shock protein gene. Cell 24:321–331

    Google Scholar 

  • Kistler J, Duncombe Y, Laemmli UK (1984) Mapping nucleolar proteins with monoclonal antibodies. J Cell Biol 99:1981–1988

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Google Scholar 

  • Lau Y-F, Werteleck W, Pfeiffer RA, Arrighi FE (1979) Cytological analysis of a 14a + variant by means of N-banding and combination of silver staining and chromosome banding. Hum Genet 46:75–82

    Google Scholar 

  • Levinger L, Varshavsky A (1982) Protein D1 preferentially binds A+T-rich DNA in vitro and is a component of Drosophila melanogaster nucleosomes containing A+T-rich satellite DNA. Proc Natl Acad Sci USA 79:7152–7156

    Google Scholar 

  • Lin S-Y, Riggs AD (1975) The general affinity of lac repressor for E. coli DNA: Implications for gene regulation in prokaryotes and eukaryotes. Cell 4:107–111

    Google Scholar 

  • Lischwe MA, Richards RL, Busch RK, Busch H (1981) Localization of phosphoprotein C23 to nucleolar structures and to the nucleolus organizer regions. Exp Cell Res 136:101–109

    Google Scholar 

  • Lischwe MA, Smetana K, Olson MOJ, Busch H (1978) Protein C23 and B23 and the major nucleolar silver staining proteins. Life Sci 25:701–708

    Google Scholar 

  • Matsui S (1974a) Structural proteins associated with ribosomal cistrons in Xenopus laevis chromosomes. Exp Cell Res 88:88–94

    Google Scholar 

  • Matsui S (1974b) Nucleolus organizer of Vicia faba chromosomes revealed by the N-banding technique. Jpn J Genet 49:93–96

    Google Scholar 

  • Matsui S, Busch H (1977) Isolation of rDNA-rich chromatin from nucleoli. Exp Cell Res 109:151–161

    Google Scholar 

  • Matsui S, Fuke M, Busch H (1977) Fidelity of rRNA synthesis by nucleoli and nucleolar chromatin. Biochemistry 16:39–45

    Google Scholar 

  • Matsui S, Sandberg AA (1985) Intranuclear compartmentalization of DNA-dependent RNA polymerases: Association of RNA polymerase I with nucleolar organizing chromosomes. Chromosoma 92:1–6

    Google Scholar 

  • Matsui S, Sandberg AA, Negoro S, Seon BK, Goldstein G (1982) A novel eukaryotic enzyme that cleaves isopeptide bonds. Proc Natl Acad Sci USA 77:1535–1539

    Google Scholar 

  • Matsui S, Sasaki M (1973) Differential staining of nucleolus organizer in mammalian chromosomes. Nature 246:148–150

    Google Scholar 

  • Matsui S, Seon BK, Sandberg AA (1979a) Disappearance of a structural chromatin protein A24 in mitosis: Implications for molecular basis of chromatin condensation. Proc Natl Acad Sci USA 76:6386–7390

    Google Scholar 

  • Matsui S, Weinfeld H, Sandberg AA (1979b) Quantitative conservation of chromatin-bound RNA polymerase I and II in mitosis. Implications for chromosome structure. J Cell Biol 80:451–464

    Google Scholar 

  • Matsuo Y, Matsui S, Nishi N, Wada F, Sandberg AA (1985) Quantitative solubilization of non-histone chromosomal proteins without denaturation using zwitterionic detergents. Anal Biochem 150:337–344

    Google Scholar 

  • McClintock B (1934) The reaction of a particular chromosomal element to the development of the nucleoli in Zea mays. Z Zellforsch 21:294–328

    Google Scholar 

  • Melli M, Whitfield C, Rao KJ, Richardson M, Bishop JO (1971) DNA-RNA hybridization in vast DNA excess. Nature New Biol 231:8–12

    Google Scholar 

  • Miller OJ, Miller DA, Dev VG, Tantravahi R, Croce CM (1976) Expression of human suppression of mouse nucleolus activity in mouse-human somatic hybrids. Proc Natl Acad Sci USA 73:4531–4535

    Google Scholar 

  • Mulvihill ER, DePennec JP, Chambon P (1982) Chicken oviduct progesterone receptor. Location of specific regions of high-affinity binding in cloned DNA fragments of hormone-responsive genes. Cell 24:621–632

    Google Scholar 

  • Nowock J, Sippel AE (1982) Specific protein-DNA interaction at four sites flanking the chicken lysozyme gene. Cell 30:607–615

    Google Scholar 

  • Olson MOJ, Guetzow K, Busch H (1981) Localization of phosphoprotein C23 in nucleoli by immunological methods. Exp Cell Res 135:259–265

    Google Scholar 

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

    Google Scholar 

  • Olson MOJ, Thompson BA (1983) Distribution of proteins among chromatin components of nucleoli. Biochemistry 22:3187–3193

    Google Scholar 

  • Payvar F, Wrange O, Carlstedt-Duke J, Okret S, Gustafsson JA, Yamamoto KR (1981) Purified glucocorticoid receptors bind selectively in vitro to a DNA fragment whose transcription is regulated by glucocorticoids in vivo. Proc Natl Acad Sci USA 78:6628–6632

    Google Scholar 

  • Ritossa FM, Spiegelman S (1965) Localization of DNA complementary to ribosomal RNA in the nucleolus organizer regions of Drosophila melanogaster. Proc Natl Acad Sci USA 53:737–745

    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 

  • Schmiady H, Munke M, Sperling K (1979) Ag-staining of nucleolus organizer regions on human prematurely condensed chromosomes from cells with different ribosomal RNA gene activity. Exp Cell Res 121:425–428

    Google Scholar 

  • Schmid M, Krone W (1974) On the relationship between the frequency of association and the nucleolar constriction of individual acrocentric chromosomes. Hum Genet 23:267–277

    Google Scholar 

  • Schwartz LB, Roeder RG (1974) Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase I from the mouse myeloma, MOPC 315. J Biol Chem 249:5898–5906

    Google Scholar 

  • Silver L, Elgin SCR (1977) Distribution patterns of three subfractions of Drosophila non-histone chromosomal proteins: Possible correlations with gene activity. Cell 11:971–983

    Google Scholar 

  • Silver LM, Elgin SCR (1978) Production and characterization of antisera against three individual NHC proteins; a case of generally distributed NHC protein. Chromosoma 68:101–114

    Google Scholar 

  • Somerville LL, Wang K (1981) The ultrasensitive silver “protein” stain also detects nanograms of nucleic acids. Biochem Biophys Res Commun 102:53–58

    Google Scholar 

  • Stumph WE, Elgin SCR, Hood L (1974) Antibodies to proteins dissolved in sodium dodecyl sulfate. J Immunol 113:1752–1756

    Google Scholar 

  • Stumph WE, Wu JR, Bonner J (1979) Determination of the size of rat ribosomal deoxyribonucleic acid repeating units by electron microscopy. Biochemistry 18:2864–2871

    Google Scholar 

  • Switzer RC, Merril CR, Shifrin S (1979) A highly sensitive stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem 98:231–237

    Google Scholar 

  • Tijan R (1978) The binding sites on SV 40 DNA for a T antigen-related protein. Cell 13:165–179

    Google Scholar 

  • van den Broek HWJ, Nooden LD, Seval S, Bonner J (1973) Isolation, purification and fractionation of non-histone chromosomal protein. Biochemistry 12:229–236

    Google Scholar 

  • von Hippel PH, Bear DG (1983) Location and recognition of specific DNA binding sites by proteins that regulate gene expression. In: Sund H, Veeger C (eds) Mobility and recognition in cell biology. Walter deGryter, Berlin, New York, pp 291–316

    Google Scholar 

  • Wallace H, Birnstiel ML (1966) Ribosomal cistrons and the nucleolar organizer. Biochim Biophys Acta 114:296–310

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Genetics and Endocrinology, Roswell Park Memorial Institute, 14263, Buffalo, NY, USA

    Sei-ichi Matsui, Lee Chai & Avery A. Sandberg

  2. Chemical Polymer Laboratory, Biotechnology Division, Phillips Research Center, Phillips Petroleum Company, 74004, Bartlesville, OK, USA

    Motohiro Fuke

  3. Department of Cell and Tumor Biology, Roswell Park Memorial Institute, 14263, Buffalo, NY, USA

    Sufian Elassouli

Authors
  1. Sei-ichi Matsui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Motohiro Fuke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lee Chai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Avery A. Sandberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sufian Elassouli
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This study was supported in part by a grant from the National Science Foundation (PCM-8115600) awarded to S.M. Parts of this study were presented at The Annual Meetings of The American Society for Cell Biology in 1978, 1983 and 1984

Rights and permissions

Reprints and permissions

About this article

Cite this article

Matsui, Si., Fuke, M., Chai, L. et al. N-band proteins of nucleolar organizers: chromosomal mapping, subnucleolar localization and rDNA binding. Chromosoma 93, 231–242 (1986). https://doi.org/10.1007/BF00292743

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00292743

Keywords

Search

Navigation