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EMBO J. Mar 1993; 12(3): 1059–1065.
PMCID: PMC413307

Visualization of focal sites of transcription within human nuclei.

Abstract

HeLa cells were encapsulated in agarose microbeads, permeabilized and incubated with Br-UTP in a 'physiological' buffer; then sites of RNA synthesis were immunolabelled using an antibody that reacts with Br-RNA. After extending nascent RNA chains by < 400 nucleotides in vitro, approximately 300-500 focal synthetic sites can be seen in each nucleus by fluorescence microscopy. Most foci also contain a component of the splicing apparatus detected by an anti-Sm antibody. alpha-amanitin, an inhibitor of RNA polymerase II, prevents incorporation into these foci; then, using a slightly higher salt concentration, approximately 25 nucleolar foci became clearly visible. Both nucleolar and extra-nucleolar foci remain after nucleolytic removal of approximately 90% chromatin. An underlying structure probably organizes groups of transcription units into 'factories' where transcripts are both synthesized and processed.

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Selected References

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  • Ascoli CA, Maul GG. Identification of a novel nuclear domain. J Cell Biol. 1991 Mar;112(5):785–795. [PMC free article] [PubMed]
  • Carmo-Fonseca M, Tollervey D, Pepperkok R, Barabino SM, Merdes A, Brunner C, Zamore PD, Green MR, Hurt E, Lamond AI. Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery. EMBO J. 1991 Jan;10(1):195–206. [PMC free article] [PubMed]
  • Carmo-Fonseca M, Pepperkok R, Sproat BS, Ansorge W, Swanson MS, Lamond AI. In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells. EMBO J. 1991 Jul;10(7):1863–1873. [PMC free article] [PubMed]
  • Carter KC, Taneja KL, Lawrence JB. Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol. 1991 Dec;115(5):1191–1202. [PMC free article] [PubMed]
  • Cook PR. The nucleoskeleton: artefact, passive framework or active site? J Cell Sci. 1988 May;90(Pt 1):1–6. [PubMed]
  • Cook PR. The nucleoskeleton and the topology of transcription. Eur J Biochem. 1989 Nov 20;185(3):487–501. [PubMed]
  • Cox RF. Quantitation of elongating form A and B RNA polymerases in chick oviduct nuclei and effects of estradiol. Cell. 1976 Mar;7(3):455–465. [PubMed]
  • Davis AH, Reudelhuber TL, Garrard WT. Varigated chromatin structures of mouse ribosomal RNA genes. J Mol Biol. 1983 Jun 15;167(1):133–155. [PubMed]
  • Dickinson P, Cook PR, Jackson DA. Active RNA polymerase I is fixed within the nucleus of HeLa cells. EMBO J. 1990 Jul;9(7):2207–2214. [PMC free article] [PubMed]
  • Elliott DJ, Bowman DS, Abovich N, Fay FS, Rosbash M. A yeast splicing factor is localized in discrete subnuclear domains. EMBO J. 1992 Oct;11(10):3731–3736. [PMC free article] [PubMed]
  • Fakan S, Puvion E. The ultrastructural visualization of nucleolar and extranucleolar RNA synthesis and distribution. Int Rev Cytol. 1980;65:255–299. [PubMed]
  • Fischer D, Weisenberger D, Scheer U. Assigning functions to nucleolar structures. Chromosoma. 1991 Dec;101(3):133–140. [PubMed]
  • Franke WW, Kleinschmidt JA, Spring H, Krohne G, Grund C, Trendelenburg MF, Stoehr M, Scheer U. A nucleolar skeleton of protein filaments demonstrated in amplified nucleoli of Xenopus laevis. J Cell Biol. 1981 Aug;90(2):289–299. [PMC free article] [PubMed]
  • Fu XD, Maniatis T. Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus. Nature. 1990 Feb 1;343(6257):437–441. [PubMed]
  • Fusconi M, Cassani F, Govoni M, Caselli A, Farabegoli F, Lenzi M, Ballardini G, Zauli D, Bianchi FB. Anti-nuclear antibodies of primary biliary cirrhosis recognize 78-92-kD and 96-100-kD proteins of nuclear bodies. Clin Exp Immunol. 1991 Feb;83(2):291–297. [PMC free article] [PubMed]
  • Haaf T, Hayman DL, Schmid M. Quantitative determination of rDNA transcription units in vertebrate cells. Exp Cell Res. 1991 Mar;193(1):78–86. [PubMed]
  • He DC, Nickerson JA, Penman S. Core filaments of the nuclear matrix. J Cell Biol. 1990 Mar;110(3):569–580. [PMC free article] [PubMed]
  • Huang S, Spector DL. Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors. Genes Dev. 1991 Dec;5(12A):2288–2302. [PubMed]
  • Jackson DA. Structure-function relationships in eukaryotic nuclei. Bioessays. 1991 Jan;13(1):1–10. [PubMed]
  • Jackson DA, Cook PR. A general method for preparing chromatin containing intact DNA. EMBO J. 1985 Apr;4(4):913–918. [PMC free article] [PubMed]
  • Jackson DA, Cook PR. Transcription occurs at a nucleoskeleton. EMBO J. 1985 Apr;4(4):919–925. [PMC free article] [PubMed]
  • Jackson DA, Cook PR. Replication occurs at a nucleoskeleton. EMBO J. 1986 Jun;5(6):1403–1410. [PMC free article] [PubMed]
  • Jackson DA, Cook PR. A cell-cycle-dependent DNA polymerase activity that replicates intact DNA in chromatin. J Mol Biol. 1986 Nov 5;192(1):65–76. [PubMed]
  • Jackson DA, Cook PR. Visualization of a filamentous nucleoskeleton with a 23 nm axial repeat. EMBO J. 1988 Dec 1;7(12):3667–3677. [PMC free article] [PubMed]
  • Jackson DA, Dickinson P, Cook PR. The size of chromatin loops in HeLa cells. EMBO J. 1990 Feb;9(2):567–571. [PMC free article] [PubMed]
  • Jackson DA, McCready SJ, Cook PR. RNA is synthesized at the nuclear cage. Nature. 1981 Aug 6;292(5823):552–555. [PubMed]
  • Jackson DA, McCready SJ, Cook PR. Replication and transcription depend on attachment of DNA to the nuclear cage. J Cell Sci Suppl. 1984;1:59–79. [PubMed]
  • Jackson DA, Yuan J, Cook PR. A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin. J Cell Sci. 1988 Jul;90(Pt 3):365–378. [PubMed]
  • Jordan EG. Interpreting nucleolar structure: where are the transcribing genes? J Cell Sci. 1991 Apr;98(Pt 4):437–442. [PubMed]
  • Lawrence JB, Singer RH, Marselle LM. Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell. 1989 May 5;57(3):493–502. [PubMed]
  • Miller OL, Jr, Bakken AH. Morphological studies of transcription. Acta Endocrinol Suppl (Copenh) 1972;168:155–177. [PubMed]
  • Nakamura H, Morita T, Sato C. Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus. Exp Cell Res. 1986 Aug;165(2):291–297. [PubMed]
  • Nakayasu H, Berezney R. Mapping replicational sites in the eucaryotic cell nucleus. J Cell Biol. 1989 Jan;108(1):1–11. [PMC free article] [PubMed]
  • Raska I, Andrade LE, Ochs RL, Chan EK, Chang CM, Roos G, Tan EM. Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies. Exp Cell Res. 1991 Jul;195(1):27–37. [PubMed]
  • Saunders WS, Cooke CA, Earnshaw WC. Compartmentalization within the nucleus: discovery of a novel subnuclear region. J Cell Biol. 1991 Nov;115(4):919–931. [PMC free article] [PubMed]
  • Scheer U, Rose KM. 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. [PMC free article] [PubMed]
  • Schmickel RD. Quantitation of human ribosomal DNA: hybridization of human DNA with ribosomal RNA for quantitation and fractionation. Pediatr Res. 1973 Jan;7(1):5–12. [PubMed]
  • Shermoen AW, O'Farrell PH. Progression of the cell cycle through mitosis leads to abortion of nascent transcripts. Cell. 1991 Oct 18;67(2):303–310. [PMC free article] [PubMed]
  • Spector DL. Higher order nuclear organization: three-dimensional distribution of small nuclear ribonucleoprotein particles. Proc Natl Acad Sci U S A. 1990 Jan;87(1):147–151. [PMC free article] [PubMed]
  • Vagner-Capodano AM, Bouteille M, Stahl A, Lissitzky S. Nucleolar ribonucleoprotein release into the nucleoplasm as nuclear bodies in cultured thyrotropin-stimulated thyroid cells: autoradiographic kinetics. J Ultrastruct Res. 1982 Jan;78(1):13–25. [PubMed]
  • Xing YG, Lawrence JB. Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation. J Cell Biol. 1991 Mar;112(6):1055–1063. [PMC free article] [PubMed]

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