Active RNA polymerases are localized within discrete transcription "factories' in human nuclei.

Nascent transcripts in permeabilized HeLa cells were elongated by approximately 30-2,000 nucleotides in Br-UTP or biotin-14-CTP, before incorporation sites were immunolabelled either pre- or post-embedding, and visualized by light or electron microscopy. Analogues were concentrated in approximately 2,100 (range 2,000-2,700) discrete sites attached to a nucleoskeleton and surrounded by chromatin. A typical site contained a cluster (diameter 71 nm) of at least 4, and probably about 20, engaged polymerases, plus associated transcripts that partially overlapped a zone of RNA polymerase II, ribonucleoproteins, and proteins rich in thiols and acidic groups. As each site probably contains many transcription units, these results suggest that active polymerases are confined to these sites, which we call transcription 'factories'. Results are consistent with transcription occurring as templates slide past attached polymerases, as nascent RNA is extruded into the factories.

[1]  P. Chambon.  9. Eucaryotic RNA Polymerases , 1974 .

[2]  S. Fakan,et al.  The ultrastructural visualization of nucleolar and extranucleolar RNA synthesis and distribution. , 1980, International review of cytology.

[3]  M. W. Clark.  Nucleolar-specific positive stains for optical and electron microscopy. , 1991, Methods in enzymology.

[4]  D. Jackson,et al.  Transcription occurs at a nucleoskeleton. , 1985, The EMBO journal.

[5]  J. Dubochet,et al.  On the preparation of cryosections for immunocytochemistry. , 1984, Journal of ultrastructure research.

[6]  D. Rawlins,et al.  Confocal microscopy and image processing in the study of plant nuclear structure , 1992 .

[7]  R. F. Cox.  Quantitation of elongating form A and B RNA polymerases in chick oviduct nuclei and effects of estradiol , 1976, Cell.

[8]  A. Sasseville,et al.  Lamin proteins form an internal nucleoskeleton as well as a peripheral lamina in human cells. , 1995, Journal of cell science.

[9]  D. Jackson,et al.  A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin. , 1988, Journal of cell science.

[10]  R. Pepperkok,et al.  Mammalian nuclei contain foci which are highly enriched in components of the pre‐mRNA splicing machinery. , 1991, The EMBO journal.

[11]  B. Lewin.  Units of transcription and translation: Sequence components of heterogeneous nuclear RNA and messenger RNA , 1975, Cell.

[12]  P. Cook,et al.  RNA is synthesized at the nuclear cage , 1981, Nature.

[13]  A. Pearse.  Histochemistry: Theoretical and Applied , 1953 .

[14]  G. A. Rice,et al.  Footprinting analysis of mammalian RNA polymerase II along its transcript: an alternative view of transcription elongation. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[15]  P. O’Farrell,et al.  Progression of the cell cycle through mitosis leads to abortion of nascent transcripts , 1991, Cell.

[16]  D. Jackson,et al.  Visualization of replication factories attached to a nucleoskeleton , 1993, Cell.

[17]  D. Spector,et al.  Dynamics of transcription and pre-mRNA splicing within the mammalian cell nucleus. , 1993, Cold Spring Harbor symposia on quantitative biology.

[18]  N. Gas,et al.  Bismuth staining of a nucleolar protein. , 1984, Experimental cell research.

[19]  T. Russo,et al.  The DNA-binding efficiency of Sp1 is affected by redox changes. , 1994, European journal of biochemistry.

[20]  P. Cook.  RNA polymerase: structural determinant of the chromatin loop and the chromosome. , 1994, BioEssays : news and reviews in molecular, cellular and developmental biology.

[21]  W. Earnshaw,et al.  Anionic regions in nuclear proteins , 1987, The Journal of cell biology.

[22]  C. F. Bennett,et al.  Nonhistone protein BA is a glutathione S-transferase localized to interchromatinic regions of the cell nucleus , 1986, The Journal of cell biology.

[23]  D. Jackson,et al.  Visualization of focal sites of transcription within human nuclei. , 1993, The EMBO journal.

[24]  Dr. Gareth Griffiths.  Fine Structure Immunocytochemistry , 1993, Springer Berlin Heidelberg.

[25]  G. Erdos.  Localization of Carbohydrate-Containing Molecules , 1986 .

[26]  A. V. Grimstone.  Molecular biology of the cell (3rd edn) , 1995 .

[27]  B. van Steensel,et al.  Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus , 1993, The Journal of cell biology.

[28]  H Nakamura,et al.  Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus. , 1986, Experimental cell research.

[29]  F S Fay,et al.  A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus. , 1993, Science.

[30]  C. Robson,et al.  Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding , 1993, Molecular and cellular biology.

[31]  D. Bregman,et al.  Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains , 1995, The Journal of cell biology.

[32]  D. Spector,et al.  Macromolecular domains within the cell nucleus. , 1993, Annual review of cell biology.

[33]  D. Jackson,et al.  Visualization of a filamentous nucleoskeleton with a 23 nm axial repeat. , 1988, The EMBO journal.

[34]  W. Jahn.  Visualization of a filamentous network in cryo sections of liver tissue. , 1980, European journal of cell biology.

[35]  S. Fakan,et al.  Perichromatin fibrils are in situ forms of nascent transcripts. , 1994, Trends in cell biology.

[36]  P. Cook,et al.  Site of transcription of ribosomal RNA and intranucleolar structure in HeLa cells. , 1994, Journal of cell science.

[37]  R. Young,et al.  RNA polymerase II. , 1991, Annual review of biochemistry.

[38]  W. Kim,et al.  Immunochemical analysis of mammalian RNA polymerase II subspecies. Stability and relative in vivo concentration. , 1986, The Journal of biological chemistry.

[39]  D. Jackson,et al.  A general method for preparing chromatin containing intact DNA. , 1985, The EMBO journal.

[40]  J. Tooze,et al.  Regulated secretion of mature cathepsin B from rat exocrine pancreatic cells. , 1991, European journal of cell biology.

[41]  L. Jong,et al.  Transcription by RNA Polymerase II and Nuclear Architecture , 1997 .