title: Preferential distribution of active RNA polymerase II molecules in the nuclear periphery.
abstract: We have combined immunogold labeling with the Miller spreading technique in order to localize proteins at the electron microscope (EM) level in whole mount nuclei from mouse and human fibroblasts. Anti-histone H1 antibody labels nuclei uniformly, indicating that the nuclear interior is accessible to both antibodies and gold conjugates. Anti-topoisomerase I antibody labels nucleoli intensely, in agreement with previous immunofluorescent and biochemical data. Two different antibodies against the large subunit of RNA polymerase II (pol II) show preferential labeling of the nuclear periphery, as do antibodies against lamin, a known peripheral nuclear protein. Treatment of cells with alpha-amanitin results in loss of virtually all RNA polymerase II staining, supporting the specificity of labeling. Finally, when nuclei are incubated in the presence of biotin-UTP (bio-UTP) under run-off transcription conditions, incorporation is preferentially located at the nuclear periphery. These results support the conclusions that transcriptionally active pol II molecules are non-uniformly distributed in fibroblast nuclei, and that their differential distribution mirrors that of total pol II.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
authors.forename: R F
authors.initials: RF
authors.lastname: Clark
authors.forename: K W
authors.initials: KW
authors.lastname: Cho
authors.forename: R
authors.initials: R
authors.lastname: Weinmann
authors.forename: B A
authors.initials: BA
authors.lastname: Hamkalo
mesh_terms: D000546:Amanitins; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives; D002466:Cell Nucleolus / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology*; D004264:DNA Topoisomerases, Type I / Q000032:analysis; D003856:Deoxyuracil Nucleotides; D006367:HeLa Cells; D006657:Histones / Q000032:analysis; D006801:Humans; D007150:Immunohistochemistry; D007739:L Cells; D051379:Mice; D016253:Microscopy, Immunoelectron / Q000379:methods*; D009687:Nuclear Proteins / Q000032:analysis*; D012319:RNA Polymerase II / Q000032:analysis*; D013347:Subcellular Fractions
chemical_list: D000546:Amanitins; D003856:Deoxyuracil Nucleotides; D006657:Histones; D009687:Nuclear Proteins; D001710:Biotin; C045931:biotin-11-dUTP; D012319:RNA Polymerase II; D004264:DNA Topoisomerases, Type I