title: In situ fluorescence visualization of bromouridine incorporated into newly transcribed nucleolar RNA.
abstract: Bromouridine-triphosphate is commonly used for in situ immunocytochemical labeling of newly synthesized RNA in living cells. While extranucleolar transcripts do not require special conditions for visualization, special treatment prior to fixation (e.g. incubation with alpha-amanitine) is necessary for immunofluorescence detection of bromouridine-labeled nucleolar RNA in previous studies. We show in the present investigation that bromouridine-triphosphate is efficiently used by both extranucleolar and nucleolar RNA polymerases in living cultured cells. The failure to detect incorporated bromouridine within nucleoli is entirely due to improper treatment of cells after bromouridine incorporation. When methanol/acetone fixation is used, fluorescence signals within nucleoli can be routinely found.
authors.affiliation: Department of Cell Biology, Academy of Sciences of the Czech Republic, Prague.
authors.forename: K
authors.initials: K
authors.lastname: Koberna
authors.forename: D
authors.initials: D
authors.lastname: Stanĕk
authors.forename: J
authors.initials: J
authors.lastname: Malínský
authors.forename: V
authors.initials: V
authors.lastname: Ctrnáctá
authors.forename: S
authors.initials: S
authors.lastname: Cermanová
authors.forename: J
authors.initials: J
authors.lastname: Novotná
authors.forename: V
authors.initials: V
authors.lastname: Kopský
authors.forename: I
authors.initials: I
authors.lastname: Raska
mesh_terms: D000818:Animals; D001976:Bromouracil / Q000031:analogs & derivatives; D002466:Cell Nucleolus / Q000378:metabolism; D002478:Cells, Cultured; D004285:Dogs; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D009697:Nucleolus Organizer Region / Q000378:metabolism*; D012318:RNA Polymerase I / Q000378:metabolism; D012335:RNA, Ribosomal / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D012335:RNA, Ribosomal; D001976:Bromouracil; D012318:RNA Polymerase I; C006824:5-bromouridine; D014529:Uridine