Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP.
In situ sites of nucleolar transcription in cells microinjected with 5-bromo-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4-90 min at 37 degrees C, fixed, and embedded in LR White resin. Postembedding immunoelectron microscopic visualization with colloidal gold has been used for localizing both Br-labeled precursor incorporated into pre-rRNA and different nucleolar transcription or processing factors. This high resolution approach allowed us to identify significant signal as early as after 4-min incubation periods following BrUTP microinjection. It revealed the dense fibrillar component (DFC) as being the first nucleolar compartment labeled with anti-bromodeoxyuridine antibody. Moreover, RNA polymerase I, nucleolar transcription factor UBF, and fibrillarin were also detected almost exclusively in this same nucleolar compartment. From 30 min onward, following microinjection, Br-labeled rRNA occurred also in the granular component. The results indicate that the DFC is the site of pre-rRNA transcription and of initial steps of pre-rRNA processing. Moreover, it demonstrates that BrUTP microinjection followed by postembedding detection of Br-labeled RNA is a useful technique for high resolution studies of structure-function associations in the nucleolus.
Cmarko
D
D
Centre of Electron Microscopy, University of Lausanne, Switzerland.
Verschure
P J
PJ
Rothblum
L I
LI
Hernandez-Verdun
D
D
Amalric
F
F
van Driel
R
R
Fakan
S
S
D002466:Cell Nucleolus / Q000648:ultrastructure*; D002868:Chromosomal Proteins, Non-Histone / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000648:ultrastructure; D006801:Humans; D008845:Microinjections; D016253:Microscopy, Immunoelectron / Q000379:methods*; D009697:Nucleolus Organizer Region / Q000648:ultrastructure*; D035463:Pol1 Transcription Initiation Complex Proteins; D012318:RNA Polymerase I / Q000648:ultrastructure; D012322:RNA Precursors / Q000648:ultrastructure; D012335:RNA, Ribosomal / Q000648:ultrastructure; D012261:Ribonucleoproteins / Q000648:ultrastructure; D014157:Transcription Factors / Q000648:ultrastructure; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014544:Uridine Triphosphate / Q000008:administration & dosage / Q000031:analogs & derivatives*; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000648:ultrastructure
D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; D012322:RNA Precursors; D012335:RNA, Ribosomal; D012261:Ribonucleoproteins; D014157:Transcription Factors; C047458:fibrillarin; C063844:transcription factor UBF; C018009:5-bromouridine triphosphate; D012318:RNA Polymerase I; D014544:Uridine Triphosphate