Visualization of focal sites of transcription within human nuclei.
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.
Jackson
D A
DA
Sir William Dunn School of Pathology, University of Oxford, UK.
Hassan
A B
AB
Errington
R J
RJ
Cook
P R
PR
D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D004261:DNA Replication; D006367:HeLa Cells; D006801:Humans; D012318:RNA Polymerase I / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism; D016196:S Phase; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000031:analogs & derivatives
D012333:RNA, Messenger; D017411:Ribonucleoproteins, Small Nuclear; C018009:5-bromouridine triphosphate; D012319:RNA Polymerase II; D012318:RNA Polymerase I; D014544:Uridine Triphosphate