101. Visualization of focal sites of transcription within human nuclei.
- Author
-
Jackson DA, Hassan AB, Errington RJ, and Cook PR
- Subjects
- Cell Nucleolus metabolism, DNA Replication, HeLa Cells, Humans, RNA Polymerase I metabolism, RNA Polymerase II metabolism, RNA, Messenger biosynthesis, Ribonucleoproteins, Small Nuclear metabolism, S Phase, Uridine Triphosphate analogs & derivatives, Cell Nucleus metabolism, Transcription, Genetic
- Abstract
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.
- Published
- 1993
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