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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.

Type

Journal article

Journal

EMBO J

Publication Date

03/1993

Volume

12

Pages

1059 - 1065

Keywords

Cell Nucleolus, Cell Nucleus, DNA Replication, HeLa Cells, Humans, RNA Polymerase I, RNA Polymerase II, RNA, Messenger, Ribonucleoproteins, Small Nuclear, S Phase, Transcription, Genetic, Uridine Triphosphate