Visualization of focal sites of transcription within human nuclei
Jackson DA., Hassan AB., Errington RJ., Cook PR.
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 BrRNA. After extending nascent RNA chains by < 400 nucleotides in vitro, ~ 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. α-amanitin, an inhibitor of RNA polymerase II, prevents incorporation into these foci; then, using a slightly higher salt concentration, ~ 25 nucleolar foci became clearly visible. Both nucleolar and extra-nucleolar foci remain after nucleolytic removal of ~ 90% chromatin. An underlying structure probably organizes groups of transcription units into 'factories' where transcripts are both synthesized and processed.