Visualization of focal sites of transcription within human nuclei

EMBO J. 1993 Mar;12(3):1059-65.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Nucleolus / metabolism
  • Cell Nucleus / metabolism*
  • DNA Replication
  • HeLa Cells
  • Humans
  • RNA Polymerase I / metabolism
  • RNA Polymerase II / metabolism
  • RNA, Messenger / biosynthesis
  • Ribonucleoproteins, Small Nuclear / metabolism
  • S Phase
  • Transcription, Genetic*
  • Uridine Triphosphate / analogs & derivatives

Substances

  • RNA, Messenger
  • Ribonucleoproteins, Small Nuclear
  • 5-bromouridine triphosphate
  • RNA Polymerase II
  • RNA Polymerase I
  • Uridine Triphosphate