title: Functional organization of human nuclei.
abstract: 1. Despite much progress in deciphering nuclear functions at the molecular level, our understanding of how these processes occur in vivo has been limited by the technologies presently available. I have used and developed a permeabilized cell system that retains most of the RNA- and DNA-polymerizing activities of HeLa cells. 2. Focal sites of transcription were visualized after incubation with bromouridine-UTP and immunolabelling with an antibody that reacts with bromouridine-RNA. Focal sites of replication were directly visualized by incubation with fluorochrome-dUTP conjugates. Approximately 300 transcription and 150 replication fluorescent foci were visualized in human cells. Foci resisted nucleolytic removal of 90% of chromatin. Experiments using laser scanning confocal microscopy show co-localization of sites of transcription with both splicing and replication sites, the latter particularly at the onset of S phase. Sites of replication were localized to discrete ovoid bodies when chromatin-depleted nuclei were visualized by thick section (resinless) electron microscopy. 3. These results suggest that active polymerases are focally concentrated (approximately 40 per focus) in 'factories' within nuclei. This higher-order organization may be important for both the initiation of replication and transcription in vivo.
authors.affiliation: Sir William Dunn School of Pathology, Oxford, U.K.
authors.forename: A B
authors.initials: AB
authors.lastname: Hassan
mesh_terms: D002453:Cell Cycle / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics* / Q000648:ultrastructure; D002843:Chromatin / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D002843:Chromatin