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.