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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.

Type

Journal article

Journal

Clin Sci (Lond)

Publication Date

07/1995

Volume

89

Pages

13 - 18

Keywords

Cell Cycle, Cell Nucleus, Chromatin, DNA Replication, HeLa Cells, Humans, Microscopy, Confocal, Microscopy, Fluorescence, Models, Genetic, Transcription, Genetic