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Related Experiment Videos

Functional organization of human nuclei

A B Hassan1

  • 1Sir William Dunn School of Pathology, Oxford, U.K.

Clinical Science (London, England : 1979)
|July 1, 1995
PubMed
Summary

Researchers visualized active transcription and replication sites within human cells using a novel permeabilized cell system. These findings reveal that nuclear polymerases concentrate in distinct

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Understanding in vivo nuclear processes like transcription and replication is limited by current technologies.
  • A novel permeabilized cell system retaining RNA and DNA polymerase activity in HeLa cells was developed.

Purpose of the Study:

  • To visualize and characterize focal sites of transcription and replication within human cells.
  • To investigate the spatial organization of nuclear processes in vivo.

Main Methods:

  • Utilized a permeabilized HeLa cell system.
  • Employed bromouridine-UTP and immunolabelling for transcription visualization.
  • Used fluorochrome-dUTP conjugates for direct replication visualization.
  • Applied laser scanning confocal microscopy and electron microscopy.

Main Results:

  • Visualized approximately 300 transcription and 150 replication fluorescent foci per cell.
  • Observed co-localization of transcription sites with splicing and replication sites.
  • Replication sites were identified as discrete ovoid bodies in chromatin-depleted nuclei.
  • Demonstrated that active polymerases concentrate in nuclear 'factories'.

Conclusions:

  • Active polymerases are focally concentrated in nuclear 'factories'.
  • This higher-order organization is crucial for the initiation of in vivo replication and transcription.
  • The developed cell system enables visualization of dynamic nuclear processes.

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