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Related Experiment Videos

Capturing nuclear sequence-specific DNA-binding proteins by using simian virus 40-derived minichromosomes.

U Nir1, E Fodor, W J Rutter

  • 1Hormone Research Institute, University of California, San Francisco 94143-0534.

Molecular and Cellular Biology
|February 1, 1988
PubMed
Summary
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Researchers identified specific DNA-binding proteins in COS-7 cell nuclei using simian virus 40 (SV40) minichromosomes. These proteins, including transcription factors AP-1 and Sp1, bind to viral DNA and regulatory elements of the rat insulin enhancer, influencing gene expression.

Area of Science:

  • Molecular Biology
  • Virology
  • Genetics

Background:

  • Simian virus 40 (SV40) minichromosomes are valuable tools for studying DNA-protein interactions in eukaryotic cells.
  • COS-7 cells are a common cell line used for gene expression studies and viral research.
  • Understanding transcription factor binding is crucial for elucidating gene regulation mechanisms.

Purpose of the Study:

  • To identify sequence-specific DNA-binding proteins in COS-7 cell nuclei.
  • To investigate the binding of these proteins to SV40 DNA and the rat insulin (rINS1) enhancer.
  • To explore the potential role of these proteins in regulating enhancer function.

Main Methods:

  • Utilized recombinant SV40 minichromosomes to isolate nuclear proteins.
  • Analyzed protein binding to SV40 DNA during late viral infection.

Related Experiment Videos

  • Examined protein interactions with the rINS1 enhancer in COS-7 cells under negative regulation.
  • Employed competition assays with the SV40 P element to characterize protein binding sites.

    • Main Results:

    • Identified transcription factors AP-1 and Sp1 stably bound to SV40 DNA.
    • Discovered two distinct proteins binding to specific regions of the rINS1 enhancer.
    • One rINS1-binding protein showed competition with the SV40 P element, suggesting a relation to AP-1.
    • Identified a second factor selectively binding to a regulatory element between -92 and -124 of the insulin enhancer.

    Conclusions:

    • Sequence-specific DNA-binding proteins, including AP-1 and Sp1, are present in COS-7 cell nuclei and interact with viral and cellular regulatory DNA.
    • The identified proteins binding to the rINS1 enhancer may play a significant role in its negative regulation.
    • Further investigation into these factors could reveal novel insights into insulin gene regulation and viral-host interactions.