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Related Experiment Videos

Engineered apoptotic nucleases for chromatin research.

Fei Xiao1, Piotr Widlak, William T Garrard

  • 1Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA.

Nucleic Acids Research
|July 13, 2007
PubMed
Summary
This summary is machine-generated.

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Researchers engineered DNA fragmentation factor (DFF) for controlled DNA cleavage in yeast. This novel tool, DFF-T, enables precise chromatin mapping and gene analysis in Saccharomyces cerevisiae.

Area of Science:

  • Molecular Biology
  • Genomics
  • Chromatin Research

Background:

  • DNA fragmentation factor (DFF) mediates internucleosomal DNA cleavage during apoptosis.
  • DFF comprises DFF45 (inhibitor) and DFF40 (endonuclease), activated by caspase-3 cleavage of DFF45.
  • Saccharomyces cerevisiae lacks DFF, but caspase-3 expression is lethal, unlike tobacco etch virus protease (TEVP).

Purpose of the Study:

  • To develop novel genomics tools for chromatin research by engineering DFF.
  • To create a DFF variant (DFF-T) controllable by TEVP for precise DNA cleavage in yeast.
  • To enable in vitro mapping of nucleosome positions and hypersensitive sites.

Main Methods:

  • Genetically engineered human and mouse DFF by inserting TEVP cleavage sites into DFF45.

Related Experiment Videos

  • Co-expressed TEVP and the engineered DFF-T in Saccharomyces cerevisiae under galactose control.
  • Created synthetic DFF genes optimized for high-level expression in E. coli and S. cerevisiae.

    • Main Results:

    • TEVP-controlled DFF-T induced nucleosomal DNA laddering and cell death in S. cerevisiae.
    • Synthetic DFF gene products demonstrated effectiveness for in vitro mapping of nucleosome positions.
    • Demonstrated utility in identifying hypersensitive sites in specific genes, such as yeast PHO5.

    Conclusions:

    • Engineered DFF-T provides a novel, TEVP-inducible system for controlled DNA cleavage in yeast.
    • Synthetic DFF genes facilitate high-level expression for advanced chromatin analysis.
    • This engineered nuclease system is a valuable tool for in vitro chromatin mapping and gene regulation studies.