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Related Experiment Videos

Phage Trojan horses: a conditional expression system for lethal genes.

J Heitman1, W Fulford, P Model

  • 1Rockefeller University, New York, NY 10021.

Gene
|December 21, 1989
PubMed
Summary
This summary is machine-generated.

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Researchers developed a conditional expression system to isolate mutants of the lethal EcoRI restriction enzyme (ENase). This method uses a filamentous phage vector to screen for ENase mutants that form turbid plaques, aiding in the study of toxic proteins.

Area of Science:

  • Molecular Biology
  • Enzymology
  • Genetics

Background:

  • The EcoRI restriction enzyme (ENase) poses a lethal threat to cells due to its DNA cleavage activity.
  • Cells typically express M.EcoRI methyltransferase (MTase) to protect their DNA by modifying recognition sites.
  • Filamentous phages normally form turbid plaques; however, expressing EcoRI ENase leads to clear plaques.

Purpose of the Study:

  • To develop a method for isolating mutants of the EcoRI restriction enzyme (ENase).
  • To create a conditional expression system for studying toxic proteins.

Main Methods:

  • Cloned the EcoRI ENase gene into a filamentous phage vector (M13mp18) under lac promoter control.
  • Screened for mutants exhibiting turbid plaque formation instead of clear plaques on an M- host.

Related Experiment Videos

  • Utilized the protective effect of M.EcoRI MTase to rescue host cells.

    • Main Results:

    • Successfully isolated EcoRI ENase mutants by screening for turbid plaques.
    • Demonstrated that expressing M.EcoRI MTase rescues host cells and restores turbid plaque formation.
    • The conditional expression system effectively identified desired mutants.

    Conclusions:

    • The developed conditional expression system is effective for isolating mutants of lethal enzymes like EcoRI ENase.
    • This system provides a valuable tool for cloning and mutating genes encoding other toxic proteins.
    • The interplay between restriction enzymes and methyltransferases can be leveraged for genetic studies.