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General selection for specific DNA-binding activities.

N Benson, P Sugiono, S Bass

    Genetics
    |September 1, 1986
    PubMed
    Summary
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    Scientists developed a novel method using a "challenge phage" to identify bacterial clones with specific DNA-binding abilities. This technique enables the selection of proteins that recognize particular DNA sequences, advancing genetic research.

    Area of Science:

    • Molecular Biology
    • Bacteriophage Genetics
    • Protein-DNA Interactions

    Background:

    • Identifying bacterial clones expressing specific DNA-binding activities is crucial for understanding gene regulation.
    • Existing methods may lack efficiency or specificity in selecting for desired DNA-binding proteins.

    Purpose of the Study:

    • To present a generalizable strategy for selecting bacterial clones with specific DNA-binding activities.
    • To demonstrate the utility of a novel 'challenge phage' vector system for this selection process.

    Main Methods:

    • A modified bacteriophage P22 vector (P22 Kn9 arc-amH1605) was engineered with synthetic DNA-binding sites.
    • These sites replaced the natural binding site controlling the P22 antirepressor (ant) gene transcription.
    • Infection with the challenge phage selects for host survival based on the presence of specific DNA-binding proteins.

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    Main Results:

    • The challenge phage system successfully selected bacterial clones expressing DNA-binding activities for E. coli Lac operator, phage lambda OL1 operator, and synthetic operators.
    • Host survival and antibiotic resistance indicated successful binding of the target DNA sequence by expressed proteins.
    • The system demonstrated its ability to select for specific protein-DNA recognition.

    Conclusions:

    • The challenge phage vector system provides an effective and generalizable method for selecting bacterial clones with specific DNA-binding activities.
    • This approach holds potential for selecting eukaryotic DNA-binding activities as well, broadening its applicability.