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Synthesis and biological activity of a lambda pseudo operator

E Kawashima, T Gadek, M H Caruthers

    Biochemistry
    |September 20, 1977
    PubMed
    Summary
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    Researchers chemically synthesized a bacteriophage lambda pseudo operator DNA sequence. This synthetic DNA, a binding site for the cI repressor protein, was successfully formed and tested.

    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genetics

    Background:

    • Bacteriophage lambda DNA contains operator sites crucial for regulating gene expression.
    • The cI repressor protein binds to these operator sites to control the phage life cycle.
    • Previous studies proposed multiple sequences for these operator sites.

    Purpose of the Study:

    • To chemically and enzymatically synthesize a representative bacteriophage lambda pseudo operator DNA.
    • To create a twofold symmetric 17 base-paired duplex representing a consensus of proposed operator sequences.
    • To investigate the binding of the cI repressor protein to the synthesized DNA duplex.

    Main Methods:

    • Chemical synthesis of deoxyoligonucleotides: d(T-A-T-C-A-C), d(C-G-C-C-G-G-T-G-A-T-A), d(T-A-T-C-A-C-C), and d(G-G-C-G-G-T-G-A-T-A).

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  • Enzymatic ligation using T4 DNA ligase to join synthesized deoxyoligonucleotides.
  • Formation of the 17 base-paired duplex: d(T-A-T-C-A-C-C-G-C-C-G-G-T-G-A-T-A) and d(T-A-T-C-A-C-C-G-G-C-G-G-T-G-A-T-A).
  • Binding assays to determine cI repressor protein interaction with the synthetic DNA.

    • Main Results:

    • Successfully synthesized and ligated deoxyoligonucleotides to form two distinct DNA duplexes.
    • The synthesized 17 base-paired duplex represents a nucleotide summation of proposed operator sites.
    • The cI repressor protein demonstrated binding to the synthesized pseudo operator DNA duplex.

    Conclusions:

    • The study successfully created a synthetic bacteriophage lambda pseudo operator DNA sequence.
    • This synthetic DNA serves as a functional binding site for the cI repressor protein.
    • The findings provide a tool for further studies on bacteriophage lambda gene regulation.