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Related Experiment Videos

Phenotypically cryptic EcoRI endonuclease activity specified by the ColE1 plasmid.

C A Miller, S N Cohen

    Proceedings of the National Academy of Sciences of the United States of America
    |March 1, 1978
    PubMed
    Summary

    Bacteria with the ColE1 plasmid produce EcoRI endonuclease but do not show restriction or modification. This suggests a control mechanism prevents the enzyme

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    Area of Science:

    • Molecular Biology
    • Genetics
    • Microbiology

    Background:

    • The ColE1 plasmid encodes an endonuclease with EcoRI specificity.
    • Bacteria harboring ColE1 typically do not exhibit EcoRI restriction or modification functions in vivo.

    Purpose of the Study:

    • To investigate the expression and activity of EcoRI endonuclease in bacteria containing the ColE1 plasmid.
    • To determine the presence or absence of a corresponding EcoRI modification system in these cells.

    Main Methods:

    • Analysis of DNA from ColE1-containing bacterial cells for modification status.
    • In vitro cleavage assays using purified EcoRI endonuclease and enzyme extracts from ColE1-carrying bacteria.
    • Detection assays for EcoRI DNA methylase activity.

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    Main Results:

    • Phage and plasmid DNA from ColE1-containing cells showed unmodified EcoRI sites.
    • These unmodified sites were susceptible to cleavage by purified EcoRI endonuclease and bacterial extracts.
    • No detectable EcoRI DNA methylase activity was associated with the ColE1 plasmid.

    Conclusions:

    • ColE1 plasmid confers a cryptic, phenotypically silent EcoRI endonuclease activity.
    • The absence of a detectable modification system suggests a regulatory mechanism prevents in vivo expression of the ColE1-determined enzyme.