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Cloning the DdeI restriction-modification system using a two-step method.

K A Howard, C Card, J S Benner

    Nucleic Acids Research
    |October 24, 1986
    PubMed
    Summary
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    The DdeI restriction-modification system from Desulfovibrio desulfuricans was successfully cloned into E. coli. A new E. coli strain was engineered to overcome viability issues with cytosine methylase activity.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Microbiology

    Background:

    • DdeI is a Type II restriction-modification system from Desulfovibrio desulfuricans.
    • It recognizes the specific DNA sequence CTNAG.
    • Restriction-modification systems are crucial for bacterial defense and DNA regulation.

    Purpose of the Study:

    • To clone the DdeI restriction-modification system into E. coli.
    • To characterize the DdeI methylase activity.
    • To develop a stable E. coli host for the DdeI system.

    Main Methods:

    • Cloning of methylase and endonuclease genes into E. coli plasmids (pBR322 and pACYC184).
    • Southern blot analysis for gene identification.
    • Screening for endonuclease activity in E. coli hosts.

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  • Engineering a new E. coli strain (ER1467) for cytosine methylase tolerance.

    • Main Results:

    • Both DdeI methylase and endonuclease genes were successfully cloned and stably maintained in E. coli on separate plasmids.
    • DdeI methylase was confirmed as a cytosine methylase.
    • Increased cytosine methylase activity reduced E. coli RR1 viability, necessitating the use of ER1467.
    • High methylation levels were essential for introducing the active endonuclease gene.

    Conclusions:

    • The DdeI restriction-modification system can be functionally cloned and maintained in E. coli.
    • A specialized E. coli strain (ER1467) is required for efficient cloning and maintenance of cytosine methylase-producing systems.
    • Successful cloning required high levels of DdeI methylation, highlighting the importance of host compatibility.