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Related Experiment Videos

Cloned restriction/modification system from Pseudomonas aeruginosa.

T R Gingeras, J E Brooks

    Proceedings of the National Academy of Sciences of the United States of America
    |January 1, 1983
    PubMed
    Summary

    Researchers cloned Pseudomonas aeruginosa restriction/modification genes into E. coli. They characterized the PaeR7 restriction endonuclease and methylase, finding the methylase modifies adenine within the recognition sequence.

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    Area of Science:

    • Molecular Biology
    • Genetics
    • Microbiology

    Background:

    • Pseudomonas aeruginosa possesses a restriction/modification system.
    • Cloning these genes into Escherichia coli allows for detailed study.

    Purpose of the Study:

    • To clone and characterize the PaeR7 restriction/modification system from Pseudomonas aeruginosa.
    • To investigate the properties of the PaeR7 endonuclease and methylase enzymes.

    Main Methods:

    • Cloning of Pseudomonas aeruginosa DNA fragments encoding PaeR7 genes into plasmid vector pBR322.
    • Construction of a subclone (pPAORM3.8) containing the complete system.
    • BAL-31 nuclease digestion to generate clones with active methylase or endonuclease genes.
    • In vivo and in vitro assays to assess enzyme activity and DNA modification/cleavage.

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  • Examination of PaeR7 enzyme properties, including recognition sequence and cleavage specificity.
  • Comparison with other restriction enzymes like Xho I and EcoRI.

    • Main Results:

    • A 3.8-kilobase fragment (pPAORM3.8) contains the complete PaeR7 restriction/modification system.
    • Clones with active methylase but inactive endonuclease modified DNA without restricting phage growth.
    • Clones with active endonuclease but inactive methylase showed endonuclease activity in vitro but did not restrict phage in vivo.
    • PaeR7 restriction endonuclease recognizes the sequence CTNAG, similar to Xho I, but exhibits different cleavage patterns on specific DNA sites.
    • PaeR7 methylase was shown to modify the adenine residue within the recognition sequence.

    Conclusions:

    • The PaeR7 restriction/modification system can be functionally expressed in Escherichia coli.
    • The PaeR7 endonuclease and methylase exhibit distinct activities and specificities.
    • The methylase's modification of adenine is crucial for protecting DNA from cleavage by the corresponding endonuclease.