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Replication of a plasmid bearing a human Alu-family repeat in monkey COS-7 cells

E M Johnson, W R Jelinek

    Proceedings of the National Academy of Sciences of the United States of America
    |July 1, 1986
    PubMed

    Insights

    Certain human Alu sequences within plasmids can act as replication origins in monkey COS-7 cells. A specific deletion in the Alu sequence prevented this DNA replication, confirming its role.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Cell Biology

    Background:

    • Human Alu sequences are abundant repetitive elements in the genome.
    • Their function, particularly in replication, remains incompletely understood.

    Purpose of the Study:

    • To investigate whether human Alu sequences can function as origins of replication.
    • To identify specific regions within Alu sequences critical for replication initiation.

    Main Methods:

    • Transformation of monkey COS-7 cells with a pBR322 plasmid (BLUR8 DNA) containing a human Alu sequence.
    • Assessment of DNA replication using Dpn I resistance assay.
    • Electron microscopy to visualize replicated molecules and map replication origins.
    • Site-directed mutagenesis to create a 16-base-pair deletion within the Alu sequence.

    Main Results:

    • 2-5% of transformed BLUR8 molecules replicated within 24 hours, as indicated by Dpn I resistance.
    • Electron microscopy confirmed circular molecules with replication bubbles centered on the Alu insert.
    • A 16-base-pair deletion in the Alu sequence abolished replication.

    Conclusions:

    • Specific human Alu sequences can function as functional origins of replication in mammalian COS-7 cells.
    • The identified region within the Alu sequence is essential for initiating DNA replication.

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