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Related Experiment Videos

SV40 T antigen binding site mutations that affect autoregulation.

D C Rio, R Tjian

    Cell
    |April 1, 1983
    PubMed
    Summary
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    Simian virus 40 (SV40) T antigen binding sites I and II are crucial for repressing early gene transcription. Mutations in these sites lead to increased viral gene expression, highlighting their role in autoregulation.

    Area of Science:

    • Molecular Biology
    • Virology
    • Gene Regulation

    Background:

    • Simian virus 40 (SV40) early transcription is regulated by the viral T antigen.
    • T antigen plays a role in both activating and repressing its own gene expression (autoregulation).

    Purpose of the Study:

    • To investigate the role of T antigen binding sites I and II in the autoregulation of SV40 early transcription.
    • To determine the functional significance of these binding sites in both in vitro and in vivo systems.

    Main Methods:

    • Site-directed mutagenesis was used to create SV40 mutants with deletions or substitutions in T antigen binding sites.
    • In vitro transcription assays were performed using cell-free extracts and purified T antigen.
    • In vivo transcription was measured in COS7 monkey cells transfected with SV40-dihydrofolate reductase (dhfr) reporter gene constructs.

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    Main Results:

    • A mutant with substitutions in T antigen binding site II (pIN4) showed normal transcription in vitro but was resistant to T antigen-mediated repression.
    • Mutant SV-dhfr vectors lacking site I or site II sequences exhibited a 3-4 fold increase in dhfr RNA levels.
    • Deletion of both sites or use of a temperature-sensitive T antigen resulted in an 8-10 fold increase in dhfr RNA.

    Conclusions:

    • T antigen binding site II is functionally important for the repression of SV40 early transcription.
    • Binding of T antigen to both sites I and II is essential for effective autoregulation of SV40 early gene expression in vivo.