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Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development
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Revertants of an S49 cell mutant that expresses altered cyclic AMP-dependent protein kinase

T van Daalen Wetters, P Coffino

    Molecular and Cellular Biology
    |October 1, 1982
    PubMed

    Insights

    Researchers isolated Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive revertants from resistant cells using a novel counter-selection method. This process confirmed the role of cAMP-dependent protein kinase (cA-PK) in mediating cellular responses to cAMP.

    Area of Science:

    • Cell Biology
    • Molecular Genetics
    • Biochemistry

    Background:

    • Somatic cell genetics relies on isolating rare mutant phenotypes.
    • Cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cA-PK) mediates cellular responses to cAMP.
    • Understanding cA-PK's function is crucial for cellular signaling research.

    Purpose of the Study:

    • To isolate and characterize Dibutyryl adenosine 3",5"-phosphate (Bt2cAMP)-sensitive revertants from a resistant S49 cell mutant.
    • To investigate the role of structural gene lesions in the regulatory subunit of cA-PK.
    • To validate a novel counter-selection technique for isolating rare cellular phenotypes.

    Main Methods:

    • Isolation of Bt2cAMP-sensitive revertants using a counter-selection strategy.
    • Employing Bt2cAMP for reversible arrest of revertants.
    • Utilizing bromodeoxyuridine, 33258 Hoechst dye, and white light for selective cell killing.
    • Assessing reversion rates and the impact of mutagens like EMS, MNNG, and ICR191.

    Main Results:

    • Successfully isolated Bt2cAMP-sensitive revertants from a resistant S49 cell mutant.
    • Demonstrated that reversion restores wild-type levels and cAMP activation constants of cA-PK.
    • Observed mutagenic effects of EMS, MNNG, and ICR191 on reversion frequency.
    • Identified MNNG-induced revertants with thermally labile cA-PK activity.

    Conclusions:

    • The counter-selection method is effective for isolating rare phenotypes in somatic cell experiments.
    • Reversion is linked to alterations in cA-PK function, supporting its role as a cAMP mediator.
    • Reversion likely results from mutational events, with ICR191 suggesting novel mechanisms for somatic cell reversion.

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