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A method for analyzing transcription using permeabilized cells.

T E Gilroy, A L Beaudet, J Yu

    Analytical Biochemistry
    |December 1, 1984
    PubMed
    Summary
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    Permeabilized Friend cells efficiently synthesize RNA, enabling studies of gene transcription. This method is more active than isolated nuclei for RNA synthesis and initiation.

    Area of Science:

    • Molecular Biology
    • Cell Biology
    • Biochemistry

    Background:

    • Friend cells are a model system for studying erythroid differentiation.
    • RNA synthesis is a fundamental process in gene expression.
    • Understanding transcription in permeabilized cells offers insights into gene regulation.

    Purpose of the Study:

    • To characterize RNA synthesis in permeabilized Friend cells.
    • To investigate the transcription of the mouse beta-major globin gene.
    • To compare the RNA synthesis activity of permeabilized cells with isolated nuclei.

    Main Methods:

    • Friend cells were permeabilized using a hypotonic solution with Tween 80.
    • RNA synthesis was measured by [3H]UTP incorporation.
    • Transcription of the beta-major globin gene was analyzed using hybridization techniques.

    Related Experiment Videos

  • RNA initiation was assessed by incorporating a thiol group at the 5' nucleotide.

    • Main Results:

    • Permeabilized cells incorporated [3H]UTP into RNA, with synthesis inhibited by actinomycin D and sensitive to KOH hydrolysis.
    • UMP incorporation kinetics varied with temperature, and alpha-amanitin inhibition differed between 23°C and 37°C.
    • Induced Friend cells showed a fivefold increase in beta-major globin gene transcription.
    • Permeabilized cells demonstrated active RNA initiation and were 7-11 times more active than isolated nuclei.

    Conclusions:

    • Permeabilized Friend cells provide a robust system for studying RNA synthesis and gene transcription.
    • This method allows for the investigation of transcriptional regulation, as exemplified by the beta-major globin gene.
    • Permeabilized cells offer a more active and comprehensive model for RNA synthesis compared to isolated nuclei.