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Related Experiment Videos

Partial purification and characterization of the mRNA complementing a temperature-sensitive S-phase cell cycle

A Fainsod, M Marcus, P F Lin

    Proceedings of the National Academy of Sciences of the United States of America
    |April 1, 1984
    PubMed
    Summary

    Researchers identified a specific messenger RNA (mRNA) that temporarily corrects a temperature-sensitive (ts) cell cycle mutation in hamster cells. This mRNA, approximately 940 nucleotides long, codes for a protein under 230 amino acids, aiding in cell cycle progression research.

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    Area of Science:

    • Cell Biology
    • Molecular Genetics
    • Biochemistry

    Background:

    • E36ts24 is a temperature-sensitive (ts) cell cycle mutant of Chinese hamster lung cells.
    • At restrictive temperatures (40.3°C), E36ts24 cells arrest at early S phase.

    Purpose of the Study:

    • To identify the genetic material responsible for complementing the ts mutation in E36ts24 cells.
    • To characterize the size and potential coding capacity of the complementing mRNA.

    Main Methods:

    • Microinjection of poly(A)+ RNA from wild-type E36 cells into arrested E36ts24 cells.
    • Assaying for transient complementation via [3H]thymidine incorporation and autoradiography.
    • Fractionation of mRNA using methylmercuric hydroxide/agarose gel electrophoresis.

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    Main Results:

    • Microinjected wild-type RNA transiently complemented the ts mutation in E36ts24 cells.
    • The complementing mRNA is approximately 940 nucleotides in length.
    • The identified mRNA can encode a protein of at most 230 amino acids.

    Conclusions:

    • A specific mRNA species is responsible for the transient complementation of the E36ts24 cell cycle mutation.
    • The size of the mRNA suggests a relatively small protein product involved in S phase regulation.
    • This study provides a partial purification (100- to 200-fold) of the active mRNA, facilitating further molecular analysis.