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Recombinant plasmids containing avian vitellogenin structural gene sequences derived from complementary DNA

T Ohno, P J Cozens, A C Cato

    Biochimica Et Biophysica Acta
    |January 1, 1980
    PubMed
    Summary

    Researchers cloned chicken vitellogenin complementary DNA (cDNA) using avian myeloblastosis virus reverse transcriptase and Escherichia coli DNA polymerase. This allows for the study of egg yolk protein precursors.

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    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genetics

    Background:

    • Vitellogenin is a precursor protein essential for egg yolk formation in avian species.
    • Understanding vitellogenin gene expression is crucial for avian reproductive biology.

    Purpose of the Study:

    • To clone and characterize complementary DNA (cDNA) encoding chicken vitellogenin.
    • To develop a tool for studying vitellogenin gene regulation and protein synthesis.

    Main Methods:

    • Messenger RNA (mRNA) for chicken vitellogenin was isolated and reverse transcribed into cDNA using avian myeloblastosis virus (AMV) reverse transcriptase.
    • Double-stranded cDNA synthesis was performed using Escherichia coli DNA polymerase I.
    • The cDNA was inserted into the pBR322 plasmid via the poly(dA)·poly(dT) tailing method and transformed into E. coli.

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  • Colony hybridization with labeled vitellogenin mRNA and subsequent screening methods (agarose gel electrophoresis, in situ hybridization) were employed to identify positive clones.

    • Main Results:

    • Successful cloning of chicken vitellogenin cDNA sequences into the pBR322 plasmid.
    • The largest cloned cDNA insert was approximately 2500 base pairs.
    • Restriction mapping indicated sequence diversity among the cloned inserts.
    • The cloned sequences hybridized to mRNA that directed the synthesis of immunoprecipitable vitellogenin in a cell-free system.

    Conclusions:

    • The study successfully generated a recombinant plasmid containing chicken vitellogenin cDNA.
    • This cloned cDNA serves as a valuable tool for further research into vitellogenin gene expression and regulation.
    • The findings contribute to the understanding of egg yolk protein synthesis at the molecular level.