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A general method for cloning eukaryotic structural gene sequences.

R Higuchi, G V Paddock, R Wall

    Proceedings of the National Academy of Sciences of the United States of America
    |September 1, 1976
    PubMed
    Summary
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    Researchers successfully inserted complementary DNA (cDNA) from rabbit globin mRNA into E. coli plasmids. This enables the study of globin gene sequences within bacteria, advancing genetic research.

    Area of Science:

    • Molecular Biology
    • Genetic Engineering
    • Biotechnology

    Background:

    • Rabbit globin messenger RNA (mRNA) is crucial for hemoglobin production.
    • Efficient methods for cloning eukaryotic genes in prokaryotic systems are essential for molecular biology research.

    Purpose of the Study:

    • To clone complementary DNA (cDNA) transcribed from rabbit globin mRNA into Escherichia coli (E. coli) plasmids.
    • To confirm the presence and analyze the characteristics of the inserted globin sequences in E. coli.

    Main Methods:

    • In vitro transcription of rabbit globin mRNA to create complementary DNA (cDNA).
    • Double-stranded cDNA synthesis and insertion into E. coli plasmids (pSC101, pMB9) using the poly(dT)/poly(dA) tailing and annealing technique.
    • Hybridization of globin RNA to E. coli DNA from clones for sequence verification.

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  • Fingerprint analysis to estimate the quantity of inserted globin sequences.

    • Main Results:

    • Successful insertion of rabbit globin cDNA into E. coli plasmids.
    • Verification of globin sequence presence in E. coli transformants via RNA-DNA hybridization.
    • Identification of the inserted sequences as rabbit beta globin chain.
    • Confirmation of an EcoRI restriction site within the inserted beta globin sequences.

    Conclusions:

    • The study demonstrates the successful cloning of functional rabbit beta globin sequences into E. coli.
    • This achievement provides a valuable tool for further investigation of globin gene structure and function.
    • The methods employed are effective for expressing eukaryotic genes in prokaryotic hosts.