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Selection of functional cDNAs by complementation in yeast.

G L McKnight, B L McConaughy

    Proceedings of the National Academy of Sciences of the United States of America
    |July 1, 1983
    PubMed
    Summary
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    Researchers created a yeast cDNA library to isolate specific genes. This method successfully identified several yeast cDNAs, demonstrating its utility for discovering rare genetic sequences.

    Area of Science:

    • Molecular Biology
    • Yeast Genetics

    Background:

    • Gene discovery and characterization are fundamental to understanding biological processes.
    • Efficient methods for isolating specific complementary DNAs (cDNAs) are crucial for genetic research.

    Purpose of the Study:

    • To construct a yeast cDNA expression library for functional screening.
    • To isolate and identify specific yeast cDNAs, including ADC1, HIS3, URA3, and ASP5, using functional complementation.
    • To assess the utility of this approach for isolating rare cDNAs.

    Main Methods:

    • Preparation of a yeast cDNA expression plasmid pool comprising approximately 40,000 members.
    • Transformation of yeast mutants with the cDNA plasmid pool.
    • Isolation of cDNAs via functional complementation of yeast mutants.

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  • Restriction enzyme analysis for genetic identification and characterization of isolated cDNAs.

    • Main Results:

    • Successfully isolated and genetically identified ADC1, HIS3, and URA3 cDNAs.
    • Confirmed the presence of 5' noncoding sequences within the URA3 cDNA.
    • Demonstrated that the relative abundance of cDNAs in the pool mirrors mRNA abundance in total poly(A)+ RNA (0.01%–1%).

    Conclusions:

    • The constructed yeast cDNA expression library is effective for isolating specific genes.
    • Functional complementation is a viable strategy for identifying cDNAs, even those present at low abundance.
    • This methodology holds promise for the isolation of rare cDNAs from higher eukaryotes.