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Cloning yeast genes by complementation.

V Lundblad1

  • 1University of California, Berkeley, California, USA.

Current Protocols in Molecular Biology
|February 12, 2008
PubMed
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This study details a method for cloning yeast genes using complementation, focusing on the cdc101-1 cell cycle mutant. The protocol ensures isolated genes are functional wild-type CDC101, not suppressors, enabling precise genetic analysis.

Area of Science:

  • Molecular Biology
  • Yeast Genetics
  • Cell Cycle Research

Background:

  • Yeast gene cloning is crucial for understanding eukaryotic gene function.
  • Complementation assays are standard for identifying functional genes.
  • Cell cycle mutants like cdc101-1 provide models for studying cell division.

Purpose of the Study:

  • To present a generalized protocol for cloning yeast genes via complementation.
  • To describe the principles of isolating and validating gene clones using a hypothetical temperature-sensitive mutation (cdc101-1).
  • To ensure isolated clones represent the wild-type gene and not phenotypic suppressors.

Main Methods:

  • Transformation of a yeast strain with a cdc101-1 mutation using a genomic DNA library.
  • Screening for temperature-resistant colonies at 37°C.

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  • Plasmid segregation analysis to confirm complementation specificity.
  • Complementation tests with integrated gene disruptions to rule out suppressor effects.

    • Main Results:

    • Successful isolation of a genomic DNA clone complementing the cdc101-1 mutation.
    • Demonstration of plasmid-specific complementation through co-segregation of phenotype and marker.
    • Confirmation that the cloned gene encodes the wild-type CDC101, not a suppressor, via complementation tests.

    Conclusions:

    • The presented protocol provides a robust method for yeast gene cloning by complementation.
    • Validation steps are essential to confirm the identity and function of cloned genes.
    • This approach is vital for dissecting gene function in yeast cell cycle regulation.