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Cloning yeast genes by complementation.

¹University of California, Berkeley, California, USA.

Current Protocols in Molecular Biology

|February 12, 2008

Summary

This summary is machine-generated.

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.

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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.
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.