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Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae
Published on: December 2, 2022
Dominant marker vectors for selecting yeast mating products
Ivan Sadowski1, Pedro Lourenco, Jennifer Parent
1Department of Biochemistry and Molecular Biology, Molecular Epigenetics, Life Sciences Center, University of British Columbia, Vancouver, BC, Canada. sadowski@interchange.ubc.ca
Yeast (Chichester, England)
|July 10, 2008
Summary
New plasmid vectors facilitate the selection of diploid yeast strains from haploid parent cells lacking compatible markers. This method utilizes dominant selectable markers and auxotrophic selection for efficient diploid identification and isolation.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Synthetic Biology
Background:
- Selecting diploid yeast strains from haploid parents typically requires compatible recessive genetic markers.
- Existing methods can be cumbersome or lack efficiency in isolating desired diploid clones.
Purpose of the Study:
- To develop novel plasmid vectors for efficient selection of yeast diploids.
- To enable diploid selection in haploid strains lacking compatible recessive markers.
Main Methods:
- Construction of plasmid vectors containing one of five dominant selectable markers (kanMX4, hphMX4, natMX4, patMX3, ZEO), a yeast origin of replication, and the URA3 gene.
- Transformation of haploid yeast strains with plasmids expressing different dominant markers.
- Selection of diploids using drug combinations targeting both markers, followed by non-selective growth.
- Curing of plasmids and selection of diploids on 5-fluoroorotic acid (5-FOA) or via replica-plating.
Main Results:
- Successful development of plasmid vectors enabling diploid selection.
- Demonstrated efficient selection of diploids by combining dominant drug resistance markers.
- Established a method for post-selection identification of diploids through plasmid curing and auxotrophic selection.
Conclusions:
- The developed plasmid vectors provide a versatile and efficient tool for yeast diploid selection.
- This approach overcomes limitations of traditional methods by utilizing dominant markers and auxotrophy.
- The system facilitates genetic studies and strain engineering in yeast.
