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Efficient construction of homozygous diploid strains identifies genes required for the hyper-filamentous phenotype in Saccharomyces cerevisiae.

Furukawa K, Furukawa T, Hohmann S - PLoS ONE (2011)

Bottom Line: Following this approach, we identified 49 suppressor mutations.Those include well-known positive regulator genes for filamentous growth signaling pathways, genes involved in mitochondrial function, DNA damage checkpoint, chromatin remodeling, and cell cycle, and also previously uncharacterized genes.Our results indicate that combinatorial use of the PGAL1-HO and PSTE18-URA3 genes is suitable to efficiently construct and select diploids and that this approach is useful for genetic studies especially when combined with large-scale screening.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology/Microbiology, University of Gothenburg, Gothenburg, Sweden. kentaro.furukawa@cmb.gu.se

ABSTRACT
Yeast cells undergo diploid-specific developments such as spore formation via meiosis and pseudohyphal development under certain nutrient-limited conditions. Studies on these aspects require homozygous diploid mutants, which are generally constructed by crossing strains of opposite mating-type with the same genetic mutation. So far, there has been no direct way to generate and select diploids from haploid cells. Here, we developed a method for efficient construction of homozygous diploids using a PGAL1-HO gene (galactose-inducible mating-type switch) and a PSTE18-URA3 gene (counter selection marker for diploids). Diploids are generated by transient induction of the HO endonuclease, which is followed by mating of part of the haploid population. Since the STE18 promoter is repressed in diploids, diploids carrying PSTE18-URA3 can be selected on 5-fluoroorotic acid (5-FOA) plates where the uracil prototrophic haploids cannot grow. To demonstrate that this method is useful for genetic studies, we screened suppressor mutations of the complex colony morphology, strong agar invasion and/or hyper-filamentous growth caused by lack of the Hog1 MAPK in the diploid Σ1278b strain background. Following this approach, we identified 49 suppressor mutations. Those include well-known positive regulator genes for filamentous growth signaling pathways, genes involved in mitochondrial function, DNA damage checkpoint, chromatin remodeling, and cell cycle, and also previously uncharacterized genes. Our results indicate that combinatorial use of the PGAL1-HO and PSTE18-URA3 genes is suitable to efficiently construct and select diploids and that this approach is useful for genetic studies especially when combined with large-scale screening.

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Effect of the PSTE18-URA3 gene on growth of haploids and diploids.(A) The haploid and diploid PSTE18-URA3 strains display opposite growth phenotypes on plates lacking uracil or containing 5-FOA. The strains were grown for 2–3 days at 30°C. (B) The growth phenotype of the diploid PSTE18-URA3 strain can revert to that of haploid after sporulation. The indicated diploid strains were sporulated, tetrads were dissected and spore progeny was grown on YPD plate for 3 days at 30°C. Then, the cells were replicated on the indicated plates and grown for 2–3 days at 30°C.
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pone-0026584-g002: Effect of the PSTE18-URA3 gene on growth of haploids and diploids.(A) The haploid and diploid PSTE18-URA3 strains display opposite growth phenotypes on plates lacking uracil or containing 5-FOA. The strains were grown for 2–3 days at 30°C. (B) The growth phenotype of the diploid PSTE18-URA3 strain can revert to that of haploid after sporulation. The indicated diploid strains were sporulated, tetrads were dissected and spore progeny was grown on YPD plate for 3 days at 30°C. Then, the cells were replicated on the indicated plates and grown for 2–3 days at 30°C.

Mentions: First, we investigated whether the PSTE18-URA3 gene allows selecting for diploid cells. We constructed wild-type PSTE18-URA3 and hog1Δ PSTE18-URA3 strains in the three cell types (MATa, MATα, and MATa/α) and grew them on SC plates lacking uracil or containing 0.1% 5-FOA. As shown in Figure 2A, the haploid and diploid PSTE18-URA3 strains showed the expected phenotypes, i.e. the haploid strains were uracil prototrophic and 5-FOA sensitive, and the diploid strains were uracil auxotrophic and 5-FOA resistant. Moreover, the haploid cells that germinated from spore progeny of the diploid PSTE18-URA3 strains displayed uracil prototrophy and 5-FOA sensitivity (Figure 2B). Taken together, these results demonstrate that the PSTE18-URA3 gene can be used as a diploid selection marker.


Efficient construction of homozygous diploid strains identifies genes required for the hyper-filamentous phenotype in Saccharomyces cerevisiae.

Furukawa K, Furukawa T, Hohmann S - PLoS ONE (2011)

Effect of the PSTE18-URA3 gene on growth of haploids and diploids.(A) The haploid and diploid PSTE18-URA3 strains display opposite growth phenotypes on plates lacking uracil or containing 5-FOA. The strains were grown for 2–3 days at 30°C. (B) The growth phenotype of the diploid PSTE18-URA3 strain can revert to that of haploid after sporulation. The indicated diploid strains were sporulated, tetrads were dissected and spore progeny was grown on YPD plate for 3 days at 30°C. Then, the cells were replicated on the indicated plates and grown for 2–3 days at 30°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198790&req=5

pone-0026584-g002: Effect of the PSTE18-URA3 gene on growth of haploids and diploids.(A) The haploid and diploid PSTE18-URA3 strains display opposite growth phenotypes on plates lacking uracil or containing 5-FOA. The strains were grown for 2–3 days at 30°C. (B) The growth phenotype of the diploid PSTE18-URA3 strain can revert to that of haploid after sporulation. The indicated diploid strains were sporulated, tetrads were dissected and spore progeny was grown on YPD plate for 3 days at 30°C. Then, the cells were replicated on the indicated plates and grown for 2–3 days at 30°C.
Mentions: First, we investigated whether the PSTE18-URA3 gene allows selecting for diploid cells. We constructed wild-type PSTE18-URA3 and hog1Δ PSTE18-URA3 strains in the three cell types (MATa, MATα, and MATa/α) and grew them on SC plates lacking uracil or containing 0.1% 5-FOA. As shown in Figure 2A, the haploid and diploid PSTE18-URA3 strains showed the expected phenotypes, i.e. the haploid strains were uracil prototrophic and 5-FOA sensitive, and the diploid strains were uracil auxotrophic and 5-FOA resistant. Moreover, the haploid cells that germinated from spore progeny of the diploid PSTE18-URA3 strains displayed uracil prototrophy and 5-FOA sensitivity (Figure 2B). Taken together, these results demonstrate that the PSTE18-URA3 gene can be used as a diploid selection marker.

Bottom Line: Following this approach, we identified 49 suppressor mutations.Those include well-known positive regulator genes for filamentous growth signaling pathways, genes involved in mitochondrial function, DNA damage checkpoint, chromatin remodeling, and cell cycle, and also previously uncharacterized genes.Our results indicate that combinatorial use of the PGAL1-HO and PSTE18-URA3 genes is suitable to efficiently construct and select diploids and that this approach is useful for genetic studies especially when combined with large-scale screening.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology/Microbiology, University of Gothenburg, Gothenburg, Sweden. kentaro.furukawa@cmb.gu.se

ABSTRACT
Yeast cells undergo diploid-specific developments such as spore formation via meiosis and pseudohyphal development under certain nutrient-limited conditions. Studies on these aspects require homozygous diploid mutants, which are generally constructed by crossing strains of opposite mating-type with the same genetic mutation. So far, there has been no direct way to generate and select diploids from haploid cells. Here, we developed a method for efficient construction of homozygous diploids using a PGAL1-HO gene (galactose-inducible mating-type switch) and a PSTE18-URA3 gene (counter selection marker for diploids). Diploids are generated by transient induction of the HO endonuclease, which is followed by mating of part of the haploid population. Since the STE18 promoter is repressed in diploids, diploids carrying PSTE18-URA3 can be selected on 5-fluoroorotic acid (5-FOA) plates where the uracil prototrophic haploids cannot grow. To demonstrate that this method is useful for genetic studies, we screened suppressor mutations of the complex colony morphology, strong agar invasion and/or hyper-filamentous growth caused by lack of the Hog1 MAPK in the diploid Σ1278b strain background. Following this approach, we identified 49 suppressor mutations. Those include well-known positive regulator genes for filamentous growth signaling pathways, genes involved in mitochondrial function, DNA damage checkpoint, chromatin remodeling, and cell cycle, and also previously uncharacterized genes. Our results indicate that combinatorial use of the PGAL1-HO and PSTE18-URA3 genes is suitable to efficiently construct and select diploids and that this approach is useful for genetic studies especially when combined with large-scale screening.

Show MeSH
Related in: MedlinePlus