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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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Morphological assay of homozygous double mutant strains which suppress enhanced morphological developments of hog1Δ/hog1Δ.CCM: complex colony morphology, IG: invasive growth, FG: filamentous growth. All other suppressor mutants identified are shown in Table 1.
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pone-0026584-g004: Morphological assay of homozygous double mutant strains which suppress enhanced morphological developments of hog1Δ/hog1Δ.CCM: complex colony morphology, IG: invasive growth, FG: filamentous growth. All other suppressor mutants identified are shown in Table 1.

Mentions: In addition to the mitochondria-related mutations, we identified 30 mutations that suppress at least two of the enhanced morphological developments of hog1Δ/hog1Δ (Table S1) and representative mutants are shown in Figure 4. Thirteen of the 30 genes have previously been reported to be involved in at least one of the three morphological developments in the S. cerevisiae Σ1278b background [14], [15], [16], [19], [20], [21], [22], [23], [24]. Those genes include the well-known STE7, KSS1, TEC1, RAS2, and FLO8 that regulate filamentous growth via the MAPK or cAMP-PKA signaling pathway. These two signaling pathways converge on the regulation of the MUC1 (also known as FLO11) gene [25], which encodes a GPI-anchored cell surface mucin required for morphological developments. DHH1 is involved in translational regulation of the Ste12 transcription factor which is regulated under the Kss1 MAPK pathway and essential for MUC1 expression [24]. GCN2 (general amino acid control system) and MSN1 (transcriptional activator) are involved in the regulation of MUC1 under certain nutrient conditions [19], [26]. Presumably, RIM9 is also important for the regulation of MUC1 through the pH-responsive Dfg16-Rim101 pathway [27]. Thus, our screen implies that impaired MUC1 expression is sufficient to suppress enhanced morphological developments of hog1Δ/hog1Δ. Indeed, deletion of the MUC1 gene in the hog1Δ/hog1Δ background lost the enhanced morphological developments and resulted in morphology similar to a muc1Δ/muc1Δ strain (Figure 4).


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)

Morphological assay of homozygous double mutant strains which suppress enhanced morphological developments of hog1Δ/hog1Δ.CCM: complex colony morphology, IG: invasive growth, FG: filamentous growth. All other suppressor mutants identified are shown in Table 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026584-g004: Morphological assay of homozygous double mutant strains which suppress enhanced morphological developments of hog1Δ/hog1Δ.CCM: complex colony morphology, IG: invasive growth, FG: filamentous growth. All other suppressor mutants identified are shown in Table 1.
Mentions: In addition to the mitochondria-related mutations, we identified 30 mutations that suppress at least two of the enhanced morphological developments of hog1Δ/hog1Δ (Table S1) and representative mutants are shown in Figure 4. Thirteen of the 30 genes have previously been reported to be involved in at least one of the three morphological developments in the S. cerevisiae Σ1278b background [14], [15], [16], [19], [20], [21], [22], [23], [24]. Those genes include the well-known STE7, KSS1, TEC1, RAS2, and FLO8 that regulate filamentous growth via the MAPK or cAMP-PKA signaling pathway. These two signaling pathways converge on the regulation of the MUC1 (also known as FLO11) gene [25], which encodes a GPI-anchored cell surface mucin required for morphological developments. DHH1 is involved in translational regulation of the Ste12 transcription factor which is regulated under the Kss1 MAPK pathway and essential for MUC1 expression [24]. GCN2 (general amino acid control system) and MSN1 (transcriptional activator) are involved in the regulation of MUC1 under certain nutrient conditions [19], [26]. Presumably, RIM9 is also important for the regulation of MUC1 through the pH-responsive Dfg16-Rim101 pathway [27]. Thus, our screen implies that impaired MUC1 expression is sufficient to suppress enhanced morphological developments of hog1Δ/hog1Δ. Indeed, deletion of the MUC1 gene in the hog1Δ/hog1Δ background lost the enhanced morphological developments and resulted in morphology similar to a muc1Δ/muc1Δ strain (Figure 4).

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