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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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Screening suppressor mutations of the complex colony morphology or hyper-filamentous growth phenotype in the hog1Δ/hog1Δ backgrounds.(A) Strategy for screening the suppressor mutations. Using the haploid hog1Δ PSTE18-URA3 strain carrying pJH283 (PGAL1-HO::TRP1) as a host strain, transposon insertion mutagenesis was performed and mutant strains defective in complex colony morphology were screened by visual inspection. The details are described in Materials and Methods. (B) One example of the screening results is shown. The candidates, smooth colony or less complex colony, were further analyzed: identification of the transposon insertion position, mating-type PCR, and morphological assay for invasive growth and filamentous growth.
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pone-0026584-g003: Screening suppressor mutations of the complex colony morphology or hyper-filamentous growth phenotype in the hog1Δ/hog1Δ backgrounds.(A) Strategy for screening the suppressor mutations. Using the haploid hog1Δ PSTE18-URA3 strain carrying pJH283 (PGAL1-HO::TRP1) as a host strain, transposon insertion mutagenesis was performed and mutant strains defective in complex colony morphology were screened by visual inspection. The details are described in Materials and Methods. (B) One example of the screening results is shown. The candidates, smooth colony or less complex colony, were further analyzed: identification of the transposon insertion position, mating-type PCR, and morphological assay for invasive growth and filamentous growth.

Mentions: Following the strategy shown in Figure 3A, a transposon insertion mutagenesis was performed using the haploid hog1Δ PSTE18-URA3 strain carrying pJH283 (PGAL1-HO) as a host strain. Since the diploid hog1Δ/hog1Δ strain displays complex colony morphology even on YPD plates while the diploid wild-type strain does not [10], [16], mutant strains defective in formation of complex colony morphology were first screened by visual inspection (Figure 3B). From more than six thousand 5-FOA resistant strains (candidates for homozygous diploids) which were obtained at 93% success rate of randomly picked transformants, we isolated more than 100 mutant strains that showed smooth- or less complex-colony morphology. The diploid state in those was confirmed by PCR analysis of the mating-type locus. We amplified the transposon insertion regions of these strains by vectorette PCR and sequencing of the PCR products identified 49 unique genes (Table 1 and Table S1). Morphological developments (complex colony morphology, invasive growth, and filamentous growth) of all 49 homozygous double mutant strains on YPD plates were characterized, and the results are discussed below.


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)

Screening suppressor mutations of the complex colony morphology or hyper-filamentous growth phenotype in the hog1Δ/hog1Δ backgrounds.(A) Strategy for screening the suppressor mutations. Using the haploid hog1Δ PSTE18-URA3 strain carrying pJH283 (PGAL1-HO::TRP1) as a host strain, transposon insertion mutagenesis was performed and mutant strains defective in complex colony morphology were screened by visual inspection. The details are described in Materials and Methods. (B) One example of the screening results is shown. The candidates, smooth colony or less complex colony, were further analyzed: identification of the transposon insertion position, mating-type PCR, and morphological assay for invasive growth and filamentous growth.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026584-g003: Screening suppressor mutations of the complex colony morphology or hyper-filamentous growth phenotype in the hog1Δ/hog1Δ backgrounds.(A) Strategy for screening the suppressor mutations. Using the haploid hog1Δ PSTE18-URA3 strain carrying pJH283 (PGAL1-HO::TRP1) as a host strain, transposon insertion mutagenesis was performed and mutant strains defective in complex colony morphology were screened by visual inspection. The details are described in Materials and Methods. (B) One example of the screening results is shown. The candidates, smooth colony or less complex colony, were further analyzed: identification of the transposon insertion position, mating-type PCR, and morphological assay for invasive growth and filamentous growth.
Mentions: Following the strategy shown in Figure 3A, a transposon insertion mutagenesis was performed using the haploid hog1Δ PSTE18-URA3 strain carrying pJH283 (PGAL1-HO) as a host strain. Since the diploid hog1Δ/hog1Δ strain displays complex colony morphology even on YPD plates while the diploid wild-type strain does not [10], [16], mutant strains defective in formation of complex colony morphology were first screened by visual inspection (Figure 3B). From more than six thousand 5-FOA resistant strains (candidates for homozygous diploids) which were obtained at 93% success rate of randomly picked transformants, we isolated more than 100 mutant strains that showed smooth- or less complex-colony morphology. The diploid state in those was confirmed by PCR analysis of the mating-type locus. We amplified the transposon insertion regions of these strains by vectorette PCR and sequencing of the PCR products identified 49 unique genes (Table 1 and Table S1). Morphological developments (complex colony morphology, invasive growth, and filamentous growth) of all 49 homozygous double mutant strains on YPD plates were characterized, and the results are discussed below.

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