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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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Strategy for construction of homozygous diploid strains.(A) The traditional method requires individual construction of MATa and MATα haploid strains carrying different selection markers (linked to mutations or each strain). The diploid strains can be selected on plates lacking nutrients or containing antibiotics. (B) The new method proposed in this study generates homozygous diploids from a single haploid strain by subsequent use of the PGAL1-HO (galactose-inducible mating-type switch) and PSTE18-URA3 (counter selection marker for diploids) genes. The diploid strains are selected on plates containing 5-FOA, where non-mated haploid strains cannot grow.
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pone-0026584-g001: Strategy for construction of homozygous diploid strains.(A) The traditional method requires individual construction of MATa and MATα haploid strains carrying different selection markers (linked to mutations or each strain). The diploid strains can be selected on plates lacking nutrients or containing antibiotics. (B) The new method proposed in this study generates homozygous diploids from a single haploid strain by subsequent use of the PGAL1-HO (galactose-inducible mating-type switch) and PSTE18-URA3 (counter selection marker for diploids) genes. The diploid strains are selected on plates containing 5-FOA, where non-mated haploid strains cannot grow.

Mentions: The yeast sexual cell types are designated a and α, which are conferred by the MATa and MATα alleles of the Mating-Type Locus (MAT), respectively [2]. In general, homozygous diploid mutant strains (i.e. MATa/α xxxΔ/xxxΔ) are constructed by crossing strains of the opposite mating-type, which need to be constructed individually. When the two haploids have different prototrophic or antibiotic resistance markers, the diploids can be easily selected on plates lacking both nutrients or containing both antibiotics because auxotrophy or antibiotic sensitivity are complemented by each genotype (Figure 1A). The HO endonuclease, which mediates mating-type switch, can be used to obtain diploids via mating of MATa and MATα cells within colonies [3]. Alternatively, zygotes (dumbbell-shaped cells) can be isolated by micromanipulation during conjugation of two cells. However, these methods are unsuitable for large-scale analysis. Thus, there has been no easy way to construct and select diploid strains from single haploids at high throughput so far.


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)

Strategy for construction of homozygous diploid strains.(A) The traditional method requires individual construction of MATa and MATα haploid strains carrying different selection markers (linked to mutations or each strain). The diploid strains can be selected on plates lacking nutrients or containing antibiotics. (B) The new method proposed in this study generates homozygous diploids from a single haploid strain by subsequent use of the PGAL1-HO (galactose-inducible mating-type switch) and PSTE18-URA3 (counter selection marker for diploids) genes. The diploid strains are selected on plates containing 5-FOA, where non-mated haploid strains cannot grow.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026584-g001: Strategy for construction of homozygous diploid strains.(A) The traditional method requires individual construction of MATa and MATα haploid strains carrying different selection markers (linked to mutations or each strain). The diploid strains can be selected on plates lacking nutrients or containing antibiotics. (B) The new method proposed in this study generates homozygous diploids from a single haploid strain by subsequent use of the PGAL1-HO (galactose-inducible mating-type switch) and PSTE18-URA3 (counter selection marker for diploids) genes. The diploid strains are selected on plates containing 5-FOA, where non-mated haploid strains cannot grow.
Mentions: The yeast sexual cell types are designated a and α, which are conferred by the MATa and MATα alleles of the Mating-Type Locus (MAT), respectively [2]. In general, homozygous diploid mutant strains (i.e. MATa/α xxxΔ/xxxΔ) are constructed by crossing strains of the opposite mating-type, which need to be constructed individually. When the two haploids have different prototrophic or antibiotic resistance markers, the diploids can be easily selected on plates lacking both nutrients or containing both antibiotics because auxotrophy or antibiotic sensitivity are complemented by each genotype (Figure 1A). The HO endonuclease, which mediates mating-type switch, can be used to obtain diploids via mating of MATa and MATα cells within colonies [3]. Alternatively, zygotes (dumbbell-shaped cells) can be isolated by micromanipulation during conjugation of two cells. However, these methods are unsuitable for large-scale analysis. Thus, there has been no easy way to construct and select diploid strains from single haploids at high throughput so far.

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