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Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae

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...

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Autores principales: Furukawa, Kentaro, Furukawa, Takako, Hohmann, Stefan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198790/
https://www.ncbi.nlm.nih.gov/pubmed/22039512
http://dx.doi.org/10.1371/journal.pone.0026584
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author Furukawa, Kentaro
Furukawa, Takako
Hohmann, Stefan
author_facet Furukawa, Kentaro
Furukawa, Takako
Hohmann, Stefan
author_sort Furukawa, Kentaro
collection PubMed
description 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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spelling pubmed-31987902011-10-28 Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae Furukawa, Kentaro Furukawa, Takako Hohmann, Stefan PLoS One Research Article 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. Public Library of Science 2011-10-21 /pmc/articles/PMC3198790/ /pubmed/22039512 http://dx.doi.org/10.1371/journal.pone.0026584 Text en Furukawa et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Furukawa, Kentaro
Furukawa, Takako
Hohmann, Stefan
Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae
title Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae
title_full Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae
title_fullStr Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae
title_full_unstemmed Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae
title_short Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae
title_sort efficient construction of homozygous diploid strains identifies genes required for the hyper-filamentous phenotype in saccharomyces cerevisiae
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198790/
https://www.ncbi.nlm.nih.gov/pubmed/22039512
http://dx.doi.org/10.1371/journal.pone.0026584
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