efficient construction of homozygous diploid strains identifies genes required for the hyper-filamentous phenotype in saccharomyces cerevisiae高效施工所需的基因纯合二倍体品种识别hyper-filamentous表型在酿酒酵母.pdfVIP

Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae Kentaro Furukawa*, Takako Furukawa, Stefan Hohmann* Department of Cell and Molecular Biology/Microbiology, University of Gothenburg, Gothenburg, Sweden 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 S1278b 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 comb