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Taking the Yeast Genome by “Storm”: Beginning to Simplify Saccharomyces cerevisiae
Author(s) -
Lin Cindy,
Tartakoff Alan
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.878.5
Subject(s) - mutagenesis , saccharomyces cerevisiae , biology , gene , mutant , insertional mutagenesis , genome , genetics , zygote , yeast , phenotype , auxotrophy , strain (injury) , embryogenesis , anatomy
80% of the 6000 genes in S. cerevisiae can be deleted without interrupting mitotic growth. This has prevented some genetic investigations. A strain in which non‐essential genes have been rendered nonfunctional would therefore be a useful tool. To generate such a strain, we randomly mutagenized S. cerevisiae with graded doses of UV irradiation, thereby avoiding the complications that accompany sequential targeted engineering. Our parental strain lacked the RAD14 gene, which is responsible for UV‐damage repair, and had its copy of Prm1 tagged with GFP. Irradiated cells were then selected by FACS for GFP expression. Because Prm1 is activated far‐downstream of the pheromone‐receptor interaction necessary for zygote formation, surviving mutagenized cells were still likely to be able to form zygotes. Additionally, the parental strain and its mutants were grown in glycerol, ensuring that surviving cells retained their mitochondria, which are essential for sporulation. After repeated iterations of UV irradiation using these mutants (“Storm mutagenesis”), we have recovered mutants that are copper‐sensitive and no longer completely self‐sufficient for amino acid synthesis. We are continuing this iterative process, using the appearance of phenotypic deficiencies such as metal sensitivity and auxotrophy as indicators of efficacy. When we obtain strains that can no longer sustain further mutagenesis without severe growth impairment, we plan to clone and sequence their genomes to learn which genes were mutated in these strains and are therefore likely to be part of the 80% that can be damaged simultaneously without impairing growth.