The effect of Bos1 mutations on yeast growth.

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The effect of Bos1 mutations on yeast growth. The effect of Bos1 mutations on yeast growth. (A) Bos1 p.Gly176Trp, p.Gly176Asp and p.Arg196del show growth impairment compared to Bos1 wild type. The yeast strain BY4742 bos1Δ containing pRS316-BOS1 was generated as previously described (Corbett et al., 2011) and transformed with pRS315 plasmids expressing Bos1 wild type or Bos1 mutants. URA3, which is only present on the pRS316 plasmid, converts 5-fluoroorotic acid (5-FOA) to cytotoxic 5-fluorouracil. Plating yeast cells on media containing 5-FOA allows for negative selection regarding RS316. Only cells that express a functional Bos1 protein and lack pRS316 can thrive on 5-FOA-containing media. Indicated strains were spotted on SD-Leu/Ura and 5-FOA plates with serial dilutions (1:10) and incubated at 30°C for 48 h. Cells transfected with pRS315-bos1-p.Arg196del showed impaired growth compared to pRS315-BOS1 wild-type strains; however, pRS315-bos1-p.Gly176Trp and -p.Gly176Asp led to severe impairment, indicating a functional impairment of the Bos1 p.Arg196del mutant and even stronger impairment of the Bos1 p.Gly176Trp and p.Gly176Asp variants. (B) Temperature-sensitive (ts) strain of S. cerevisiae was transformed with different variants of Bos1. At room temperature, growth was detected for all variants, including the negative control. At 24°C, reduced growth was detected for Bos1 p.Gly176Trp and p.Gly176Asp; only a few remaining colonies were detected for the negative control. At 30°C, only the wild type and the p.Arg196del enabled the yeast to grow. Finally, at 37°C, also yeasts transformed with the p.Arg196del variant lacked the ability to grow completely. Jörn M. Völker et al. Dis. Model. Mech. 2017;10:1391-1398 © 2017. Published by The Company of Biologists Ltd