cerevisiae. As opposed to a single “”snapshot”" observations, we used a more informative time-course design investigating selected gene expression response from initial (0 h), early growth (1 and 6 h),

exponential/log phase (24 h), and entering stationary phase (48 h) relative HM781-36B to the cell growth stage under the ethanol challenge. The dynamics of gene expression over time closely correlated with metabolic profiles and cell growth phenotypes between the two strains. This allowed identification of at least 82 candidate and key genes for ethanol tolerance and subsequent ethanol fermentation under the ethanol stress. Among which, 36 genes were the first report by the present study. Our results also suggest a potential key regulatory role of Msn4p for ethanol-tolerance among other transcription factor and regulatory elements. The newly developed data acquisition and analysis standard for qRT-PCR array assays using the robust mRNA as the PCR Ct reference provided reliable means to safeguard data fidelity and allowed unification of gene expression data for comparable analysis. Housekeeping genes are commonly

used as quality controls for qRT-PCR but vary under different experimental conditions [42, 47]. Among numerous systems developed [41–45], the universal RNA controls have been shown another successful applications under ethanol stress conditions AICAR order in this study. An extended adaptation and applications of such methods for consistent quantitative gene expression analyses are expected in the future. Genes associated with ethanol stress were mostly reported based on snapshots of gene expression response in yeast [11–13, 15]. In this study, we investigated a time-course study comparing cell growth, viability, glucose-to-ethanol conversion, and gene expression dynamics for two closely related strains. This allowed assessment of phenotype associations and identification of legitimate candidate genes for ethanol tolerance. As demonstrated by this study, the parental strain showed

briefly induced expression of numerous genes before becoming repressed and BAY 80-6946 purchase unable Megestrol Acetate to establish a viable culture under the ethanol challenge. Uncovered by the expression dynamics of the tolerant strain, we are able to distinguish ethanol-tolerance candidate genes and tolerance-response from the transient stress-response in yeast. For example, unlike many heat shock protein genes in parental strain becoming repressed after 6 h, these genes in the tolerant Y-50316 showed continued inductions through 48 h. This indicated that the continued expression of those heat shock protein genes after 6 h is critical for the ethanol tolerance in yeast. Heat shock proteins, mainly act as chaperones, insuring properly folding or refolding of nascent or denatured proteins and enzymes to maintain functional conformation [48–50].