To identify genes that may regulate the development of ethanol tolerance, we determined gene expression levels in the heads of flies exposed for 30 minutes to either 60% ethanol vapor or humidified air as a control (Fig. 2). We captured the dynamic effects of ethanol exposure on gene expression levels by performing a time-course microarray analysis on samples collected before, immediately after, and at 6 additional time-points extending to 3.5 hours following the termination of ethanol exposure. Gene expression changes were quantified using a linear models approach and ranked by the magnitude of the difference in expression change for ethanol versus air exposure (Table S1; Smyth, 2004). Genes with significant expression changes (p < 0.05, 1,807 genes) were then clustered into groups with similar patterns of expression. This identified 9 distinct patterns of gene expression that included changes in expression induced by exposure to ethanol, air, or both (Fig. 2B). Gene expression changes following ethanol exposure were generally of greater magnitude than following air exposure. However, some genes (e.g., Cp1 and Cyp4p2) showed greater air regulation, and others (CG32602 and
