Fig. 2.

Time course microarray analysis of gene expression levels following a single exposure to a moderate dose of ethanol. (A) Treatment scheme for microarray analysis. Fourteen groups of 200 flies were exposed to either 60% ethanol vapor or humidified air for 30 minutes, allowed to recover for 0 to 210 minutes and then frozen at the time-point indicated by the triangles. Two additional groups were frozen as no exposure controls (time 0). RNA extracted from the heads of the resulting 16 samples was hybridized to Drosophila 2.0 microarrays (Affymetrix). (B) Clustered gene expression patterns (rows) for exposure to ethanol vapor (left) and humidified air (right) at the given time-points (columns), represented as the medoid values for each cluster. Green and magenta indicate reduced and increased expression relative to the averaged unexposed controls, respectively. (C to F) Example expression level time courses from the microarray analysis (C and E) and qPCR confirmations using RNA isolated from a separate exposure series (D and F) for the genes tyramine decarboxylase 1 (Tdc1; C and D) and Sir2 (E and F). Data are expressed as fold change relative to unexposed controls. Time of the 30-minute ethanol or air exposure is indicated as a box on the horizontal axis. E (red) and A (blue) indicate gene expression levels in ethanol- and air-exposed samples, respectively. Error bars indicate standard deviation of 3 sample replicates for qPCR. (G) All genes (rows) in cluster 1. Genes in area bracketed by red bars are predominantly olfactory specific, showing a large, coordinate decrease in expression of many, but not all, olfactory specific genes. (H) Overlap of genes identified in this and 2 previous microarray studies (Morozova et al., 2006 and Urizar et al., 2007) of gene regulation by ethanol exposure in Drosophila.