BMC Genomics 2008, 9:627 http://www.biomedcentral.com/1471-2164/9/627 Comparison of the left (anaerobic) and middle (antimy- cin A + air) heat maps in Figure 3A shows that most of the genes that transiently responded to anaerobiosis ( C N2 1 – lated networks involved in respiration and mitochondrial energy production ( C N2 5 and C A-N2 2) and delayed yet chronic down-regulation of networks involved in mito- 4 and C N2 7 – 9) responded in a similar manner to chondrial ribosomal biogenesis ( C N2 6 and C A-N2 4) that antimycin A treatment in air. In contrast, few of those that exhibited a delayed, chronic response ( C N2 5 – 6 and C N2 10 – 13) were differentially expressed in response to inhibitor treatment in air. These trends are almost totally reversed for the N2 + antimycin A treatment (right heat- map in Figure 3A) as can be easily seen by comparing the distribution of the significance indicators (black bars) among gene clusters for each treatment. In regard to the recovered gene networks, comparison of Figure 3A with 3B indicates the same or similar TFMs and MIPS functional categories were enriched in clusters of genes that transiently responded. From this and previous studies of the anaerobic response [5,6], a common picture of gene network activity emerges. For example, both anaerobiosis and antimycin A treatment in air result in the transient down-regulation of MCB- and SCB-regulated networks ( C N2 1 and C A-O2 1) involved in processes asso- ciated with the G1/S transition of the cell cycle (e.g., DNA synthesis & replication). In addition, Abf1 and PAC/ RRPE-associated networks ( C N2 2 – 4 and C A-O2 2 & 3) involved in energetically costly programs of rRNA tran- scription/processing, translation, and ribosomal biogen- esis are apparently also down-regulated. Finally, both treatments apparently result in the transient induction of Msn2/4-regulated networks ( C N2 8 – 10 and C A-O2 4 & 5) involved in carbohydrate and reserve energy (trehalose and glycogen) metabolism/transport and autophagy. In contrast, networks that more chronically responded to anaerobiosis, in particular those with identifiable heme- responsive transcription factor binding sites (eg., Hap1 for C N2 5, and Rox1 and Upc2 for C N2 10 – 13), failed to respond to the antimycin A treatment in air (Figure 3B) but were recovered from clustering the response to antimycin A treatment in N2 (Hap1 in C A-N2 2, Rox1 in C A-N2 7, and Upc2 in C A-N2 9 in Figure 3C). A compari- son of these heatmaps shows the response of these net- works is kinetically very similar in the two anaerobic datasets. Similarities included a slightly delayed (ca. 0.08 generations) yet chronic down-regulation of Hap1-regu- were significantly enriched for 3'-Puf3 sites. Both treat- ments also resulted in the delayed yet chronic de-repres- sion of Rox1-regulated networks ( C N2 11 – 13 and C A-N2 7) involved in carbohydrate import/utilization and redox balance and delayed activation of Upc2-regulated ones ( C N2 11 – 13 and C A-N2 9) involved in sterol and cell wall homeostasis. Although there are differences in terms of specific transcription factor binding sites and functional categories enriched from clustering the two anaerobic datasets, it is clear that the networks that more chronically respond to anaerobiosis are not affected by the presence of the respiratory inhibitor. Overall, it is apparent from these comparisons that the normoxic addition of antimycin A to respiratory compe- tent cells and oxygen deprivation elicits a kinetically sim- ilar response in the same gene networks (MCB/SCB, Abf1/ PAC/RRPE and Msn2/4). Moreover, pretreatment of aero- bic cells with antimycin A abrogates the transient response of a large number of networks that acutely respond to anoxia yet has not affect heme-regulated ones (e.g., Hap1, Rox1 and Upc2) that more chronically respond. These results show that the acute and chronic phases of the anaerobic response in catabolite non-repressed (galac- tose) cells are separable and, thus, are likely triggered by different sensing and signaling pathways: the acute tran- sient phase from the respiratory inhibition and, we hypothesize, associated changes in cellular energetics and/or growth rate, and the chronic phase from oxygen deprivation. This interpretation is further corroborated by previous studies that show this transient "stress response" to anaerobiosis is absent in catabolite-repressed (glucose) cells [5]. Under both conditions, respiration is inhibited or repressed during the transition to anaerobiosis: here by chemical means and in previous studies by the presence of glucose. Thus, in both cases, the aerobic to anaerobic tran- sition proceeds rather smoothly, that is without a major disruption in catabolism or measured growth rate as the cells ferment the available carbon source in both the aer- obic and anaerobic phases. In contrast, cells grown on galactose in the absence of respiratory inhibitor are forced to switch from respiro-fermentative to strictly fermenta- tive metabolism and, in so doing, undergo a substantial decrease in growth rate (here from a 2.4 h mass doubling time to 4 h). Although several recent studies have sug- gested the changes in the activity of these general stress- responsive gene networks result simply from changes in Page 7 of 14 (page number not for citation purposes)