BMC Genomics 2007, 8:271 http://www.biomedcentral.com/1471-2164/8/271 examined (-6076 in human, -5981 in dog, -6524 in macaque, -7701 in cow, -6593 in mouse and -6649 in rat). When compared with human, dog and macaque, the whole region in cow was located further from the tran- scription start site (TSS). Nevertheless, it was not dis- rupted as in rodents. Mulan and zPICTURE also detected this fragmentation of the rodent sequence (Fig. 1A and data not shown), expanding their extension through the use of a gap-permitted alignment algorithm. AVID detected the conserved sequence of the -5 kb to -6.5 kb human NF-κB enhancer region in dog, cow and macaque, but did not detect the similar but fragmented sequences in rodents. This is probably due to the global alignment strategy used in AVID, which assumes that the order of the functionally conserved regions has been preserved [20]. Evolutionary analysis the proximal promoter and enhancer regions reveals differences in rodents We calculated the evolutionary distance between every pair of species in the proximal promoter, in the enhancer region and in the fifth intron (the last for reference, as an approximation to the neutral rate of substitution). After- wards, we computed the branch length for each sequences from the unique common ancestor for each trio of species using two phylogenetic schemes (see Methods). The results are shown in Figure 2, and they reveal that the proximal promoter and the enhancer have a similar branch length in primates and dog (cow presenting a branch even shorter in the enhancer). However, in mouse and rat the enhancer seems to evolve almost to the same rate than intron sequences. This result suggests that selec- tive pressures are almost absent in the rodent enhancers, probably due to a loss of function in this region. Alterna- tively, the high efficiency of the rodent proximal promoter contributed to the loss of biological relevance of the enhancer regions (see Discussion). Bioinformatic analysis of κB sites and other inflammation- related TFBSs The 11 kb NOS-2 promoter sequences were scanned with Match (TRANSFAC Professional 8.2) to ensure that the published κB sites were identified. Interestingly, high quality matrices from TRANSFAC (minimizing false posi- tives) did not detect any κB sites in the human promoter, and only detected one in mouse (at -998 bp, described to be functional) and one in rat (at -2.46 kb), which has never been described in the literature. In view of these data, the promoters were scanned with all TRANSFAC ver- tebrate matrices. The results are summarized in Table 1. Four human elements were identified correctly; as Klein- ert and co-workers pointed out [8] the functionality of the human κB at -119 is controversial. Three other function- ally recognized human κB sites were not detected. In mouse and rat we found several sites, including the two functionally recognized murine κB sites. Strikingly, this SpFerigoleumcrtoievte2rcoanndsteranihnatnscdeerduced from distances in proximal Selective constraints deduced from distances in proximal promoter and enhancer. Branch lengths derived from the evolutionary divergence of the proximal promoter (1 kb), the alignable enhancer and the fifth intron of the NOS-2 gene were compared for each one of the spe- cies. Two alternative and comparable evolutionary schemes using human-mouse-cow and macaque-rat-dog species were used. Branch length to species i (Li) was derived from pair- wise distances (Di-j) according to an exact equation (see methods). See text for further explanation. analysis did not detect the described downstream and upstream κB sites in rat (highly similar to the mouse func- tional sites). Moreover, we found six putative sites in the rat promoter between -1601 and -2460. This is the region described by Zhang and co-workers to be important for full induction of NOS-2 by LPS and/or cytokines [14], although these authors' data indicated involvement of ele- ments other than κB [21]. Since this approach still failed to detect previously recog- nized human κB sites, we defined a consensus κB sequence covering all functional κB elements described in NOS-2 promoters (NFKB-NOS2, see Methods). Scanning of NOS-2 promoters of human, dog, rat and mouse for this consensus sequence obviously detected all known human κB sites but the incidence of putative sites was extremely high: there are 79 sites that match that consen- sus sequence in human, 80 in chimpanzee, 71 in mouse, and 85 in rat (data not shown). NOS-2 is positively regulated by NF-κB, but also by IFN-γ dependent transcription factors (TFs) and AP-1, all of them key TFs for the regulation of the inflammatory response. In order to gain insight in the evolution of NOS- 2 regulatory TFBSs, further analysis of NF-κB, AP-1 and IFN-γ regulated TFs was performed using MultiTF and TRANSFAC matrices. MultiTF is a phylogenetic footprint- Page 4 of 10 (page number not for citation purposes)