BMC Genomics 2007, 8:271 http://www.biomedcentral.com/1471-2164/8/271 sequences) suggesting lower selective constraint in these species. These conserved distal sequences have not been previ- ously described in rodents. MEME alignment with the κB sites from the human sequence identified three putative κB sites, but none showed any binding activity in EMSA (Fig. 3). The mouse κB-like probes differed from the homologous human sequences as follows: mouse -5.2 κB- like, 18 bp out of 28 bp equal to human (18/28); mouse -5.8 κB-like, 20/28; and mouse -8.3 κB-like, 13/26. Thus, these mouse sequences probably do not bind NF-κB because, despite the similarity of the regions where they are locate, the nucleotide substitutions in mouse sites are critical for NF-κB binding in EMSA. The effects of these sequence variations in the κB sites have been studied in depth and their affinity appears to be dependent on the nature of the co-activators engaged in the stabilization and activity of the NF-κB complexes [2]. These negative results are in agreement with the accepted idea that only the proximal 1000 bp of the 5'-flanking sequence is necessary for mouse NOS-2 promoter induc- tion by NF-κB, while human NOS-2 requires the distal enhancer. The MultiTF results showed that not only is the overall structure of the human NF-κB enhancer region entirely conserved in dog, cow and primates, but also that some inflammatory TFBSs sites not present in rodents are conserved in the other species. We cannot be sure that mice lost these hypothetical ancestral κB sites, or whether the human κB sites were gained during evolution, but the analysis of multi-conserved TFBSs (Fig. 4) supports the first possibility. The functionality of these putative κB sites in macaque, dog and cow needs to be tested before mak- ing any conclusion, but if the sites do retain activity, that would support the hypothesis of a loss of functionality during the evolution of rodents. Controversy exists about the functionality of the human proximal κB site located at -115 (additional file 1). Taylor and co-workers concluded that this site is non-functional, while others have reported a certain relevance (see [8]). We have found that this region is very conserved indeed (it corresponds to the multi-conserved κB site indicated after alignment of the human, dog and rodent NOS-2 pro- moters; additional file 2). Whether it is functional or not remains unclear, but it is certain that this region is less functional than the homologous site in mouse and rat. The (A)n simple repeat we found downstream of human and chimp putative -115 κB sites is absent in dog, mouse and rat (alignments in the additional files 2 and 3). The insertion of this element in primates may account for the reduced NF-κB activity of this site, as the insertion of transposable elements is an important evolutionary mechanism that can affect gene regulation [26]. Func- tional experiments and analysis of available sequences from more species will give us more data to test this hypothesis. In this regard, it should be mentioned that pathogens, as expected, have exerted a significant evolu- tionary pressure on NOS-2 inducibility, at least in mam- malian macrophages. Although some controversy still persists [27-30], in vivo data demonstrate that non-rodent species display functional NOS-2 expression after infec- tion with a wide array of pathogens. The relevant issue is the unusual deviation in the upstream region of rodent NOS-2 orthologues vs. other mammalian species. Solely evolutionary distances cannot explain this divergence since the same region is well-conserved in the more dis- tant dog and horse orthologues. Availability of more NOS-2 upstream sequences from mammalian species might contribute to understand the evolutionary pressure exerted by pathogens. MultiTF was a very powerful tool for this analysis because it can search common TFBSs in alignments of more than two sequences, and it allowed us to use TRANSFAC matri- ces and a consensus sequence defined by ourselves. How- ever, bioinformatic discovery of putative TFBSs based on weight matrices (such as those in the TRANSFAC data- base) have serious drawbacks [31]. Attempts to minimize false positives and the use "high quality" matrices can fail to identify sites that have been experimentally demon- strated, as was the case with our analysis of the NOS-2 promoter. Indeed, phylogenetic footprinting of human- rodent alignments showed an acceptable degree of confi- dence [32] that is not the case for the NOS-2 promoter. Given these limitations, bioinformatic analysis of pro- moters needs to be complemented with functional data from wet-lab work. Even for very well known promoters, such as those studied in this paper, the functional data available for these types of studies is scarce. The results presented here represent a first basic approach to the study of vertebrate evolution of NF-κB regulated genes. Conclusion The analysis of the promoter sequences of these inducible genes reveal differences in the degree of conservation, with NOS-2 showing divergence between rodents and other mammals in the location and functionality of con- served regions containing putative κB and IFN-γ elements. These data that are in agreement with the refractoriness in the induction of NOS-2 in primates and other non-rodent species. Methods Sequence data retrieval Sequences of NOS-2 (NOS2A) were obtained from EnsEMBL [33]. The human gene sequence was down- loaded from the Human Genome, and the NOS-2 ortho- logues in other species were directly selected from the Page 7 of 10 (page number not for citation purposes)