BMC Genomics 2007, 8:271 http://www.biomedcentral.com/1471-2164/8/271 Human GeneView page. In all cases, we examined the 10 kb region upstream from TSS at base +1 and the 1 kb downstream of this position. The EnsEMBL gene identi- ties and chromosome positions sequence are listed in the additional data file 3. Sequences were obtained from EnsEMBL v22.1, except for dog sequences (v27.1 – Dec2004) and macaque and cow (v36 – Dec2005). EnsEMBL gene discrepancies: The rat NOS-2 5'-UTR was not annotated in EnsEMBL, but we used the TSS described [34]. The start of transcription of mouse NOS-2 in EnsEMBL is 25 bp upstream with respect to the one first published [11,35]; it should be noted that although this site has been widely used in the literature, we used the TSS given for the EnsEMBL gene (ENSMUSG00000020826). Dog NOS-2 is only annotated in EnsEMBL as a fragment, and the exact transcription start site is uncertain (there are differences in the 5' end between NP_001003186 [Gen- Bank:NP_001003186] and the genomic sequence in EnsEMBL (data not shown). We decided to position the dog NOS-2 +1 site further upstream than in the annotated gene in EnsEMBL. The same decision was made with macaque NOS-2. These decisions were based on the high genomic sequence similarity between dog (and macaque) sequence in this region and the start site of the human NOS-2 gene. The dog and macaque NOS-2 base positions are therefore only estimates; there are no experimental data on the dog or rhesus monkey NOS-2 promoter, and all results from these sequences presented in this work should be considered as a tentative approximation based on sequence comparison. Sequence alignment methods We compared the different promoters with MEME [36], AVID at mVISTA [37-39], BlastZ with zPICTURE [40], and Mulan [41]. Mulan is based in the tba method [42]. We masked repetitive elements with RepeatMasker (A.F.A. Smit & P. Green, unpublished data, [43]) with the Mulan default settings, and we also used these masked sequences with all other methods. For clarity, we used the human sequence as the reference in AVID/mVISTA, zPICTURE and Mulan. The following settings were established in the analysis. MEME: one occurrence of a single motif per sequence among all sequences; motifs between 6 and 120 bp, 10 motifs to find. mVISTA: window length 90 bp, 65% minimum similarity. zPICTURE and Mulan: evolutionary conserved region (ECR) length 90 bp, 65% minimum similarity. Evolutionary analysis of the NOS-2 promoter Calculation of the pairwise evolutionary distances between species was done for each one of the sequences (intron, enhancer an proximal promoter) by means of the Tajima-Nei method [44] using MEGA 3.1 program [45]. For each one of the sequences analysed, distances were transformed into branch lengths in a star-like unrooted tree by means of the following equation [46]: La = (Dab + Dac - Dbc)/2, where La is the length of the branch from the common ancestor leading to a and Dab, Dac, Dbc are the distances between species a and b, a and c, and b and c, respectively. Two alternative unrooted trees (trios of species) were considered: 1- human (H), mouse (M) and cow (C) and 2- macaque (Mc), rat (R) and dog (D). Since two of the species belongs to the same monophyletic group in both trees (primates and rodents), the internal node represents to the same common ancestor in both diagrams and consequently, branch lengths can be com- pared between sequences within species. It is important to emphasize that we are not considering that the phyloge- netic relationships of these species is a star topology. We used this exact equation for determining the branch lengths of the three species, because the unique way to arrange three species in a phylogenetic tree is a star topol- ogy. TFBSs prediction Match [47] was used, with false positives minimized with TRANSFAC Professional 8.2. MutiTF is integrated in Mulan [41], and uses an older version of TRANSFAC Pro- fessional 7.3. We used the default settings (optimized for function) with TRANSFAC matrices. NFKB-NOS2 consen- sus site was obtained from the alignment of all known functional κB sites in NOS-2 promoters of human, rat and mouse: GGRDNNNYYY. Protein extracts and EMSAs Murine peritoneal macrophages were treated with 200 ng/ ml of LPS for 1 h. Nuclear extracts and EMSAs were pre- pared as previously described [48]. The oligonucleotide sequences for binding were the following. Mouse -103 κB (-87 in [11,35]): sense, tcga TGC TAG GGG GAT TTT CCC TCT CTC TGT; antisense, tcga ACG ATC CCC CTA AAA GGG AGA GAG ACA. Mouse "-5,2 κB-like": sense, gatc CTG CCT GGG CAT GTC CAA CAA AAA CCC C; antisense gatc GGG GTT TTT GTT GGA CAT GCC CAG GCA G. Mouse "-5,8 κB-like": sense, gatc CTG GAG GCA GTT TCC AGA AGC ATG; antisense, gatc CAT GCT TCT GGA AAC TGC CTC CAG. Mouse "-8,3 κB-like": sense, gatc GAG GGC CAG TGG AGG GCC GGC AAG AG; antisense, gatc CTC TTG CCG GCC CTC CAC TGG CCC TC. Authors' contributions DR participated in the design and carried out the sequence analysis and the experimental section. JMV and HD partic- ipated in the bioinformatic and statistical analysis. LB conceived of the study and participated in the design and coordination. All authors read and approved the final manuscript. Page 8 of 10 (page number not for citation purposes)