Nucleic Acids Research, 2007, Vol. 35, Web Server issue W247 from this approach. This brings the total number of orthologs to 15 162. Despite improvements in EnsEMBL’s ortholog prediction, this is only 1079 more orthologs than were present in our previous database build. Based on the small incremental increases in mapped orthologs, we may be nearing the upper bound for the number of genes in human and mouse that are truly orthologous and detectable by sequence conservation. Detailed descriptions of transcription start region (TSR) determination and the distribution of TSRs for human and mouse genes are available as Supplementary Data. For each human/mouse orthologous pair, we determine the coordinates of the longest region from the UCSC genome alignments (6) spanning all transcripts plus an additional 10 kb of upstream sequence. The orthologous sequences are retrieved and re-aligned using ORCA, a pairwise global progressive alignment algorithm (1) to optimally align short, conserved blocks within longer global alignments. If possible, TSRs from human and mouse are paired in the alignment. We apply three dynamically computed and progressively more stringent conservation thresholds corresponding to the top 10, 20 and 30% of all 100-bp non-coding windows, each with a minimum percent identity of 70, 65 and 60%, respectively. Of the 15 162 orthologous gene pairs supplied as input to the oPOSSUM pipeline, 15 121 (99.7%) successfully align, and 15 027 (99.1%) have non-exonic conserved regions above 60% nucleotide identity. This is a signiﬁcant improvement over the previous version of oPOSSUM. Caenorhabditis elegans/Caenorhabditis briggsae. To facil- itate transcriptional regulatory analysis of the numerous gene expression studies performed in C. elegans, we have implemented a worm version of oPOSSUM. While the database structure and pipeline procedure are very similar to that used for the human/mouse database, there are small modiﬁcations that allow for mapping of genes to their operons, as deﬁned by Blumenthal et al. (7). In addition, nucleotide identity thresholds for conserved regions were reduced to 60, 55 and 50% for the top 10, 20 and 30% of non-coding windows, respectively, to account for the greater sequence divergence between C. elegans and C. briggsae compared to human and mouse. The set of orthologs for C. elegans and C. briggsae is deﬁned by one-to-one InParanoid clusters (8) from WormBase (WS160) (9). After ﬁltering overlapping genes, 10592 orthologous gene pairs (of which, 2140 genes are in operons) remain for alignment. Alignments are performed on the orthologous gene sequences plus 2 kb of upstream sequence (relative to the start codon) for C. elegans, and 4 kb of upstream sequence for C. briggsae. Annotations are not as mature for C. briggsae, and the longer upstream region aids in the alignment of the worm promoter sequences. Alternative promoters have not been considered in this ﬁrst version; however, should CAGE data or other reliable means for annotating TSSs in worms become available, eﬀorts will certainly be made to include them. Of the 10 592 worm orthologs, 9331 (88%) successfully align. Yeast. The analysis of yeast promoters is simpliﬁed by the more compact nature of the yeast genome. This characteristic diminishes the requirement for comparative methods to reduce the search space and noise inherent in larger genomes. Computational methods using S. cerevisiae sequences alone have successfully been used to identify regulatory elements associated with known sets of related genes (10,11). We opted to exclude phylogenetic footprinting for yeast, and instead, select promoter sequences corresponding to the 50 untranslated region 1000 bp immediately upstream of the start codon of each open reading frame (ORF). Note that for all applications, users have the option to further restrict the search space if they wish. The sequences were downloaded from the Saccharomyces Genome Database (12). TFBS prediction For the metazoan species, we search for matches to TFBS proﬁles contained in the JASPAR CORE and JASPAR PhyloFACTS database collections (13,14). Additionally, we include a set of proﬁles compiled for C. elegans TFs from literature review for Worm SSA (Table S2). Binding sites are predicted for the sequences using the TFBS suite of Perl modules for regulatory sequence analysis (15). A predicted binding site for a given TF model is reported if the site occurs in the promoters of both orthologs above a threshold PSSM score of 70% and at equivalent positions in the alignment. Overlapping sites for the same TF are ﬁltered such that only the highest scoring motif is kept. The genomic location, proﬁle score, motif orientation and local sequence conservation level of each TFBS match in orthologous genes are stored in the respective species databases. For S. cerevisiae, we compiled a collection of yeast-speciﬁc TFBS motifs from both the Yeast Regulatory Sequence Analysis (YRSA) system (16) and the literature (Table S3), and record the genomic location, proﬁle score and motif orientation for each prediction. Based on the observation that members of the same structural family of TFs often bind to similar sequences, plant and insect matrices are available for inclusion in the analysis. The MADS family of TFs is an excellent example of conservation of binding domains between plants and vertebrates (17,18), and there are numerous examples of conservation of binding domains across vertebrates, ﬂies and worms. Thus, in cases where a proﬁle for the TF of interest is not available in the database, oPOSSUM can still provide insights into the underlying regulation by suggesting a particular TF family that may be involved. RESULTS AND DISCUSSION Examples of applications Each oPOSSUM component was validated on sets of reference genes. The results of all validations are available as Supplementary Data (Tables S4–S13). In the interest of space, selected examples are described for each system. Human SSA. Wonsey and Follettie (19) performed a microarray analysis of genes that are transcriptionally