BMC Genomics 2006, 7:133 http://www.biomedcentral.com/1471-2164/7/133 Table 9: Number of di -nucleotide appearances in the consensus sequences of major Alu subfamilies. ALU ALU NAM lengt E h AluJo AluJb AluSc AluSg AluSp AluSq AluSx AluY 283 283 280 281 284 284 283 282 AA AC AG AT CA CC CG CT GA GC GG GT TA TC TG TT 11 13 15 15 19 19 15 15 12 13 14 14 14 14 14 15 28 25 23 23 21 22 23 23 8 7 9 8 10 8 8 8 18 18 17 17 17 18 17 16 23 24 21 22 23 23 23 21 21 22 24 25 23 21 24 25 19 18 18 17 17 18 18 18 23 22 23 22 22 20 22 23 33 31 29 30 28 28 30 29 29 32 30 32 33 34 32 34 11 13 12 12 11 13 12 12 8 6 7 7 7 7 7 8 13 14 16 15 15 15 15 15 17 18 16 15 16 17 16 15 8 6 5 6 7 6 6 4 To scan for BMs we downloaded 410 vertebrate PSSMs from the MathInspector [30] database, with two addi- tional PSSMs ($PITX2_Q2 and &LUN1_01) taken from the TRANSFAC database [31]. The coordinates of the upstream regions of 21000 human genes were taken from [52]. The annotations of the U133 Array (Affymetrix, Santa Clara, CA) were used in order to remove multiple upstream regions for different splice var- iants of the same gene, (a single upstream region was asso- ciated with each gene symbol). We also removed from our database those Refseq genes that have duplicates on chro- mosomes X and Y, and those with upstream regions that were not fully sequenced. All this left 13686 promoters of different Refseq genes for analysis. The coordinates of Alu elements and other transposable elements in the human genome were extracted from the file chromOut.zip, which was downloaded from [52]. This is the output file of RepeatMasker [53]. The sequences of the Alu that reside in promoters were then extracted from the human genome, by intersecting these Alu coordinates with the upstream sequences of our 13686 genes. Similarly, for mouse we downloaded from [54] the files upstream5000.zip which contained the coordinates and sequences of the regions upstream to the TSS of mouse genes, and the RepeatMasker output file of the mouse genome, chromOut.zip. We used annotations of Mouse 430_2 Array (Affymetrix, Santa Clara, CA) in order to remove multiple promoters. In order to annotate genes to biological processes and molecular functions we used the DAVID database (based on the GO annotation) [55]. Calculating scores and probabilities For each PSSM we calculated the maximal score, on both sense and antisense orientations. In order to find the probability of getting some maximal score for a BM on an Alu sequence we produced 100,000 random 281 bp long sequences, of di-nucleotide distribu- tion similar to that of the AluSx consensus sequence. The next step was to calculate the maximal score for a given PSSM for each random sequence. The p-value for each given score was the percent of random sequences that con- tained a target site with higher score. Although, such pro- cedure might seem to be biased towards AluSx, it can be seen in Tables 8 and 9 that the subfamilies on which we focus in this research have similar nucleotide and di- nucleotide contents. Note that since we did not use a twice repeated random sequence of 140 nucleotides, our p-val- ues are an upper bound to the true ones. Orthologous genes in mouse and human We compared gene symbols of human and mouse and got about 5400 orthologous gene pairs and their respective promoters in human and mouse. The Pearson's correla- tion coffeeicient between the Alu and B1 densities on human and mouse genes was calculated over these 5400 pairs of promoters. Authors' contributions PP participated in the conception of the study, carried out the mapping of the Alu elements and BSs in the upstream of genes, analyzed the data, wrote the initial draft of the manuscript. ED conceived of the study, and participated in its design coordination and supervision, helped to draft and edited the manuscript. Page 13 of 15 (page number not for citation purposes)