Bernard et al. BMC Genomics 2010, 11:166 http://www.biomedcentral.com/1471-2164/11/166 Gene structure and gene function All information about gene structure was obtained from FLAGdb++ [59] including: (i) the median of the lengths of frames, coding sequences (CDSs), 3’UTRs and 5’UTRs, first introns and cumulated introns; and (ii) the percentage of intron-less genes. Information about gene function was obtained from the TAIR Gene Ontology (GO) categories [41,60]. We used the GO Biological Function and GO Cellular Com- ponent categories. For a given gene set, we calculated the percentage of genes within that category relative to the remaining A. thaliana genes with an experimentally supported TSS. Finally, we only considered previously annotated genes, i.e. we excluded the “unknown” and the “other” categories. Gene expression The transcriptome data used in this work were obtained using the CATMA v2 microarray [61]. They include 522 hybridized samples extracted from 40 different pro- jects covering 12 organ types. Expression data were downloaded from CATdb [50] containing all the tran- scriptome data generated by the CATMA-URGV plat- form. They had also been deposited in either the NCBI Gene Expression Omnibus [62] or the European Bioin- formatics Institute ArrayExpress [63] repositories. Out of the 14927 A. thaliana genes with an experimentally supported TSS, we analyzed the expression data of 11161 genes specifically relating to one CATMA probe. Each gene was spotted according to (i) the percentage of samples in which it had been detected, called the hybridization percentage and (ii) its median expression level. We performed a third order linear regression allowing us to cluster 4371 genes with a High level of Expression (HE) and 6790 with a Low level of Expres- sion level (LE). Different hybridization ranges were also defined according to two inflections in the linear regres- sion curve. The Small Range (SR) class included 4510 genes characterized by a hybridisation percentage of less than 15% and the Wide Range (WR) class included 1241 genes characterized by a hybridisation percentage of over 85%. Second, we built a 60% confidence interval allowing the identification, within each class, of the highest and the lowest expression levels (respectively HE+ and LE+). Orthologous genes We selected those A. thaliana and O. sativa orthologous gene pairs being Bidirectional Best Hits [64] with BLASTp [65] resulting in 5805 orthologous pairs of genes having an experimentally supported TSS. The pre- sence of a PLM in both genomes led to the distinction between the presence of this PLM within (i) A. thaliana Page 13 of 15 promoters but not in their respective O. sativa ortholo- gues; (b) O. sativa promoters but not in their respective A. thaliana orthologues; or (c) both orthologous genes. For each PLM-class, we performed comparisons between expected and observed conservation within orthologous gene pairs. The c/(a+c) ratio indicates the level of observed conservation in O. sativa with respect to A. thaliana. The expected conservation value is given by the ratio b/(5805-a-c), i.e. the presence of a given PLM-class in O. sativa orthologous genes from the PLM-class-less A. thaliana genes. Statistical analysis Statistical analyses were performed with the R statistical software [66]. We used R for (i) the regression analysis leading to PLM identification (Figure 1) and the charac- terization of expression categories (Figure 7), and for (ii) motif distributions (Figures 3 and 6). We performed Fisher exact one-sided tests using the Bonferroni correc- tion to compare percentages between two independent samples. We searched for statistical decreases or increases in the percentages of one set of genes (i) in GO annotation categories (Table 1), (ii) in intron-less (Table 2, last row), (iii) in expression categories (Table 3) and (iv) with a given motif (Table 4). Structural gene features, even after log modification, cannot be assumed to be normally distributed. We therefore performed Wilcoxon-Mann-Whitney one-sided tests using the Bon- ferroni correction to compare two independent struc- tural gene feature distributions. We considered the hypothesis that structural data might result in higher or lower values for a given gene set compared to all other genes, i.e. the 14927 genes minus the considered gene set (Table 2, first rows). Each p value less than 5% with the Bonferroni correction was considered significant. Additional file 1: Effect on the score of an extension by pyrimidines of TC[-39,-26]-PLMs. First column, motifs that do not exhibit an increase in their SMS (in parentheses) when extended. Second column, motifs that, when extended exhibit an increase in their original SMS due to the generation of an other 6-base-long motif (underlined sequence) characterized by a higher SMS (underlined SMS). Note that the SMS of the 7- or 8-base-long motifs are all lower than the SMS of the underlined 6-base-long motifs. Third column, motifs that can be extended in 7- or 9- base-long motifs. Note that the two long extensions are made of three repeats of TCT. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 166-S1.XLS ] Abbreviations CATMA: Complete Arabidopsis Transcriptome MicroArray; TFBS: Transcription Factor Binding Site; GO: Gene Ontology; HE: High Expression; LE: Low Expression; PLM: Preferentially Located Motif; SMS: Score of Maximal Square relative to the base line; SR: Small Range; TBP: TATA-binding protein; TSS: Transcriptional Start Site; WR: Wide Range;