BMC Genomics 2009, 10:435 http://www.biomedcentral.com/1471-2164/10/435 Our analysis indicated that TPA orthologs have higher sequence homology in comparison to their syntenic flanking regions. Average sequence identities among dif- ferent syntenic regions in human and rhesus monkey are as follows: 75.0% (s.d. 12.6%) in the 5' areas, 81.0% in the 3' areas (s.d. 13.5%), and 86.7% (s.d. 9.6%) in the TPA sequences. The difference in the sequence identities between pseudogenes and the respective flanking regions is statistically significant (Wilcoxon signed rank test, P- value: < 5e-50). In the majority of cases (~86%, 293/341), the percentage sequence identity between orthologous TPA sequences is greater than that of the flanking regions. This suggests that significant selection pressure exists to maintain them. We note that similar analysis comparing FmNiaugmumrbeearl2sof shared orthologs between human and other Number of shared orthologs between human and other mammals. (a) For the 68 conserved TPAs (orthologs in > = 2 mammals); (b) for all conserved TPAs between human and the other mammals. The shared number of cases between dog and human is highlighted in red to indi- cate that this number is higher than for human/rodents. the human and chimpanzee genomes is not informative because the species are too similar. Also, comparisons of the human genome with the other mammals in our panel are not informative either, because the appropriate regions cannot be aligned accurately or significantly. Conserved TPAs tend to be GC rich We examined whether there existed any sequence feature that distinguished conserved TPAs from the rest of the human pseudogene population. A positive finding would indicate that these pseudogenes are not evolving neu- trally. It has been widely observed that genes tend to be GC-rich in comparison to non-transcribed genomic seg- ments [31]. Here, we examined whether TPAs and anno- tations of non-transcribed pseudogenes showed any difference in the GC contents relative to their flanking regions (GCdiff). Pseudogene populations from a variety of organisms have been shown to relax to the composi- tion of intergenic DNA over evolutionary time [32,33]. Here, the GC content of neutrally-evolving pseudogenes would be expected to relax to that of the background genomic GC content. Interestingly, GC content calcula- tions revealed that 84% (327/391) of the human TPAs derived from the VEGA pseudogene data set, that are con- served in rhesus monkey, have GC content greater than their flanking regions (Table 2). This compares to 74% (5289/7154) for the non-transcribed cases (Table 2). This difference is statistically significant (χ2 test, d.f. = 1, P- value < 10-4). A similar result is obtained if we examine the whole population of TPAs, or also if we just examine transcription of conserved processed pseudogenes. There is however no such significant differences for transcribed pseudogenes of the 'nonprocessed' and 'unclassified' pseudogene categories (Table 2). This shows that there is a greater tendency for the conserved TPAs to be GC-rich than for non-transcribed cases, and that this tendency arises primarily because of transcription of processed pseudogenes. This finding on GC content is further evi- dence that such transcribed pseudogenes are not evolving neutrally. Such GC trends can be explained by selection for transcriptional efficiency, as noted above [31]. We checked whether the age of pseudogenes could be causing the GC content differences noticed above. To do this, we looked at the GCdiff in the following (VEGA) sub- sets of TPAs, i.e.: (i) TPAs unique to humans; (ii) TPAs conserved in rhesus monkey only; (iii) TPAs conserved in more divergent mammals such as mouse, rat, dog and cow. We found that 82.5% (381/462) of set (i) have GCdiff > 0 (i.e., GC content of pseudogene greater than that of the flanking region). Similar percentages were observed in the other classes: 84.1% (317/377) in set (ii), and 77.78% (21/27) in set (iii). There was no statistically significant difference in the GCdiff between any two of the classes (χ2 test, P-value > 0.55), suggesting that age of pseudogenes Page 5 of 14 (page number not for citation purposes)