BMC Genomics 2009, 10:435 http://www.biomedcentral.com/1471-2164/10/435 However, the above finding indicates a general selection pressure against antisense transcribed pseudogenes, thus generally limiting the possibilities for complementary hybridization with transcripts and RNA elements from homologous genes. general, although approximately half (742/1750) of the human TPAs are syntenically conserved in rhesus mon- key, only 3% are syntenically conserved in mouse. This suggests that a large number of human transcribed pseu- dogenes are primate-specific. We performed a similar survey in the mouse genome for TPAs. Surprisingly, in the mouse genome, we found a very low percentage of TPAs, in comparison to the human genome (<2%) (P << 0.001 for the likelihood of the number in mouse, given the human percentage as an expectation, using binomial statistics). This is despite these two animals having similar amounts of pseudogene annotation data (Table 1), and transcript data (203,785 transcript sequences in total for human, and 203,550 for mouse). This indicates that transcribed pseudogenes are rarer in mice than in humans. Identification of orthologous pseudogenes in mammals Transcription of pseudogenes per se does not necessarily indicate functionality. It has been shown that transcrip- tional activation at a particular genomic locus has a ripple effect on the neighboring loci [21]. It is therefore possible that many transcribed pseudogenes arise simply because of this. However, of the various identified human TPAs in our present study, those that are evolutionarily conserved across mammals due to natural selection are more likely to be biologically functional. Therefore, we set out to identify a list of such orthologous transcribed pseudo- genes that have conserved in ≥2 of our panel of mammals. Certain gene families are known to spawn large numbers of pseudogenes. Examples include olfactory receptors [22], ribosomal-protein genes [23], human thioredoxin and glutaredoxin [24], ABC transporters [25], and heat shock proteins [26]. In such cases, identifying orthologs in other mammals using the standard bi-directional best-hit procedure is problematic, since the rates of sequence evo- lution may vary in different lineages and genomic regions. Furthermore, such a procedure does not work well for pseudogenes, since these sequences are not evolving like protein-coding genes, which are under strong purifying selection. Because of this, the best match obtained using blastp to a pseudogene query is expectedly the parent pro- tein-coding gene or a pseudogene recently evolved from the parent gene. Thus, the standard bi-directional best-hit procedure alone is not sufficient. Therefore, here, we have used synteny information between two organisms to pin- point pseudogene orthologs. We have used synteny maps along with homology-based searches to identify con- served orthologs in five mammals (rhesus monkey, mouse, rat, dog and cow) (see Methods for details). We identified a set of 68 human TPAs that are conserved in at least two of these mammals, representing potentially functional candidates (see Additional file 2: Table S1). In A multiple sequence alignment of orthologous sequences for an example taken from Additional file 2: Table S2, is shown in Figure 1(a). The corresponding phylogenetic tree is drawn in Figure 1(b). This example is a human pseudogene named 'urn:lsid:pseudogene.org:9606.Pseudog- ene:4346', that is homologous to human ADP-ribose pyrophosphatase. In this case, one can see clearly that dis- ablements at several positions in the alignment are con- served in divergent species, and parsimoniously would be assigned in the ancestral sequence. Also, in this phyloge- netic tree, dog clusters closer to primates, than rodents do; this may be due to variance in local genomic mutation rates. Interestingly, we find that a significantly higher number of human transcribed pseudogenes were con- served in dog, compared to in mouse (Fisher's exact test, P-value: 0.0086) (Figure 2). There is some debate regard- ing whether human is phylogenetically closer to rodents than to dog, although most data analysis indicates a rodent-primate grouping [27-29]. Conserved transcribed pseudogenes are overrepresented on chromosome X It is noteworthy that the highest number of conserved TPAs is on the human chromosome X (13 out of the total of 68; Figure 3 and Additional file 2: Table S2), followed by 11 cases on chromosome 6. There is also a significant over-representation of human conserved TPAs on these chromosomes after normalizing for the chromosome size and dosage in gametes (χ2 test, d.f. = 1, P-value < 10-3). Furthermore, it is chromosome X that is consistently and most significantly overrepresented in the whole popula- tion of TPAs, and in the datasets of TPAs that are con- served in monkey and in mouse (calculations not shown). This finding is in line with the observation that over the course of evolution there has been some extensive gene trafficking to/from the mammalian X chromosome [30]. Analysis of TPAs for selection to maintain them Human TPAs from the VEGA data set, that have synteni- cally-conserved orthologs in rhesus monkey, were ana- lyzed for significant selection pressure to maintain them. This was assessed through comparison of the nucleotide percentage sequence identity between orthologs, with the highest nucleotide percentage sequence identity for the immediately flanking genomic regions, as illustrated in Figure 4. We chose the VEGA data set for this analysis, since the genomic coordinates of this pseudogene annota- tion data set are more precisely annotated. 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