BMC Genomics 2009, 10:435 http://www.biomedcentral.com/1471-2164/10/435 in two chief ways: (i) from retrotransposition, i.e., reverse transcription of a cellular messenger RNA, followed by reintegration into the genomic DNA [3-5], or (ii) from decay of genes that originated (however long ago) from duplication [1,6]. These genomic entities have generally been believed to be non-functional. Historically, there were some early individual reports of transcribed pseudo- genes in the scientific literature [2,7,8]. Examples included: human leukocyte interferon (LeIFN) pseudog- ene [9], glyceraldehyde-3-phosphate dehydrogenase pseudogene [10], glucocerebrosidase pseudogene [11], steroid 21-hydrolase pseudogene [7], glutamine syn- thetase pseudogene [12], tumor repressor ΨPTEN [13]. More recently, genome-wide screens have detected tran- scription evidence for many retropseudogenes (>200) in humans [14-17]. In mouse oocytes, transcribed pseudo- genes have been shown to play a significant role in the generation of small interfering RNAs (siRNAs) [18,19], which regulate the expression of homologous genes. Collectively, these reports indicate that an unknown cohort of human transcribed pseudogenes could be potentially functional in regulation of gene transcription. A key indicator of such function is significant conserva- tion in other mammalian genomes. Svensson et al. [20] have explored conservation of apparent pseudogenes in three mammals (human, chimpanzee and mouse). Their analysis revealed 30 cases of transcribed pseudogenes that are preserved in mouse. Here, we analyze the distribution of transcribed pseudogene annotations (TPAs) in the human and mouse genomes, examine their conservation in an expanded panel of mammals (rhesus monkey, mouse, rat, dog and cow), and assess evidence for signifi- cant selection pressures. TPAs that are conserved in rhesus monkey show evidence of significant selection pressure, despite also displaying codon substitution patterns char- acteristic of non-protein-coding sequences. Also, we have discovered a short-list of 68 putative human transcribed pseudogenes that are syntenically conserved in at least Table 1: Percentages of TPAs in human and mouse. Dataset VEGA Transcribed (human) Total = 866/8160 (10.6%) two other mammals from our panel. These sequences rep- resent a strong subset of candidates for further investiga- tion as functional transcribed pseudogenes. Results & Discussion Derivation of transcribed pseudogene annotations (TPAs) in the human genome To identify transcribed pseudogenes, transcript sequences from the Unigene, RefSeq and H-InvDB databases were mapped onto the human genome and were examined for overlap with pseudogene annotations. These pseudogene annotations were taken from the VEGA http:// vega.sanger.ac.uk/ and http://pseudogene.org websites (see Methods for details). We pooled these datasets with re- mappings of: (i) 'disrupted mRNAs' (dmRNAs) [16], and (ii) transcribed processed pseudogenes [14], from previ- ous analyses [14,16]. Overlap of the transcripts with pseu- dogenes was verified through using the positions of mid- sequence disablements (frameshifts and premature stop codons) as 'anchors'. That is, at least one disablement position was required to occur in both the genomic DNA and the transcript sequence (see Methods for further details). We found that ~11.5% (1750/15000) of human pseudo- genes are transcribed (after correcting for pseudogene annotation overlaps within and between the various data sets) [see Additional file 1]. Table 1 summarises the num- bers of transcribed pseudogene annotations (TPAs) in dif- ferent categories and data sets. The number of processed pseudogenes that were identified to be transcribed is 3-4 times higher than in our previous analysis [14]. Interest- ingly, in humans, regardless of category, only a small frac- tion of TPAs are transcribed completely in the antisense direction (~3-6%). Such a finding of significant avoidance of antisense transcription (Table 1) is surprising, espe- cially for retropseudogenes. Retrotransposed sequences are inserted back into the genomic DNA irrespective of the position of existing local promoters. Thus, one would expect equal numbers of sense and antisense transcripts. Transcribed (mouse) Total = 71/4187 (1.7%) # Processed = 383/3737 (10.24%) # Non-processed = 71/1078 (6.58%) http:// Pseudogene.or g(excluding ambiguous pseudogenes) 828 (sense) 38 (antisense) 371 (sense) 12 (antisense) 61 (sense) 4 (antisense) 49 (sense) 22 (antisense) Total = 1035/13354 (7.75%) # Processed = 767/11072 (6.93%) # Non-processed = 268/2282 (11.74%) Total = 65/15064 (0.5%) 977 (sense) 58 (antisense) 724 (sense) 43 (antisense) 253 (sense) 15 (antisense) 53 (sense) 12 (antisense) Page 2 of 14 (page number not for citation purposes)