BMC Genomics 2008, 9:122 http://www.biomedcentral.com/1471-2164/9/122 served tORFs. Conservation of such sequences at the amino acid level may reflect a functional role. A related comparative genomics approach, supported by simula- tion-based statistics, has recently been published [8]. Based on conservation between human and mouse, Chung et al. convincingly demonstrate that these frameshifted ORFs (which they name alternative reading frames, or ARFs) are highly unlikely to occur by chance. In our study, the term 'matreshka' was coined to describe the overlapping tORFs, in analogy with Russian dolls, as one protein can be thought of as 'hiding' another. It should be kept in mind, however, that all matreshkas reported here are observations based on mRNA sequence. Translation of these sequences in vivo has not yet been experimentally confirmed. Results and Discussion Matreshkas are defined here as overlapping, frameshifted ORFs (relative to a known ORF) in transcripts, which are well conserved at the amino acid level. Matreshkas may potentially represent alternative translation products like Alex, or suggest the existence of functional frameshifted splice variants. To obtain matreshka predictions, we trans- lated in silico all frame +1 and -1 ORFs from known human, mouse and rat "parent" transcripts, and applied a conservation filter (Fig. 2). Open reading frame translation The first step was to build Human-mouse-rat ortholog tri- plets, from which frame +1 and -1 ORFs were extracted (frames +1 and -1 are also sometimes known as frames 2 and 3, respectively). Ortholog sets were based on a varia- tion of best reciprocal BLASTP hits, which favoured hits with a higher percentage identity (see Methods). This method yielded 9163 triplets. The coding nucleotide sequence was retrieved for each ortholog, and all trans- lated ORFs (tORFs) at least 50 amino acids (aa) long were extracted computationally. The matreshka identification pipeline was run twice, each time varying the tORF extraction method. In an effort to identify Alex-like cases, the first run retained only tORFs beginning with methionine (labelled here 'tORFM'). The second run was somewhat more comprehensive, includ- ing translated ORFs starting with any amino acid (labelled 'tORFX'). It should be kept in mind that although the tORFM set is contained within the tORFX set, the derived matreshka sets only partially overlap, due to the proper- Transcripts Human tORFs Matreshkas Mouse Orthologous triplet Rat Translation of all ORFs from frames +1 and -1 Conservation filter FMiagturerseh2ka bioinformatics pipeline Matreshka bioinformatics pipeline. A) Human, rat and mouse transcripts were assembled into orthologous triplets, and all frame +1 and -1 ORFs greater than 50aa in length translated in silico (frames +1 and -1 are sometimes also known as frames 2 and 3, respectively). A conservation filter was then applied to yield matreshka predictions. Page 3 of 16 (page number not for citation purposes)