BMC Genomics 2008, 9:122 http://www.biomedcentral.com/1471-2164/9/122 Redundant methionine-start ORFs (tORF ) M Translation of e.g., frame +1 of parent sequence Non-redundant ORFs beginning with any amino acid (tORF ) X matreshkaM Conservation filter MLSAHWNMGPFMBDCL MGPFMBDCL MBDCL Method variation 1 Method variation 2 matreshkaX ...*APQRYMLSAHWNMGPFMBDCL*... APQRYMLSAHWNMGPFMBDCL Conservation filter FOiRguFrtera8nslation method ORF translation method. Two variations on the ORF translation step in Fig. 2 were performed. In an effort to identify Alex-like proteins which start with methionine, a first run of the analysis pipeline generated redundant (overlapping) methio- nine-start ORFs. A second, more comprehensive run generated all ORFs regardless of the first amino acid. derived from tORFM), for instance, might be translated from leaky ribosomal scanning events. A strong Kozak motif could support initiation of translation at these loca- tions [14]. We therefore scanned the nucleotide context around the ATG start for Kozak motifs. The pattern [AG]NNATGG (where N is any nucleotide, and letters in square brackets indicate alternative bases at a given posi- tion) was used, derived from published data on initiator codons from highly curated sequences [15]. The signifi- cant enrichment of Kozak motifs in the longest matreshkaM was ascertained with the Exact Binomial Test using R software [30], which was also used for the χ2 test in Table 1. Matreshkas were additionally scanned for sig- nal peptide motifs using SignalP v.2.0 [31]. BLASTP searches of human matreshkas against UniProt (UniRef100, last updated 13th Sep 2005) were carried out with an e-value threshold of 0.0001. To check for repeat- ing sequence elements, the EMBOSS command line pro- grams Equicktandem and Etandem were used with default settings to scan matreshka nucleotide sequences for tan- dem repeats. SpotFire DecisionSite v. 8.0 (Spotfire Inc., MA, USA), a data visualisation tool, was used to rapidly select the most interesting matreshka candidates. Matreshka amino acid composition analysis was per- formed using the EMBOSS program pepstats [28,32]. Page 14 of 16 (page number not for citation purposes)