Table 1. Relative length of Xist elements. Species Gene size Length of homologous exons C.f. B.t. H.s. P.t. R.n. M.m. M.r. consensus consensus 37592 34934 32063 32050 22898 22786 21161 30297 Ex1 15480 18693 11333 11316 Ex1 9430 9483 7939 11506 Ex1 pEx2 Ex2 96 90 90 90 83 91 84 93 Ex2 Ex2 59 - 64 64 - - - 58 Ex3 Ex3 140 137 137 137 Ex3 137 132 138 138 Ex4 Ex4 211 210 209 209 Ex4 213 211 213 209 Ex5 pEx5 162 164 161 144 Ex5 141 147 103 185 Ex6 Ex5 130 131 164 164 Ex6 154 155 134 169 Ex7 Ex6 4561 4524 4543 4541 Ex7 4610 4521 4361 4678 Ex8 Ex7 195 156 146 146 146 141 134 151 Ex9 pEx7 Ex8 370 372 378 378 Ex8 387 340 384 374 Ex10 16188 10457 14838 14861 7597 7565 7671 12736 21404 24477 17225 17189 15301 15221 13490 17561 Notes. Pseudoexon (pEx) means a part of an intronic sequence which is present in other species as an exon. Abbreviations: exons (Ex) and pseudoexons (pEx), in Canis familiaris (C.f.), Bos taurus (B.t.), Homo sapiens (H.s.), Pan troglodytes (P.t.), Rattus norvegicus (R.n.), Mus musculus (M.m.), Microtus rossiaemeridionalis (M.r.), and consensus Xist gene; (–) the exon is missing. Length is shown in base pairs. doi:10.1371/journal.pone.0002521.t001 A Dual Origin of the Xist Gene Total intron length Total exon length tandem repeats detectable in the structure of the gene. Based on the analysis of the Xist gene consensus, we have reconstructed an ancestral structure of this gene, which may have existed 100 million years ago (Mya) in the eutherian ancestor, and traced its evolution to contemporary eutherian species. Results Xist consensus To examine further the origin of the Xist gene we have generated an Xist gene consensus from genomic sequences of rat, bovine, canine, and human Xist/XIST, belonging to four eutherian orders (Rodentia, Cetartiodactyla, Carnivora, and Primates, respectively). Based on PipMaker [5] comparison we removed all species-specific repeats (e.g. species-specific SINEs, tandem repeats, pseudogenes etc) from the annotated Xist sequences and then aligned the sequences by ClustalX [6]. The resulting alignment was then manually corrected. The Xist consensus sequences were generated by the AnnHyb program (http://www. bioinformatics.org/annhyb). The resulting Xist gene consensus contains 10 exons and 500-bp flanking regions at the 59 and 39 ends. The size of the genomic locus is about 30 kb (Table 1, Fig. 1). The correspondence between exons in consensus, human and mouse Xist is shown in table 2. The consensus includes all known types of tandem repeats (A–F) (Table 3), which are essential components of the gene. Additionally we detected a previously unrecognized repeat H (Table 3) and mobile elements (Fig. S1) in the Xist consensus sequence, that were reconstructed from fragments found in the nucleotide sequences of the gene in different species. The Xist gene consensus sequence was used in further analyses described below. Homology of Xist and Lnx3 exons It has been shown previously that chicken Lnx3 exons 4 and 11 have similarity to human XIST exons 4 and 6 [4]. Using FASTA, SSEARCH and WUBLAST programs we performed a more comprehensive comparison between the chicken Lnx3 gene and human XIST to obtain additional evidence that Lnx3 is the precursor of Xist. In addition to the previously found similarity we discovered that Lnx3 exons 3 and 5 are similar to human XIST introns 3 and 4 (59 and 60%, respectively). Interestingly, human XIST intron 4 corresponds to rodent Xist exon 5, which displays 65% homology to Lnx3 exon 5 (Fig. 2, Fig. S2). We also discovered additional similarity between the XIST promoter region and Lnx3 exons 1 and 2 (Fig. 2, Fig. S2). Moreover, the same fragment of ancient LINE, belonging to the L3 family (widespread in the genomes of the majority of vertebrate species), is located at the orthologous position in Lnx3 and the Xist consensus 59 region. The similarity between the chicken mobile element and mammalian L3 consensus is 64.7%. Comparison between the Xist consensus described above and chicken Lnx3 revealed the same homologous regions as in human XIST (Fig. 2, Fig. S2). The P-value indicates that the similarities are statistically significant (Table 4). Therefore, our data substantiates and adds to previous observations that eutherian Xist consensus has extended homology to Lnx3 in the promoter region, exons 1, 5–7, and intron 4. Contribution of transposons to the formation of the Xist gene We compared consensus exon sequences with the database of chordate mobile elements using the program WUBLAST 2.0 and elevated search sensitivity by setting the word size W = 3 instead of the default value W = 10. We identified a short fragment of MIR3 in Xist exon 1. The consensus exon 2 (m2/ph2) is similar to the ancient L3CR element (Fig. S3, Table 2). Unexpectedly, exon 3 of the consensus, which corresponds to human exon 2, has similarity to an L1 fragment and is present in the spliced transcript (ACC M97168). The boundaries of this human exon are inside the L1 sequence. i.e., human exon 2 is composed of a 64 nt fragment of a mobile element which is included in mature XIST RNA (Fig. 1). Presumably, a similar phenomenon takes place in the splicing of canine Xist RNA too (Fig. 1). Consensus exon 4 (m3/h3) is also similar to L1 sequence (Fig. S3). A considerable part of exon 8 (h6/m7) is similar to mobile elements of different classes (SINEs, LINEs, and DNA transposons) (Fig. 1). Exon 9 (pm7/h7) shows 64% similarity to endogenous retroviruses. Exon 10 (m8/h8) displays the highest similarity to DNA transposons. Almost the entire exon displays a 61% similarity to the hAT-10_XT element and the central part, 66% similarity to the ZOMBI transposon (Fig. S3). The degree of sequence identity of consensus exons 2, 4, PLoS ONE | www.plosone.org 2 June 2008 | Volume 3 | Issue 6 | e2521