Alongside the formation of pseudoexons, which arise due to mutation to intron-exon sites (ph2, man; pEx2 - chimpanzee), we observed exons shortening at their 39 ends due to the formation of new 39–59 exon/intron junctions, for example m7 (mouse) and vole exon 7 [15]. A similar phenomenon was reported previously for Xist exon 4 in mole, dog and cow [16]. Thus, the exon-intron structure of the XIST/Xist gene is not stringently conserved or evolutionarily stable. It displays lability and exon–intron transitions, presumably connected with species- specific patterns related to its function. A characteristic of the Xist gene is the presence of both ancient and young species-specific mobile elements in the processed transcripts. A similar feature is observed in other XIC genes, Enox(Jpx), which also produce nuclear RNA. The processed transcript of the Enox(Jpx) gene includes SINEs, LINEs, and even processed pseudogenes [9]. Presumably, the integration of mobile elements into exons accompanies the overall evolution of these genes and continues in contemporary Eutheria. Possibly, this is a general mechanism involved in the formation and rearrangement of genes encoding large nuclear regulatory RNAs. Despite the drastic evolutionary rearrangements of the Xist gene structure in different species, the 42% GC content in exons is a conserved characteristic of this gene in mammals and, presumably, is maintained by selection. The introns contain less GC pairs compared to the exons and display a considerable variation between orders. The total GC content in Xist gene is similar to the overall XIC region and varies in the range of 39.8–42.2% (Tables 4). The region of the minimal promoter differs drastically—it is enriched with GC nucleotides (52.4–56.4%). The 39-flanking region of rodents is richer in its GC content compared with both the gene itself and its 59-flanking region. In contrast, the 39-flanking region in human and bovine displays a lower GC content compared with the gene; in addition, a decrease in the GC content is evident from the 59- to the 3;-flanking region (Tables 4). On the other hand, the localization and structure of Xist CpG islands in the species compared are different. The detected CpG islands in human and bovine are mainly associated with SINE and LINE repeats. The absence of conservation in the localization of CpG islands and their divergence in the case of similar localization in the Xist gene of different species may indicate that species-specific mechanisms are involved in the regulation of its activity. Thus, we have proposed a mechanism whereby the Xist gene may have originated. We suggest that the Xist gene lost the function of the protein-coding gene Lnx3 and no longer contained any extended ORFs. However, due to transposon insertions and their partial subsequent amplification, new functional domains formed (for example, repeat A in exon 1 [17]). These domains then became necessary for the transcriptional silencing of X chromosome genes. We suggest that this example of how a protein-coding gene loses its protein-coding function by mutation and then gains a new function due to transposon integration is not an exceptional case, but is a more wide-spread phenomenon applying to other non-coding RNA genes and pseudogenes with new functions. Materials and Methods The following sequences extracted from the corresponding databases of sequenced genomes at the UCSC Genome Bioinfor- matics Site (http://genome.ucsc.edu/) were used: human Mar. 2006 (hg18) assembly range = chrX:72449111-74160153; chim- panzee Mar 2006 (panTro2) assembly range = chrX:75160560- 75392648; dog May 2005 (canFam2) assembly canFam1_dna range = chrX:60100000-60735000; mouse Feb 2006 (mm8) as- sembly range = chrX: 99509137-100404904; rat June 2003 (rn3) A Dual Origin of the Xist Gene assembly range = chrX:91358074-91899712; chicken Feb. 2004 (galGal2) assembly range = chr4:11820349-12295219; opossum assembly MonDom 4.0, January 2006 from Ensembl (http:// www.ensembl.org/index.html); vole Microtus rossiaemeridionalis ACC AJ310130; and bovine ACC AJ421481. Computer analysis was conducted using modern versions of the following software packages: BLAST ( [18], http://www.ncbi.nlm. nih.gov/) for searching for homologous sequences; Tandem Repeat Finder 4 [19] for searching for tandem repeats; RepeatMasker [20] for searching for mobile elements; Fasta [21], CLUSTALX [6] for aligning two and more sequences (the programs and data are available at http://genome.ucsc.edu/, http://www.ensembl.org/, and http://bio.cse.psu.edu/) and (Pip- Maker, [5]) for genomic analysis of extended loci. Comparative analysis of the Xist gene of ten eutherian species belonging to four orders (Rodentia, Cetartiodactyla, Carnivora, and Primates) was conducted. The structure of the Xist gene in four closely related vole species was determined earlier [15]. As vole Xist genes are very similar, the sequence of M. rossiaemer- idionalis was used in the present study. We identified chimpanzee, dog, and rat orthologs of the Xist gene in the data available from genomic sequencing projects of the three species by pairwise alignments with the known human, mouse, and bovine sequences [2]. The exon–intron structures of dog, chimpanzee, and bovine Xist genes were determined by comparing with the human and mouse genes. Comparison with genomic sequences demonstrated that the exon–intron boundaries in bovine and chimpanzee genes coincide with numerous spliced ESTs. In dog only one EST is located in the region of Xist gene. Comparison with the genomic sequence demonstrated that it contained exons 2, 4, 5, and beginning of exon 6 (according to human XIST gene). This data cannot be regarded as final, because other species display various rare variants of EST splicing, including the loss of certain exons. The Xist sequences of different mammalian species were analyzed for the presence of both tandem and dispersed repeats using the TRF, RepeatMasker and the repeat database RepBase12.03. (http://www.girinst.org/repbase/index.html) [22]. To search for the homology between considerably divergent and evolutionarily distant sequences (for example, human–chicken), we used the programs WUBLAST with the parameter W = 3 and FASTA34 with the algorithm Smith–Waterman or SSEARCH34 and the parameters f = 214, g = 22. The similarity between Xist exons and mobile elements was searched for using the same programs and parameters. Each individual exon was compared with the database of chordate mobile elements, which was obtained by consolidating the human, primate, Mammalia, Rodentia, and Vertebrata databases (http://www.girinst.org/repbase/index. html). CpG islands of at least 200 bp and with a GC percentage exceeding 50% and with an observed/expected CpG ratio that is greater than 60 bp were detected using the FIND-CPG program from the PipTools [23]. To test the statistical significance of the obtained alignments the standard PRSS program from the FASTA software package was used (Table 4). As E-value and z- score estimated by the WUBLAST, FASTA and SSEARCH programs depend on sequence length and the size of search database, we assessed the significance of alignments by P-value calculated in the PRSS program from FASTA packed and selected the most significant alignments. The modified Nei-Gojobori method as implemented in the MEGA program [24] and a maximum likelihood method as implemented in the CODEML program [25] were used to estimate the numbers of synonymous and nonsynonymous substitutions per site. Phylogenetic trees based on multiple alignments of nucleotide sequences were constructed using the PLoS ONE | www.plosone.org 9 June 2008 | Volume 3 | Issue 6 | e2521