A Dual Origin of the Xist Gene Table 5. GC-content (%) in various elements of bovine, human, mouse and vole Xist genes. Spe-cies 59-flank B.t. H.s. M.m. M.r. R.n. size 20000 18355 21461 11778 18165 G+C% 41.3 42.7 42.1 43.2 41,5 Pmin size 109 101 104 105 101 G+C% 53.2 56.4 54.8 53.3 54.0 Exons size 24470 16974 15080 13357 15155 G+C% G+C% 41,4 42.1 41.6 42.4 42.7 Introns 37.6 37.2 40.6 41.9 41,6 size 10464 15089 7706 7805 7743 39-flank G+C% 40.0 39.4 45.3 44.7 44,3 size 16000 9582 15759 11452 14325 Gene Xist G+C% 40.3 39.8 41.3 42.2 42,3 size 34934 32063 22786 21162 22898 Notes. Abbreviations: Ex, exon; In, intron; Pmin, minimal promoter; H.s., human; C.f., dog; M.r. vole Microtus rossiaemeridionalis; B.t., bovine; R.n., rat. doi:10.1371/journal.pone.0002521.t005 likely that the ancestral Xist contained a single copy of the C repeat as it is present in a single copy in the majority of species analysed (vole, bovine, canine, human) and is tandemly repeated in mouse and rat only. The proportion of mobile elements in the ancestral Xist gene is relatively low, representing only 4.39% of the total gene length (Table S2). In the Xist gene of contemporary eutherian species the proportion of mobile elements is higher than in the consensus gene and varies from 7.9 to 15.9% (Table S2). The promoter region of Xist is among the most conserved regions of the gene, showing a degree of homology between 82% and 98% for different species. Rodents are the exception, as the degree of homology for the promoter region is only 68% when compared with members of the other orders (Table S3). Probably, exon 3 of the Xist consensus is retained in dog, chimpanzee and human but was deleted together with surrounding sequences in other species studied. The scenario for consensus exon 3 is consistent with evolutionary tree data that suggests that dog and primates diverged from the common ancestor earlier than primates and rodents [7]. The sequences corresponding to consensus exon 2 and 6 are retained as Xist exons in as evolutionarily distant taxa as rodent and insectivore [16], but are part of intronic sequences in other species. Conversely, consensus exon 9 which is retained in the majority of species is an intron in rodents (Fig. 1, Fig. 3). During evolution the length of Xist RNA in different species changed dramatically. This occurred mainly due to formation of new exon–intron boundaries (for example, exon 2 of Xist in rat, exon 7 of Xist in mouse and vole), and variation in length of exon 1 by differential amplification of tandem repeats (Tables 1, 3, S1) and insertions of species-specific SINEs (Fig. 1, Table S2). However, despite almost twofold differences in exon size in various species, the total GC content in exons is conserved, varying from 41.4 to 42.6% (Table 5). The GC content in introns is more variable ranging from 37.2–41.9%. The time of Xist gene emergence To estimate the approximate time of Xist gene emergence, we aligned the nucleotide sequences of chicken and opossum Lnx3 genes (Gglnx3 and Mdlnx3) with human and bovine Xist genes (Fig. S5). This alignment was constructed with preservation of the Lnx3 reading frame (all the deletions/insertions causing frameshift were discharged from the alignment). Xist is not a protein-coding gene; however, this alignment allows the number of potential synony- mous and nonsynonymous substitutions to be assessed, as all the stop codons in the Xist gene were removed. Figure 4 shows the phylogenetic trees constructed for the synonymous and nonsynonymous substitutions. As anticipated, the Figure 4. Tentative time frame of Xist origin. Minimum evolution trees constructed using (a) synonymous and (b) nonsynonymous substitutions (modified Nei-Gojobori method, p-distance) [24]. Arrow indicates an approximate time of Xist gene origin, assuming that this gene would remain a protein-coding gene and have a similar evolutionary rate as Mdlnx3 gene. Scale is shown nucleotide differences per site. doi:10.1371/journal.pone.0002521.g004 PLoS ONE | www.plosone.org 6 June 2008 | Volume 3 | Issue 6 | e2521