X-chromosome inactivation, which occurs in female eutherian mammals is controlled by a complex X-linked locus termed the X-inactivation center (XIC). Previously it was proposed that genes of the XIC evolved, at least in part, as a result of pseudogenization of protein-coding genes. In this study we show that the key XIC gene Xist, which displays fragmentary homology to a protein-coding gene Lnx3, emerged de novo in early eutherians by integration of mobile elements which gave rise to simple tandem repeats. The Xist gene promoter region and four out of ten exons found in eutherians retain homology to exons of the Lnx3 gene. The remaining six Xist exons including those with simple tandem repeats detectable in their structure have similarity to different transposable elements. Integration of mobile elements into Xist accompanies the overall evolution of the gene and presumably continues in contemporary eutherian species. Additionally we showed that the combination of remnants of protein-coding sequences and mobile elements is not unique to the Xist gene and is found in other XIC genes producing non-coding nuclear RNA.

The Xist locus plays a central role in the regulation of X chromosome inactivation in mammals, although its exact mode of action remains to be elucidated. Evolutionary studies are important in identifying conserved genomic regions and defining their possible function. Here we report cloning, sequence analysis, and detailed characterization of the Xist gene from four closely related species of common vole (field mouse), Microtus arvalis. Our analysis reveals that there is overall conservation of Xist gene structure both between different vole species and relative to mouse and human Xist/XIST. Within transcribed sequence, there is significant conservation over five short regions of unique sequence and also over Xist-specific tandem repeats. The majority of unique sequences, however, are evolving at an unexpectedly high rate. This is also evident from analysis of flanking sequences, which reveals a very high rate of rearrangement and invasion of dispersed repeats. We discuss these results in the context of Xist gene function and evolution.

X inactivation, the transcriptional silencing of one of the two X chromosomes in female mammals, achieves dosage compensation of X-linked genes relative to XY males. In eutherian mammals X inactivation is regulated by the X-inactive specific transcript (Xist), a cis-acting non-coding RNA that triggers silencing of the chromosome from which it is transcribed. Marsupial mammals also undergo X inactivation but the mechanism is relatively poorly understood. We set out to analyse the X chromosome in Monodelphis domestica and Didelphis virginiana, focusing on characterizing the interval defined by the Chic1 and Slc16a2 genes that in eutherians flank the Xist locus. The synteny of this region is retained on chicken chromosome 4 where other loci belonging to the evolutionarily ancient stratum of the human X chromosome, the so-called X conserved region (XCR), are also located. We show that in both M. domestica and D. virginiana an evolutionary breakpoint has separated the Chic1 and Slc16a2 loci. Detailed analysis of opossum genomic sequences revealed linkage of Chic1 with the Lnx3 gene, recently proposed to be the evolutionary precursor of Xist, and Fip1, the evolutionary precursor of Tsx, a gene located immediately downstream of Xist in eutherians. We discuss these findings in relation to the evolution of Xist and X inactivation in mammals.

A DNA library derived from the B chromosome of Podisma kanoi was obtained by chromosome microdissection. A total of 153 DNA clones were isolated from the microdissected DNA library. Twenty of them were sequenced. A comparison of B chromosome DNA sequences with sequences of other species from the DDBJ/GenBank/EMBL database ( http://www.ddbj.nig.ac.jp/ ) was performed. Different patterns of signals were observed after FISH with labeled cloned DNA fragments. FISH signals with cloned DNA fragments painted either whole Bs or their different regions. Some clones also gave signals in pericentromeric regions of A chromosomes. Other cloned DNA fragments gave only background-like signals on A and B chromosomes. Comparative FISH analysis of B chromosomes in Podisma kanoi and P. sapporensis with DNA probes derived from the Bs of these species revealed homologous DNA that was confined within pericentromeric and telemetric regions of the B chromosome in P. kanoi. In contrast to the B chromosomes in P. sapporensis containing large regions enriched with rDNA, only a small cluster of rDNA was detected in one of the examined B chromosomes in P. kanoi. The data strongly suggest an independent origin of B chromosomes in two closely related Podisma species.

The derivation of stable multipotent trophoblast stem (TS) cell lines from preimplantation, and early postimplantation mouse embryos has been reported previously. FGF4, and its receptor FGFR2, have been identified as embryonic signaling factors responsible for the maintenance of the undifferentiated state of multipotent TS cells. Here we report the derivation of stable TS-like cell lines from the vole M. rossiaemeridionalis, in the absence of FGF4 and heparin. Vole TS-like cells are similar to murine TS cells with respect to their morphology, transcription factor gene expression and differentiation in vitro into derivatives of the trophectoderm lineage, and with respect to their ability to invade and erode host tissues, forming haemorrhagic tumours after subcutaneous injection into nude mice. Moreover, vole TS-like cells carry an inactive paternal X chromosome, indicating that they have undergone imprinted X inactivation, which is characteristic of the trophoblast lineage. Our results indicate that an alternative signaling pathway may be responsible for the establishment and stable proliferation of vole TS-like cells.