X chromosome inactivation (X-inactivation) is triggered by X-linked noncoding Xist RNA, which is expressed asymmetrically from one of the two X chromosomes in females and coats it in cis to induce chromosome-wide silencing. Xist RNA is thought to play a role as a platform in recruiting proteins involved in gene silencing and heterochromatinization, which mediate serial changes in epigenetic modification of the chromatin. During the last two decades, many proteins have been shown to be enriched on the inactivated X chromosome in mouse and human. Although the biological significance of most of them for X-inactivation has not been fully established, extensive studies of these proteins should provide a better understanding of the molecular basis of how X-inactivation mediated by Xist RNA is regulated. Here, we review the potential roles of some of these proteins in the stepwise process of Xist RNA-mediated chromosome silencing.
Wheat-rye translocations are widely used in wheat breeding to confer resistance against abiotic and biotic stress. Studying gene expression in wheat-rye translocations is complicated due to the presence of homoeologous genes in hexaploid wheat and high levels of synteny between wheat and rye chromatin. To distinguish transcripts expressed from each of the three wheat genomes and those from rye chromatin, genomic probes generated from diploid progenitors of wheat and rye were synthesized on a custom array. A total of 407 transcripts showed homoeologous genome (‘A’, ‘B’ or ‘D’ genome)- or rye genome (‘R’)-specific differential expression, based on unequal values of probe hybridization. In a 2BS.2RL wheat-rye translocation, thirteen of the 407 transcripts showed preferential expressions from rye chromatin. As well as quantifying variation in homoeologous transcript in wheat-rye translocations, this study also provides a potential aid to examine the contribution of the subgenomes to complex allohexapolyploids.
Green fluorescent protein (GFP), fused to the N or C terminus of a protein of interest, is widely used to monitor the localization and mobility of proteins in cells. RAD51 is an essential protein that functions in mitotic DNA repair and meiotic chromosome segregation by promoting the homologous recombination reaction. A previous genetic study with Arabidopsis thaliana revealed that GFP fused to the C terminus of RAD51 (RAD51-GFP) inhibits mitotic DNA repair, but meiotic homologous recombination remained unaffected. To determine how the C-terminal GFP specifically inhibits mitotic DNA repair by RAD51, we purified rice RAD51A1-GFP and RAD51A2-GFP, and performed biochemical analyses. Interestingly, purified RAD51A1-GFP and RAD51A2-GFP are proficient in DNA binding and ATP hydrolysis. However, nucleoprotein complexes containing single-stranded DNA and RAD51A1-GFP or RAD51A2-GFP are significantly defective in binding to the second DNA molecule (secondary DNA binding), and consequently fail to catalyze homologous pairing. In contrast, RAD51A1-GFP and RAD51A2-GFP efficiently stimulated homologous pairing promoted by the meiosis-specific RAD51 isoform DMC1. These biochemical characteristics are well conserved in human RAD51-GFP. Therefore, GFP fused to the C terminus of RAD51 abolishes the homologous pairing activity of RAD51 by disrupting secondary DNA binding, but does not affect its DMC1-stimulating activity.
Despite remarkable recent progress in the analysis of plant genome organization and chromosome structure, there is a need for methods enabling DNA sequences to be mapped by fluorescence in situ hybridization (FISH) at high spatial resolution. We sorted mitotic metaphase chromosomes of wheat by flow cytometry and observed the occurrence of hyperexpanded chromosomes among them. However, this phenomenon was not reproducible in subsequent experiments. An investigation into the procedures of flow cytometry revealed that the hyperexpansion of chromosomes became reproducible when the concentration of formaldehyde used in sample fixation was reduced. We conducted FISH analysis with 45S rDNA, 5S rDNA and wheat centromeric repeat sequences as probes on flow-sorted chromosomes and also on chromosomes from squash preparations. We measured the length of chromosomes 1B and 6B, identified by FISH. On average, the hyperexpanded 1B and 6B chromosomes were 7.26 and 7.53 times longer, respectively, than the same chromosomes from the squash preparations. The most stretched 1B and 6B chromosomes both exceeded 100 micrometers.
The schizothoracine fish, also called snow trout, are members of the Cyprinidae, and are the most diversified teleost fish in the Qinghai-Tibetan Plateau (QTP). Clarifying the evolutionary history of the schizothoracine fish is therefore important for better understanding the biodiversity of the QTP. Although morphological and molecular phylogenetic studies have supported the monophyly of the Schizothoracinae, a recent molecular phylogenetic study based on the mitochondrial genome questioned the monophyly of this taxon. However, the phylogenetic analysis of that study was on the basis of only three schizothoracine species, and the support values were low. In this report, we inferred the phylogenetic tree on the basis of mitochondrial genome data including 21 schizothoracine species and five closely related species, and the polyphyletic origins of the Schizothoracinae were strongly supported. The tree further suggests that the Schizothoracinae consists of two clades, namely the “morphologically specialized clade” and the “morphologically primitive clade”, and that these two clades migrated independently of each other to the QTP and adapted to high altitude. We also detected in their mitochondrial genomes strong signals of positive selection, which probably represent evidence of high-altitude adaptation. In the case of the morphologically specialized clade, positive selection mainly occurred during the Late Paleocene to the Early Oligocene. Its migration also seems to have occurred in the Early Eocene, and this timing is consistent with the drastic uplifting of the QTP. On the other hand, positive selection in the morphologically primitive clade has mainly occurred since the Late Miocene. Because its members are thought to have migrated to the QTP recently, it is possible that they are now undergoing high-altitude adaptation.