In virtually all eukaryotic organisms, telomeric DNA is composed of a variable number of short direct repeats. While the primary sequence of telomeric repeats has been determined for a great variety of species, the actual physical DNA structure at the ends of a bona fide metazoan chromosome with a centromere is unknown. It is shown here that an overhang of the strand forming the 3' ends of the chromosomes, the G-rich strand, is found at mammalian chromosome ends. Moreover, on at least some telomeres, the overhangs are > or = 45 bases long. Such surprisingly long overhangs were present on chromosomes derived from fully transformed tissue culture cells and normal G0-arrested peripheral leukocytes. Thus, irrespective of whether the cells were actively dividing or arrested, a very similar terminal DNA arrangement was found. These data suggest that the ends of mammalian and possibly all vertebrate chromosomes consist of an overhang of the G-rich strand and that these overhangs may be considerably larger than previously anticipated.

Genomes undergo large structural changes that alter their organisation. The chromosomal regions affected by these rearrangements are called breakpoints, while those which have not been rearranged are called synteny blocks. We developed a method to precisely delimit rearrangement breakpoints on a genome by comparison with the genome of a related species. Contrary to current methods which search for synteny blocks and simply return what remains in the genome as breakpoints, we propose to go further and to investigate the breakpoints themselves in order to refine them.

The human X and Y chromosomes evolved from an ordinary pair of autosomes, but millions of years ago genetic decay ravaged the Y chromosome, and only three per cent of its ancestral genes survived. We reconstructed the evolution of the Y chromosome across eight mammals to identify biases in gene content and the selective pressures that preserved the surviving ancestral genes. Our findings indicate that survival was nonrandom, and in two cases, convergent across placental and marsupial mammals. We conclude that the gene content of the Y chromosome became specialized through selection to maintain the ancestral dosage of homologous X-Y gene pairs that function as broadly expressed regulators of transcription, translation and protein stability. We propose that beyond its roles in testis determination and spermatogenesis, the Y chromosome is essential for male viability, and has unappreciated roles in Turner's syndrome and in phenotypic differences between the sexes in health and disease.

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.

The development of appropriate expression vectors for large scale protein production constitutes a critical step in recombinant protein production. The use of conventional expression vectors to obtain cell lines is a cumbersome procedure. Often, stable cell lines produce low protein yields and production is not stable over the time. These problems are due to silencing of randomly integrated expression vectors by the surrounding chromatin. To overcome these chromatin effects, we have employed a Bacterial Artificial Chromosome (BAC) as expression vector to obtain stable cell lines suitable for protein production.

Evidence from inbred strains of mice indicates that a quarter or more of the mammalian genome consists of chromosome regions containing clusters of functionally related genes. The intense selection pressures during inbreeding favor the coinheritance of optimal sets of alleles among these genetically linked, functionally related genes, resulting in extensive domains of linkage disequilibrium (LD) among a set of 60 genetically diverse inbred strains. Recombination that disrupts the preferred combinations of alleles reduces the ability of offspring to survive further inbreeding. LD is also seen between markers on separate chromosomes, forming networks with scale-free architecture. Combining LD data with pathway and genome annotation databases, we have been able to identify the biological functions underlying several domains and networks. Given the strong conservation of gene order among mammals, the domains and networks we find in mice probably characterize all mammals, including humans.

Chromosome pairing is an essential meiotic event that ensures faithful haploidization and recombination of the genome. Pairing of homologous chromosomes is facilitated by telomere-led chromosome movements and formation of a meiotic bouquet, where telomeres cluster to one pole of the nucleus. In metazoans, telomere clustering is dynein and microtubule dependent and requires Sun1, an inner nuclear membrane protein. Here we provide a functional analysis of KASH5, a mammalian dynein-binding protein of the outer nuclear membrane that forms a meiotic complex with Sun1. This protein is related to zebrafish futile cycle (Fue), a nuclear envelope (NE) constituent required for pronuclear migration. Mice deficient in this Fue homologue are infertile. Males display meiotic arrest in which pairing of homologous chromosomes fails. These findings demonstrate that telomere attachment to the NE is insufficient to promote pairing and that telomere attachment sites must be coupled to cytoplasmic dynein and the microtubule system to ensure meiotic progression.

The regulation of microRNAs (miRNAs) is a complicated process requiring a large number of molecular events to be coordinated in both space and time. It is not known whether this complicated regulation process constrains the organization of target genes on mammalian chromosomes. We performed a genome-wide analysis to provide a better picture of chromosomal organization of miRNA target genes. Our results showed clustering of the target genes (TGs) of miRNAs on mammalian chromosomes, and further revealed that the particular gene organization is constrained by miRNA regulation. The clustering pattern of TGs provides an insight into the complexity of miRNA regulation.

Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest.

The pluripotent state of embryonic stem cells (ESCs) is produced by active transcription of genes that control cell identity and repression of genes encoding lineage-specifying developmental regulators. Here, we use ESC cohesin ChIA-PET data to identify the local chromosomal structures at both active and repressed genes across the genome. The results produce a map of enhancer-promoter interactions and reveal that super-enhancer-driven genes generally occur within chromosome structures that are formed by the looping of two interacting CTCF sites co-occupied by cohesin. These looped structures form insulated neighborhoods whose integrity is important for proper expression of local genes. We also find that repressed genes encoding lineage-specifying developmental regulators occur within insulated neighborhoods. These results provide insights into the relationship between transcriptional control of cell identity genes and control of local chromosome structure.

The spindle generates force to segregate chromosomes at cell division. In mammalian cells, kinetochore-fibers connect chromosomes to the spindle. The dynamic spindle anchors kinetochore-fibers in space and time to move chromosomes. Yet, how it does so remains poorly understood as we lack tools to directly challenge this anchorage. Here, we adapt microneedle manipulation to exert local forces on the spindle with spatiotemporal control. Pulling on kinetochore-fibers reveals the preservation of local architecture in the spindle-center over seconds. Sister, but not neighbor, kinetochore-fibers remain tightly coupled, restricting chromosome stretching. Further, pulled kinetochore-fibers pivot around poles but not chromosomes, retaining their orientation within 3 μm of chromosomes. This local reinforcement has a 20 s lifetime, and requires the microtubule crosslinker PRC1. Together, these observations indicate short-lived, specialized reinforcement in the spindle center. This could help protect chromosome attachments from transient forces while allowing spindle remodeling, and chromosome movements, over longer timescales.

Although more than thirty mammalian genomes have been sequenced to draft quality, very few of these include the Y chromosome. This has limited our understanding of the evolutionary dynamics of gene persistence and loss, our ability to identify conserved regulatory elements, as well our knowledge of the extent to which different types of selection act to maintain genes within this unique genomic environment. Here, we present the first MSY (male-specific region of the Y chromosome) sequences from two carnivores, the domestic dog and cat. By combining these with other available MSY data, our multiordinal comparison allows for the first accounting of levels of selection constraining the evolution of eutherian Y chromosomes. Despite gene gain and loss across the phylogeny, we show the eutherian ancestor retained a core set of 17 MSY genes, most being constrained by negative selection for nearly 100 million years. The X-degenerate and ampliconic gene classes are partitioned into distinct chromosomal domains in most mammals, but were radically restructured on the human lineage. We identified multiple conserved noncoding elements that potentially regulate eutherian MSY genes. The acquisition of novel ampliconic gene families was accompanied by signatures of positive selection and has differentially impacted the degeneration and expansion of MSY gene repertoires in different species.

Cohesins are multi-subunit protein complexes that regulate sister chromatid cohesion during mitosis and meiosis. Here we identified a novel kleisin subunit of cohesins, RAD21L, which is conserved among vertebrates. In mice, RAD21L is expressed exclusively in early meiosis: it apparently replaces RAD21 in premeiotic S phase, becomes detectable on the axial elements in leptotene, and stays on the axial/lateral elements until mid pachytene. RAD21L then disappears, and is replaced with RAD21. This behavior of RAD21L is unique and distinct from that of REC8, another meiosis-specific kleisin subunit. Remarkably, the disappearance of RAD21L at mid pachytene correlates with the completion of DNA double-strand break repair and the formation of crossovers as judged by colabeling with molecular markers, γ-H2AX, MSH4, and MLH1. RAD21L associates with SMC3, STAG3, and either SMC1α or SMC1β. Our results suggest that cohesin complexes containing RAD21L may be involved in synapsis initiation and crossover recombination between homologous chromosomes.

The X and Y sex chromosomes of placental mammals show hallmarks of a tumultuous evolutionary past. The X Chromosome has a rich and conserved gene content, while the Y Chromosome has lost most of its genes. In the Transcaucasian mole vole Ellobius lutescens, the Y Chromosome including Sry has been lost, and both females and males have a 17,X diploid karyotype. Similarly, the closely related Ellobius talpinus, has a 54,XX karyotype in both females and males. Here, we report the sequencing and assembly of the E. lutescens and E. talpinus genomes. The results indicate that the loss of the Y Chromosome in E. lutescens and E. talpinus occurred in two independent events. Four functional homologs of mouse Y-Chromosomal genes were detected in both female and male E. lutescens, of which three were also detected in the E. talpinus genome. One of these is Eif2s3y, known as the only Y-derived gene that is crucial for successful male meiosis. Female and male E. lutescens can carry one and the same X Chromosome with a largely conserved gene content, including all genes known to function in X Chromosome inactivation. The availability of the genomes of these mole vole species provides unique models to study the dynamics of sex chromosome evolution.

Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle. The critical role of CDK2 in the progression of meiosis was demonstrated in a single mammalian species, the mouse. We used immunocytochemistry to study the localization of CDK2 during meiosis in seven rodent species that possess hetero- and homomorphic male sex chromosomes. To compare the distribution of CDK2 in XY and XX male sex chromosomes, we performed multi-round immunostaining of a number of marker proteins in meiotic chromosomes of the rat and subterranean mole voles. Antibodies to the following proteins were used: RAD51, a member of the double-stranded DNA break repair machinery; MLH1, a component of the DNA mismatch repair system; and SUN1, which is involved in the connection between the meiotic telomeres and nuclear envelope, alongside the synaptic protein SYCP3 and kinetochore marker CREST. Using an enhanced protocol, we were able to assess the distribution of as many as four separate proteins in the same meiotic cell. We showed that during prophase I, CDK2 localizes to telomeric and interstitial regions of autosomes in all species investigated (rat, vole, hamster, subterranean mole voles, and mole rats). In sex bivalents following synaptic specificity, the CDK2 signals were distributed in three different modes. In the XY bivalent in the rat and mole rat, we detected numerous CDK2 signals in asynaptic regions and a single CDK2 focus on synaptic segments, similar to the mouse sex chromosomes. In the mole voles, which have unique XX sex chromosomes in males, CDK2 signals were nevertheless distributed similarly to the rat XY sex chromosomes. In the vole, sex chromosomes did not synapse, but demonstrated CDK2 signals of varying intensity, similar to the rat X and Y chromosomes. In female mole voles, the XX bivalent had CDK2 pattern similar to autosomes of all species. In the hamster, CDK2 signals were revealed in telomeric regions in the short synaptic segment of the sex bivalent. We found that CDK2 signals colocalize with SUN1 and MLH1 signals in meiotic chromosomes in rats and mole voles, similar to the mouse. The difference in CDK2 manifestation at the prophase I sex chromosomes can be considered an example of the rapid chromosome evolution in mammals.

Meiosis in human oocytes is a highly error-prone process with profound effects on germ cell and embryo development. The synaptonemal complex protein 3 (SYCP3) transiently supports the structural organization of the meiotic chromosome axis. Offspring derived from murine Sycp3(-)(/)(-) females die in utero as a result of aneuploidy. We studied the nature of the proximal chromosomal defects that give rise to aneuploidy in Sycp3(-)(/)(-) oocytes and how these errors evade meiotic quality control mechanisms. We show that DNA double-stranded breaks are inefficiently repaired in Sycp3(-)(/)(-) oocytes, thereby generating a temporal spectrum of recombination errors. This is indicated by a strong residual gammaH2AX labeling retained at late meiotic stages in mutant oocytes and an increased persistence of recombination-related proteins associated with meiotic chromosomes. Although a majority of the mutant oocytes are rapidly eliminated at early postnatal development, a subset with a small number of unfinished crossovers evades the DNA damage checkpoint, resulting in the formation of aneuploid gametes.

Mammalian sex chromosomes originated from a pair of autosomes, and homologous genes on the sex chromosomes (gametologs) differentiated through recombination arrest between the chromosomes. It was hypothesized that this differentiation in eutherians took place in a stepwise fashion and left a footprint on the X chromosome termed "evolutionary strata." The evolutionary stratum hypothesis claims that strata 1 and 2 (which correspond to the first two steps of chromosomal differentiation) were generated in the stem lineage of Theria or before the divergence between eutherians and marsupials. However, this prediction relied solely on the molecular clock hypothesis between pairs of human gametologs, and molecular evolution of marsupial sex chromosomal genes has not yet been investigated. In this study, we analyzed the following 7 pairs of marsupial gametologs, together with their eutherian orthologs that reside in stratum 1 or 2: SOX3/SRY, RBMX/Y, RPS4X/Y, HSFX/Y, XKRX/Y, SMCX/Y (KDM5C/D, JARID1C/D), and UBE1X/Y (UBA1/UBA1Y). Phylogenetic analyses and estimated divergence time of these gametologs reveal that they all differentiated at the same time in the therian ancestor. We have also provided strong evidence for gene conversion that occurred in the 3' region of the eutherian stratum 2 genes (SMCX/Y and UBE1X/Y). The results of the present study show that (1) there is no compelling evidence for the second stratum in the stem lineage of Theria; (2) gene conversion, which may have occurred between SMCX/Y and UBE1X/Y in the eutherian lineage, potentially accounts for their apparently lower degree of overall divergence.

During meiosis, homologous chromosomes undergo a dramatic movement in order to correctly align. This is a critical meiotic event but the molecular properties of this 'chromosomal dance' still remainunclear. We identified DEB-1 - an orthologue of mammalian vinculin - as a new component of the mechanistic modules responsible for attaching the chromosomes to the nuclear envelope as apart of the LINC complex. In early meiotic nuclei of C. elegans, DEB-1 is localized to the nuclear periphery and alongside the synaptonemal complex of paired homologues. Upon DEB-1 depletion, chromosomes attached to SUN-1 foci remain highly motile until late pachytene. Although the initiation of homologue pairing started normally, irregularities in the formation of the synaptonemal complex occur, and these results in meiotic defects such as increased number of univalents at diakinesis and high embryonic lethality. Our data identify DEB-1 as a new player regulating chromosome dynamics and pairing during meiotic prophase I.

Sex chromosomes recombine restrictly in their homologous area, the pseudoautosomal region (PAR), represented by PAR1 and PAR2, which behave like an autosome in both pairing and recombination. The PAR1, common to most of the eutherian mammals, is located at the terminus of the sex chromosomes short arm and exhibit recombination rates ~20 times higher than the autosomes. Here, we assessed the interspecific evolutionary genomic dynamics of 15 genes of the PAR1 across 41 mammalian genera (representing six orders). The strong negative selection detected in most of the assessed groups reinforces the presence of evolutionary constraints, imposed by the important function of the PAR1 genes. Indeed, mutations in these genes are associated with various diseases in humans, including stature problems (Klinefelter Syndrome), leukemia and mental diseases. Yet, a few genes exhibiting positive selection (ω-value >1) were depicted in Rodentia (ASMT and ZBED1) and Primates (CRLF2 and CSF2RA). Rodents have the smallest described PAR1, while that of simian primates/humans underwent a 3 to 5 fold size reduction. The assessment of the PAR1 genes synteny revealed differences among the mammalian species, especially in the Rodentia order where chromosomic translocations from the sex chromosomes to the autosomes were observed. Such syntenic changes may be an evidence of the rapid evolution in rodents, as previous referred in other papers, also depicted by their increased branch lengths in the phylogenetic analyses. Concluding, we suggest that genome migration is an important factor influencing the evolution of mammals and may result in changes of the selective pressures operating on the genome.

Mammalian X chromosomes are mainly euchromatic with a similar size and structure among species whereas Y chromosomes are smaller, have undergone substantial evolutionary changes and accumulated male specific genes and genes involved in sex determination. The pseudoautosomal region (PAR) is conserved on the X and Y and pair during meiosis. The structure, evolution and function of mammalian sex chromosomes, particularly the Y chromsome, is still poorly understood because few species have high quality sex chromosome assemblies.