Cohesive Site Research Articles

Exploring the cohesive and social potential of a territorial ecomuseum in eastern sicily

ABSTRACT Thinking ethnographically with the work of Alberto Magnaghi, this paper explores the tensions and nuances of constructing ‘territory’ throuxgh the making of an ecomuseum in the Simeto River Valley in southern Italy. Conceptualised by Hugues de Varine and Henri Rivière, ecomuseums are community-based processes for reconstructing the material and immaterial signs of the past to revive a shared sense of heritage. Based on our engagement in a long-term action research process, interviews, focus groups, participation in itineraries and coordination activities, we trace how a group of actors involved in the Simeto Ecomuseum (SiEco) conceptualise different relationships to the territory and articulations of heritage to nurture futures in the Simeto Valley. We explore their diverse expressions of place attachment and political consciousness throughout this process. We suggest that ecomuseums can act as sites of social cohesion, if they generate a shared understanding and care of territory. However, this is complicated by diverse relationships to physical and cultural spaces, made visible in the ways boundaries are (re)made and unmade and in the difficulties of maintaining ongoing and inclusive collective processes over time. We call for the problematisation of the meanings and actions of ‘territory’, community and inclusion in the processes of cultural heritage-making.

Meiosis-specific decoupling of the pericentromere from the kinetochore.

The primary constriction site of the M-phase chromosome is an established marker for the kinetochore position, often used to determine the karyotype of each species. Underlying this observation is the concept that the kinetochore is spatially linked with the pericentromere where sister-chromatids are most tightly cohered. Here, we found an unconventional pericentromere specification with sister chromatids mainly cohered at a chromosome end, spatially separated from the kinetochore in Peromyscus mouse oocytes. This distal locus enriched cohesin protectors, such as the Chromosomal Passenger Complex (CPC) and PP2A, at a higher level compared to its centromere/kinetochore region, acting as the primary site for sister-chromatid cohesion. Chromosomes with the distal cohesion site exhibited enhanced cohesin protection at anaphase I compared to those without it, implying that these distal cohesion sites may have evolved to ensure sister-chromatid cohesion during meiosis. In contrast, mitotic cells enriched CPC only near the kinetochore and the distal locus was not cohered between sister chromatids, suggesting a meiosis-specific mechanism to protect cohesin at this distal locus. We found that this distal locus corresponds to an additional centromeric satellite block, located far apart from the centromeric satellite block that builds the kinetochore. Several Peromyscus species carry chromosomes with two such centromeric satellite blocks. Analyses on three Peromyscus species revealed that the internal satellite consistently assembles the kinetochore in both mitosis and meiosis, whereas the distal satellite selectively enriches cohesin protectors in meiosis to promote sister-chromatid cohesion at that site. Thus, our study demonstrates that pericentromere specification is remarkably flexible and can control chromosome segregation in a cell-type and context dependent manner.

Sister chromatid cohesion is mediated by individual cohesin complexes.

Eukaryotic genomes are organized by loop extrusion and sister chromatid cohesion, both mediated by the multimeric cohesin protein complex. Understanding how cohesin holds sister DNAs together, and how loss of cohesion causes age-related infertility in females, requires knowledge as to cohesin's stoichiometry in vivo. Using quantitative super-resolution imaging, we identified two discrete populations of chromatin-bound cohesin in postreplicative human cells. Whereas most complexes appear dimeric, cohesin that localized to sites of sister chromatid cohesion and associated with sororin was exclusively monomeric. The monomeric stoichiometry of sororin:cohesin complexes demonstrates that sister chromatid cohesion is conferred by individual cohesin rings, a key prediction of the proposal that cohesion arises from the co-entrapment of sister DNAs.

The Lies of the Land: The Alluvial Formalities of Gothic East Anglia

East Anglia is an evasive region; with its stretches of grey shingle that give way to silt and water, isolated marshes and great, flat panoramas that are literally falling into the sea. This article will show that East Anglia is a broader and more cohesive site of Gothic tradition and possibility than has previously been recognized, even if that possibility is found both textually and topographically in the incohesive, the ephemeral and the immaterial. We will also suggest that the short form is how this has so far been achieved – most famously in the short ghostly tales of M. R. James; more recently in Matthew Holness's unsettling short story ‘Possum’ (2013) and his 2018 film of the same name – and is, in fact, the most appropriate form for this act of textual production.

Gendered isolation, idealised communities and the role of collective power in West Bengal self-help groups

Empowerment and power have long been fervently contested in feminist theory and geography. Much debate focuses on whether empowerment is mainly monetary or more holistic and emancipatory. In both cases, most of the literature centres on individual empowerment and internal ‘power within’. Much less attention, however, has been given to the idea of ‘power with’ and how friendship, solidarity and emotional connections are fundamental to empowerment and yet are highly vulnerable to outside influence. Drawing on field research from Self-Help Groups in West Bengal, this article highlights how the formation of ‘power with’ is particularly important in contexts of gendered isolation, where patriarchy is enacted through the spatial control of women and removal of women from the public sphere, illustrating that communities in the Global South are not necessarily sites of collective unity and cohesion. These findings contest both idealised notions of community and mainstream theories of power and empowerment and in turn, provide crucial insights for feminist geography and power theory.

Strategies for purification of the bacteriophage HK97 small and large terminase subunits that yield pure and homogeneous samples that are functional

Packaging the viral genome in the head of double-stranded DNA viruses, such as bacteriophages, requires the activity of a terminase. The bacteriophage terminase consists of a small terminase subunit (TerS), which binds the viral DNA, and a large terminase subunit (TerL) that possesses the ATPase and nuclease activities for packaging the DNA in the phage head. Some phages require additional components for DNA packaging, such as the HNH endonuclease gp74 in the bacteriophage HK97. Gp74 enhances the activity of terminase-mediated digestion of the cohesive (cos) site that connects individual genomes in phage concatemeric DNA, a pre-requisite to DNA packaging, and this enhancement requires an intact HNH motif in gp74. Testing of whether gp74 alters the terminase DNA binding or enzymatic activities requires obtaining isolated samples of pure TerS and TerL, which has been challenging owing to the poor solubility of these proteins. To this end, we developed methods to obtain purified TerS and TerL proteins that are active. TerS is expressed solubly in E. coli as a fusion with SUMO, which can be removed during purification to yield a TerS nonamer (TerS9). Homogenous samples of a TerL monomer are also obtained, but the homogeneity of the sample depends on the solution conditions, as seen for other terminases. DNA binding, ATPase, and nuclease assays demonstrate that our preparations of TerS9 and TerL are functional, and that they also function with gp74. Purified TerS9 and TerL enable studies into the molecular basis by which gp74 regulates terminase activity in phage maturation.

Dynamics of sister chromatid resolution during cell cycle progression.

Faithful genome transmission in dividing cells requires that the two copies of each chromosome's DNA package into separate but physically linked sister chromatids. The linkage between sister chromatids is mediated by cohesin, yet where sister chromatids are linked and how they resolve during cell cycle progression has remained unclear. In this study, we investigated sister chromatid organization in live human cells using dCas9-mEGFP labeling of endogenous genomic loci. We detected substantial sister locus separation during G2 phase irrespective of the proximity to cohesin enrichment sites. Almost all sister loci separated within a few hours after their respective replication and then rapidly equilibrated their average distances within dynamic chromatin polymers. Our findings explain why the topology of sister chromatid resolution in G2 largely reflects the DNA replication program. Furthermore, these data suggest that cohesin enrichment sites are not persistent cohesive sites in human cells. Rather, cohesion might occur at variable genomic positions within the cell population.

Population genetic structure of the endangered Sierra Nevada yellow-legged frog (Rana sierrae) in Yosemite National Park based on multi-locus nuclear data from swab samples

The mountain yellow-legged species complex (Rana sierrae and Rana muscosa) has declined precipitously in distribution and abundance during the last century. The two primary threats are chytrid epidemic-associated population collapses and predation from the introduction of non-native trout. Widespread declines have occurred throughout the ranges of these species, including populations of R. sierrae in Yosemite National Park. A clear picture of genetic structure of remaining Yosemite R. sierrae populations is critical to short-term management and conservation. We conducted a population genetics study that included samples from 23 geographic sites distributed throughout the range of R. sierrae in Yosemite NP. We used minimally-invasive swab samples to collect genetic data from mitochondrial and nuclear DNA via sequencing (43 transcriptome-derived markers) and analyzed the distribution of genetic variation in a geographic context. Our mtDNA analysis partially confirmed previous results suggesting that two haplotype groups occur in Yosemite: one haplotype group contained high bootstrap support for monophyly while the other did not. However, increased geographic sampling demonstrated that the two haplotypes are not completely geographically partitioned into the two main drainages (Merced drainage and Tuolumne drainage) as previously postulated. Our nuclear DNA analysis revealed a general pattern of genetic isolation by distance, where genetic differentiation was correlated with geographic distance between sites. In addition, our analyses suggested that three clusters of genetically cohesive sites occur in the study area. Understanding population genetic patterns of variability will inform management strategies such as translocations, reintroductions, and monitoring for this endangered frog. Lastly, our next generation sequencing enabled approach allowed us to obtain multi-locus data from minimally-invasive swab samples. Thus researchers can now leverage extensive archives of swab samples (initially collected for pathogen testing) to study host genetics in previously surveyed amphibian populations.

Determinants of Sir2-Mediated, Silent Chromatin Cohesion.

Cohesin associates with distinct sites on chromosomes to mediate sister chromatid cohesion. Single cohesin complexes are thought to bind by encircling both sister chromatids in a topological embrace. Transcriptionally repressed chromosomal domains in the yeast Saccharomyces cerevisiae represent specialized sites of cohesion where cohesin binds silent chromatin in a Sir2-dependent fashion. In this study, we investigated the molecular basis for Sir2-mediated cohesion. We identified a cluster of charged surface residues of Sir2, collectively termed the EKDK motif, that are required for cohesin function. In addition, we demonstrated that Esc8, a Sir2-interacting factor, is also required for silent chromatin cohesion. Esc8 was previously shown to associate with Isw1, the enzymatic core of ISW1 chromatin remodelers, to form a variant of the ISW1a chromatin remodeling complex. When ESC8 was deleted or the EKDK motif was mutated, cohesin binding at silenced chromatin domains persisted but cohesion of the domains was abolished. The data are not consistent with cohesin embracing both sister chromatids within silent chromatin domains. Transcriptional silencing remains largely intact in strains lacking ESC8 or bearing EKDK mutations, indicating that silencing and cohesion are separable functions of Sir2 and silent chromatin.

Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres, and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division.

Transcription facilitates sister chromatid cohesion on chromosomal arms.

Cohesin is a multi-subunit protein complex essential for sister chromatid cohesion, gene expression and DNA damage repair. Although structurally well studied, the underlying determinant of cohesion establishment on chromosomal arms remains enigmatic. Here, we show two populations of functionally distinct cohesin on chromosomal arms using a combination of genomics and single-locus specific DNA-FISH analysis. Chromatin bound cohesin at the loading sites co-localizes with Pds5 and Eso1 resulting in stable cohesion. In contrast, cohesin independent of its loader is unable to maintain cohesion and associates with chromatin in a dynamic manner. Cohesive sites coincide with highly expressed genes and transcription inhibition leads to destabilization of cohesin on chromatin. Furthermore, induction of transcription results in de novo recruitment of cohesive cohesin. Our data suggest that transcription facilitates cohesin loading onto chromosomal arms and is a key determinant of cohesive sites in fission yeast.

Post-Assembly Stabilization of Rationally Designed DNA Crystals.

This manuscript reports an effort to stabilize self-assembled DNA crystals. Owing to their weak inter-unit cohesion, self-assembled DNA crystals are fragile, which limits the potential applications of such crystals. To overcome this problem, another molecule was introduced, which binds to the cohesive sites and stabilizes the inter-unit interactions. The extra interactions greatly improve the stability of the DNA crystals. The original DNA crystals are only stable in solutions of high ionic strength (e.g., ≥1.2 M (NH4)2SO4); in contrast, the stabilized crystals can be stable at ionic strengths as low as that of a 0.02 M solution of (NH4)2SO4. The current strategy is expected to represent a general approach for increasing the stability of self-assembled DNA nanostructures for potential applications, for example, as structural scaffolds and molecular sieves.

Post‐Assembly Stabilization of Rationally Designed DNA Crystals

AbstractThis manuscript reports an effort to stabilize self‐assembled DNA crystals. Owing to their weak inter‐unit cohesion, self‐assembled DNA crystals are fragile, which limits the potential applications of such crystals. To overcome this problem, another molecule was introduced, which binds to the cohesive sites and stabilizes the inter‐unit interactions. The extra interactions greatly improve the stability of the DNA crystals. The original DNA crystals are only stable in solutions of high ionic strength (e.g., ≥1.2 M (NH4)2SO4); in contrast, the stabilized crystals can be stable at ionic strengths as low as that of a 0.02 M solution of (NH4)2SO4. The current strategy is expected to represent a general approach for increasing the stability of self‐assembled DNA nanostructures for potential applications, for example, as structural scaffolds and molecular sieves.

Centromere-independent accumulation of cohesin at ectopic heterochromatin sites induces chromosome stretching during anaphase.

Author SummaryDuring cell division, chromosomes acquire their characteristic X-shaped morphology by having well-resolved chromosome arms while still remaining connected at the heterochromatic regions around the centromere. This connection is mediated by the cohesin complex, a “molecular glue” that keeps the two DNA molecules stuck together and ensures that the chromosomes are properly segregated. However, it is unclear how important it is for efficient chromosome segregation that these cohesive forces are specifically positioned near the centromere. In this study, we tested several strains of the fruit fly Drosophila melanogaster carrying chromosomal rearrangements in which long stretches of heterochromatin from near the centromere have been misplaced within distant euchromatic regions. We find that such inappropriately located heterochromatin is enough to promote increased levels of cohesin complex loading and the formation of additional constrictions, regardless of proximity to the centromere. Importantly, we further show that as cell division proceeds and the sister chromatids move to opposite poles of the cell, the presence of ectopic heterochromatin (and hence ectopic cohesion) leads to significant chromosome stretching due to impaired resolution of the ectopic cohesion sites. These results highlight the possibility that chromosome rearrangements involving heterochromatin regions near the centromeres, often seen in many cancers, can induce additional errors in cell division and thereby compromise genetic stability.

Principal $$\infty $$ ∞ -bundles: presentations

We discuss two aspects of the presentation of the theory of principal infinity-bundles in an infinity-topos, introduced in [NSSa], in terms of categories of simplicial (pre)sheaves. First we show that over a cohesive site C and for G a presheaf of simplicial groups which is C-acyclic, G-principal infinity-bundles over any object in the infinity-topos over C are classified by hyper-Cech-cohomology with coefficients in G. Then we show that over a site C with enough points, principal infinity-bundles in the infinity-topos are presented by ordinary simplicial bundles in the sheaf topos that satisfy principality by stalkwise weak equivalences. Finally we discuss explicit details of these presentations for the discrete site (in discrete infinity-groupoids) and the smooth site (in smooth infinity-groupoids, generalizing Lie groupoids and differentiable stacks). In the companion article [NSSc] we use these presentations for constructing classes of examples of (twisted) principal infinity-bundles and for the discussion of various applications.

CDC-48/p97 is required for proper meiotic chromosome segregation via controlling AIR-2/Aurora B kinase localization in Caenorhabditis elegans

CDC-48/p97 is a AAA (ATPases associated with diverse cellular activities) chaperone involved in protein conformational changes such as the disassembly of protein complexes. We previously reported that Caenorhabditis elegans CDC-48.1 and CDC-48.2 (CDC-48s) are essential for the progression of meiosis I metaphase. Here, we report that CDC-48s are required for proper chromosome segregation during meiosis in C. elegans. In wild-type worms, at the diakinesis phase, phosphorylation of histone H3, one of the known substrates of aurora B kinase (AIR-2), on meiosis I chromatids correlated with AIR-2 localization at the cohesion sites of homologous chromatids. Conversely, depletion of CDC-48s resulted in a significant expansion of signals for AIR-2 and phosphorylated histone H3 over the entire length of meiotic chromosomes, leading to defective chromosome segregation, while the total amount of AIR-2 in lysates was not changed by the depletion of CDC-48s. The defective segregation of meiotic chromosomes caused by the depletion of CDC-48s was suppressed by the simultaneous depletion of AIR-2 and is similar to that observed following the depletion of protein phosphatase 1 (PP1) phosphatases. However, the amount and localization of PP1 were not changed by the depletion of CDC-48s. These results suggest that CDC-48s control the restricted localization of AIR-2 to the cohesion sites of homologous chromatids in meiosis I.

Arabidopsis sister chromatids often show complete alignment or separation along a 1.2-Mb euchromatic region but no cohesion “hot spots”

Sister chromatid cohesion is a prerequisite for correct segregation and possibly other functions of replicated chromosomes. Except for yeast, no details are known about arrangement of cohesion sites along interphase chromosomes. Within nuclei of several higher plants, sister chromatids are frequently not aligned at various positions along chromosome arms. Therefore, we tested whether preferential alignment positions ("cohesion hot spots") and constant extension of and distances between aligned sites occur in plants. Along a approximately 1.2-Mb contig from the bottom arm of chromosome 1, the sister chromatid positions of 13 individual BAC inserts were found to be aligned for approximately 67-77% of homologues in 4C Arabidopsis thaliana nuclei. The differences between the 13 BAC positions were not significant at the P < 0.01 level. This suggests variability of alignment positions between cells and indicates the absence of cohesion "hot spots". Similar as for single BACs, FISH with the entire contig indicated complete alignment for approximately 69% and complete separation of sister chromatids for approximately 31% of homologues in 4C nuclei. Partial alignment or separation was barely detectable. When three BAC inserts from a 760-kb region were tested simultaneously, alignment or separation of only the central BAC occurred in 3.3% and 3.5% of replicated chromosomes, respectively. Thus, we assume that sister chromatids can be separated or aligned within a Mb range in differentiated cells. However, the minimum extension of aligned sites or distances between them may (in rare cases) fall below ~500 kb.

Using Public Relations and Marketing to Generate Referrals and Sales

This article addresses how home health care managers and owners can use marketing and public relations to generate sales leads. It lays out a process: Develop your brand and key messages and then use marketing and public relations tactics, including a cohesive Web site, to reach your target audiences. The key is generating leads through relationship building by identifying referral sources and making certain they know how your company stands out from the crowd. After marketing the firm to the people who are actually making referrals, track the results. The article provides many examples of tips and ideas for generating leads and turning them into sales. A case study presents information relative to how these tips were applied at Kansas City Home Care.

Establishment of Sister Chromatid Cohesion at the S. cerevisiae Replication Fork

Two identical sister copies of eukaryotic chromosomes are synthesized during S phase. To facilitate their recognition as pairs for segregation in mitosis, sister chromatids are held together from their synthesis onward by the chromosomal cohesin complex. Replication fork progression is thought to be coupled to establishment of sister chromatid cohesion, facilitating identification of replication products, but evidence for this has remained circumstantial. Here we show that three proteins required for sister chromatid cohesion, Eco1, Ctf4, and Ctf18, are found at, and Ctf4 travels along chromosomes with, replication forks. The ring-shaped cohesin complex is loaded onto chromosomes before S phase in an ATP hydrolysis-dependent reaction. Cohesion establishment during DNA replication follows without further cohesin recruitment and without need for cohesin to re-engage an ATP hydrolysis motif that is critical for its initial DNA binding. This provides evidence for cohesion establishment in the context of replication forks and imposes constraints on the mechanism involved.

Sister chromatid separation at human telomeric regions.

Telomeres are nucleoprotein complexes located at chromosome ends, vital for preserving chromosomal integrity. Telomeric DNA shortens with progressive rounds of cell division, culminating in replicative senescence. Previously we have reported, on the basis of fluorescent in situ hybridization, that several human telomeric regions display solitary signals (singlets) in metaphase cells of presenescent fibroblasts, in comparison to other genomic regions that hybridize as twin signals (doublets). In the current study, we show that an additional 12 out of 12 telomeric regions examined also display metaphase singlet signals in pre-senescent cells, and that excess telomere-metaphase singlets also occur in earlier passage cells harvested from elderly individuals. In cancer cell lines expressing telomerase and in pre-senescent fibroblasts ectopically expressing hTERT, this phenomenon is abrogated. Confocal microscope image analysis showed that the telomere metaphase singlets represent regions that have replicated but not separated; this is presumably because of persistent cohesion. The introduction of mutations that interfere with the normal dissolution of cohesion at the metaphase to anaphase transition induced the cut (chromosomes untimely torn) phenotype in early passage fibroblasts, with predominantly telomeric rather than centromeric DNA, present on the chromatin bridges between the daughter nuclei. These results suggest that telomeric regions in animal cells may potentially be sites of persistent cohesion, and that this cohesion may be the basis for an observed excess of fluorescent in situ hybridization metaphase singlets at telomeres. Persistent cohesion at telomeres may be associated with attempted DNA repair or chromosomal abnormalities, which have been described in pre-senescent cells.