DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alíeles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA ...methylation were isolated through forward-or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context-and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alíeles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alíeles for chromomethylase CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alíeles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.
Security happens in the future. Threats to our security concern the potential of a future event, of possibility and uncertainty. Fear, omnipresent in popular culture is thereby non-uniform. Like time ...itself, it intensifies and softens, accelerates and slows, and disrupts and destabilises as a function of many variables. This article re-interprets the phenomenon of insecurity by reading it together with Heidegger’s analytic of time as a function of our proximity to being as fundamental ontological question, one which unfolds in the form of a threatening future.
Cytoplasmic dynein is a dimeric AAA(+) motor protein that performs critical roles in eukaryotic cells by moving along microtubules using ATP. Here using cryo-electron microscopy we directly observe ...the structure of Dictyostelium discoideum dynein dimers on microtubules at near-physiological ATP concentrations. They display remarkable flexibility at a hinge close to the microtubule binding domain (the stalkhead) producing a wide range of head positions. About half the molecules have the two heads separated from one another, with both leading and trailing motors attached to the microtubule. The other half have the two heads and stalks closely superposed in a front-to-back arrangement of the AAA(+) rings, suggesting specific contact between the heads. All stalks point towards the microtubule minus end. Mean stalk angles depend on the separation between their stalkheads, which allows estimation of inter-head tension. These findings provide a structural framework for understanding dynein's directionality and unusual stepping behaviour.
One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. ...Here, we show that purified human SNM1A (hSNM1A), which exhibits a 5'-3' exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replication-associated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.
For nearly 50 years, analyses of coral physiology have used small coral fragments (nubbins) to make inferences about larger colonies. However, scaling in corals shows that linear extrapolations from ...nubbins to whole colonies can be misleading, because polyps in nubbins are divorced of their morphologically complex and physiologically integrated corallum. We tested for the effects of integration among branches in determining size-dependent calcification of the coral
spp. under elevated
. Area-normalized net calcification was compared between branches (nubbins), aggregates of nubbins (complex morphologies without integration) and whole colonies (physiologically integrated) at 400 versus approximately 1000 µatm
. Net calcification was unaffected by
, but differed among colony types. Single nubbins grew faster than whole colonies, but when aggregated, nubbins changed calcification to match whole colonies even though they lacked integration among branches. Corallum morphology causes the phenotype of branching corals to differ from the summation of their branches.
Experiments with coral fragments (i.e., nubbins) have shown that net calcification is depressed by elevated PCO2. Evaluating the implications of this finding requires scaling of results from nubbins ...to colonies, yet the experiments to codify this process have not been completed. Building from our previous research demonstrating that net calcification of Pocillopora verrucosa (2-13 cm diameter) was unaffected by PCO2 (400 µtam and 1,000 µatm) and temperature (26.5°C and 29.7°C) we sought generality to this outcome by testing how colony size modulates PCO2 and temperature sensitivity in a branching acroporid. Together these taxa represent two of the dominant lineages of branching corals on Indo-Pacific coral reefs. Two trials conducted over two years tested the hypothesis that the seasonal range in seawater temperature (26.5°C and 29.2°C) and a future PCO2 (1,062 µatm versus an ambient of 461 µatm) affect net calcification of an ecologically relevant size range (5-20 cm diameter) of colonies of Acropora hyacinthus. Like P. verrucosa, the effects of temperature and PCO2 on net calcification (mg d-1) of A. verrucosa were not statistically detectable. These results support the generality of a null outcome on net calcification of exposing intact colonies of branching corals to environmental conditions contrasting seasonal variation in temperature and predicted future variation in PCO2. While there is a need to expand beyond an experimental culture relying on coral nubbins as tractable replicates, rigorously responding to this need poses substantial ethical and logistical challenges.
Coupling between ATPase and track binding sites is essential for molecular motors to move along cytoskeletal tracks. In dynein, these sites are separated by a long coiled coil stalk that must mediate ...communication between them, but the underlying mechanism remains unclear. Here we show that changes in registration between the two helices of the coiled coil can perform this function. We locked the coiled coil at three specific registrations using oxidation to disulfides of paired cysteine residues introduced into the two helices. These trapped ATPase activity either in a microtubule-independent high or low state, and microtubule binding activity either in an ATP-insensitive strong or weak state, depending on the registry of the coiled coil. Our results provide direct evidence that dynein uses sliding between the two helices of the stalk to couple ATPase and microtubule binding activities during its mechanochemical cycle.
Dynein ATPases are the largest known cytoskeletal motors and perform critical functions in cells: carrying cargo along microtubules in the cytoplasm and powering flagellar beating. Dyneins are ...members of the AAA+ superfamily of ring-shaped enzymes, but how they harness this architecture to produce movement is poorly understood. Here, we have used cryo-EM to determine 3D maps of native flagellar dynein-c and a cytoplasmic dynein motor domain in different nucleotide states. The structures show key sites of conformational change within the AAA+ ring and a large rearrangement of the “linker” domain, involving a hinge near its middle. Analysis of a mutant in which the linker “undocks” from the ring indicates that linker remodeling requires energy that is supplied by interactions with the AAA+ modules. Fitting the dynein-c structures into flagellar tomograms suggests how this mechanism could drive sliding between microtubules, and also has implications for cytoplasmic cargo transport.
Display omitted
► Structural changes in axonemal and cytoplasmic dynein motors seen by cryo-EM ► Movement is amplified by a hinge action within dynein's linker domain ► The linker is a stable structure that is actively remodeled by dynein's AAA+ modules ► Dynein's neck subdomain interacts with AAA4 and is an intrinsic site of flexibility
Dyneins are AAA+ motor proteins that power both microtubule-based transport and flagellar motility. Now, Roberts et al. present cryo-EM structures of both classes of dynein, revealing nucleotide-induced bending and indicating that dynein generates movement by using the AAA+ ring to actively remodel its own linker.
"This book presents the theory and practice of impact assessment tailored to new border control technologies that are increasingly employed at state borders with the aim of facilitating border ...checks. Experience has shown that their use often comes into conflict with societal values such as the respect for fundamental rights to privacy and personal data protection. As a result, there is a growing need to accommodate two requirements, the first being the deployment of new border control technologies and the second being the respect for relevant societal values. This book introduces a tool that seeks to accommodate both requirements: impact assessment. Impact assessment is an evaluation technique used to analyse the potential future consequences of a given measure for societal values. The main objective of the assessment process is to support informed decision-making about whether or not, and under what conditions, to deploy a given measure. Border Control and New Technologies. Addressing Integrated Impact Assessment is addressed predominantly to border control authorities in the European Union and in the Schengen Area who wish to ensure that new technologies for controlling state borders respect the principles of democracy, the rule of law and human rights. The book will be of interest also for border control officials elsewhere in the world as well as for anyone dealing with the theory and practice of impact assessment."
The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. As part of a ...drug discovery program targeting EZH2, we have identified highly potent, selective, SAM-competitive, and cell-active EZH2 inhibitors, including GSK926 (3) and GSK343 (6). These compounds are small molecule chemical tools that would be useful to further explore the biology of EZH2.
PDF
