117 research outputs found
No Evidence for âBreak-Induced Replicationâ in a Higher Plant â But Break-Induced Conversion May Occur
âBreak-induced replicationâ (BIR) is considered as one way to repair DNA double-strand breaks (DSBs). BIR is defined as replication of the proximal break-ends up to the end of the broken chromosome using an undamaged (homologous) double-stranded template and mimicking a non-reciprocal translocation. This phenomenon was detected by genetic experiments in yeast. BIR is assumed to occur also in mammals, but experimental evidence is not yet at hand. We have studied chromosomes of the field bean, Vicia faba L., with respect to the occurrence of BIR after DSB induction during S and G2 phase. Simultaneous incorporation of the base analog ethynyldeoxyuridine (EdU) revealed no chromosomal replication pattern indicative of BIR. Thus, if occurring at all, BIR does not play a major role in DSB repair in higher plants with large chromosome arms. However, the frequency of interstitial asymmetric EdU incorporation within heterochromatic regions, visible on metaphase chromosomes, increased after chromosome breakage during S and G2 phase. Such asymmetric labeling could be interpreted as conservative replication up to the next replicon, circumventing a DSB, and yielding an interstitial conversion-like event
Image analysis workflows to reveal the spatial organization of cell nuclei and chromosomes
Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images
Odor-Induced Multi-Level Inhibitory Maps in Drosophila
Optical imaging of intracellular Ca2+ influx as a correlate of neuronal excitation represents a standard technique for visualizing spatiotemporal activity of neuronal networks. However, the information-processing properties of single neurons and neuronal circuits likewise involve inhibition of neuronal membrane potential. Here, we report spatially resolved optical imaging of odor-evoked inhibitory patterns in the olfactory circuitry of Drosophila using a genetically encoded fluorescent Cl- sensor. In combination with the excitatory component reflected by intracellular Ca2+ dynamics, we present a comprehensive functional map of both odor-evoked neuronal activation and inhibition at different levels of olfactory processing. We demonstrate that odor-evoked inhibition carried by Cl- influx is present both in sensory neurons and second-order projection neurons (PNs), and is characterized by stereotypic, odor-specific patterns. Cl--mediated inhibition features distinct dynamics in different neuronal populations. Our data support a dual role of inhibitory neurons in the olfactory system: global gain control across the neuronal circuitry and glomerulus-specific inhibition to enhance neuronal information processing
The 'broken society' election: class hatred and the politics of poverty and place in Glasgow East
This paper considers some of the ways in which representations of people experiencing poverty and disadvantaged places continue to be informed by ideas of individual inadequacy, dependency and disorder. Drawing on media reportage of poverty during the Glasgow East by-election in July 2008, it argues not only that people defined as 'poor' and locales that are severely disadvantaged continue to be 'othered' through such narratives, but also that this provides a clear indication of the ways in which the politics of poverty and state welfare are increasingly being fought-out in the media. It is argued that such misrecognition amounts to social injustice and stands in the way of progressive approaches to poverty and social welfar
Odor-Induced Multi-Level Inhibitory Maps in Drosophila
Grabe V, Schubert M, Strube-Bloss M, et al. Odor-Induced Multi-Level Inhibitory Maps in Drosophila. eNeuro. 2019;7(1): ENEURO.0213-19.2019
Air drag coefficient of textile-covered elastic cylinders â preliminary aerodynamic studies
This paper presents preliminary experimental results on the influence on the aerodynamic drag of a cylinder from the cylinder type (i.e., rigid or soft) and its textile surface. Both a rigid cylinder and a soft-body cylinder, with a gelatin layer, each with five different textile surfaces were measured in the wind tunnel using force measurement technology. The drag coefficient was determined for several Reynolds numbers. The study shows that the elasticity of a cylinder has a significant influence on the drag force and the airflow type. However, the influence of the soft-body cylinder depends on the respective fabric. With the given measurements, no exact statements can yet be made to quantify the influence. This influence must be studied independently and in conjunction with the textile surface in order to gain understanding of the overall system of airflow, textile and elastic body
Conserved redox-dependent DNA binding of ROXY glutaredoxins with TGA transcription factors
peer-reviewedThe Arabidopsis thaliana CCâtype glutaredoxin (GRX) ROXY1 and the bZIP TGA transcription factor (TF) PERIANTHIA (PAN) interact in the nucleus and together regulate petal development. The CCâtype GRXs exist exclusively in land plants, and in contrast to the ubiquitously occurring CPYC and CGFS GRX classes, only the CCâtype GRXs expanded strongly during land plant evolution. Phylogenetic analyses show that TGA TFs evolved before the CCâtype GRXs in charophycean algae. MpROXY1/2 and MpTGA were isolated from the liverwort Marchantia polymorpha to analyze regulatory ROXY/TGA interactions in a basal land plant. Homologous and heterologous protein interaction studies demonstrate that nuclear ROXY/TGA interactions are conserved since the occurrence of CCâtype GRXs in bryophytes and mediated by a conserved ROXY Câterminus. Redox EMSA analyses show a redoxâsensitive binding of MpTGA to the cisâregulatory asâ1âlike element. Furthermore, we demonstrate that MpTGA binds together with MpROXY1/2 to this motif under reducing conditions, whereas this interaction is not observed under oxidizing conditions. Remarkably, heterologous complementation studies reveal a strongly conserved land plant ROXY activity, suggesting an ancestral role for CCâtype GRXs in modulating the activities of TGA TFs. Superâresolution microscopy experiments detected a strong colocalization of ROXY1 with the active form of the RNA polymerase II in the nucleus. Together, these data shed new light on the function of ROXYs and TGA TFs and the evolution of redoxâsensitive transcription regulation processes, which likely contributed to adapt land plants to novel terrestrial habitats
Arabidopsis NSE4 Proteins Act in Somatic Nuclei and Meiosis to Ensure Plant Viability and Fertility
The SMC 5/6 complex together with cohesin and condensin is a member of the
structural maintenance of chromosome (SMC) protein family. In non-plant organisms
SMC5/6 is engaged in DNA repair, meiotic synapsis, genome organization and
stability. In plants, the function of SMC5/6 is still enigmatic. Therefore, we analyzed
the crucial d-kleisin component NSE4 of the SMC5/6 complex in the model plant
Arabidopsis thaliana. Two functional conserved Nse4 paralogs (Nse4A and Nse4B)
are present in A. thaliana, which may have evolved via gene subfunctionalization.
Due to its high expression level, Nse4A seems to be the more essential gene,
whereas Nse4B appears to be involved mainly in seed development. The morphological
characterization of A. thaliana T-DNA mutants suggests that the NSE4 proteins are
essential for plant growth and fertility. Detailed investigations in wild-type and the
mutants based on live cell imaging of transgenic GFP lines, fluorescence in situ
hybridization (FISH), immunolabeling and super-resolution microscopy suggest that
NSE4A acts in several processes during plant development, such as mitosis, meiosis
and chromatin organization of differentiated nuclei, and that NSE4A operates in a
cell cycle-dependent manner. Differential response of NSE4A and NSE4B mutants
after induced DNA double strand breaks (DSBs) suggests their involvement in DNA
repair processes
The MCM-Binding Protein ETG1 Aids Sister Chromatid Cohesion Required for Postreplicative Homologous Recombination Repair
The DNA replication process represents a source of DNA stress that causes potentially spontaneous genome damage. This effect might be strengthened by mutations in crucial replication factors, requiring the activation of DNA damage checkpoints to enable DNA repair before anaphase onset. Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest. This arrest correlated with a partial loss of sister chromatid cohesion. The lack-of-cohesion phenotype was intensified in plants without functional CTF18, a replication fork factor needed for cohesion establishment. The synergistic effect of the etg1 and ctf18 mutants on sister chromatid cohesion strengthened the impact on plant growth of the replication stress caused by ETG1 deficiency because of inefficient DNA repair. We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress. Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein
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