TY - JOUR A1 - Mao, Hailiang A1 - Nakamura, Moritaka A1 - Viotti, Corrado A1 - Grebe, Markus T1 - A Framework for Lateral Membrane Trafficking and Polar Tethering of the PEN3 ATP-Binding Cassette Transporter JF - Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants N2 - The outermost cell layer of plants, the epidermis, and its outer (lateral) membrane domain facing the environment are continuously challenged by biotic and abiotic stresses. Therefore, the epidermis and the outer membrane domain provide important selective and protective barriers. However, only a small number of specifically outer membrane-localized proteins are known. Similarly, molecular mechanisms underlying the trafficking and the polar placement of outer membrane domain proteins require further exploration. Here, we demonstrate that ACTIN7 (ACT7) mediates trafficking of the PENETRATION3 (PEN3) outer membrane protein from the trans-Golgi network (TGN) to the plasma membrane in the root epidermis of Arabidopsis (Arabidopsis thaliana) and that actin function contributes to PEN3 endocytic recycling. In contrast to such generic ACT7-dependent trafficking from the TGN, the EXOCYST84b (EXO84b) tethering factor mediates PEN3 outer-membrane polarity. Moreover, precise EXO84b placement at the outer membrane domain itself requires ACT7 function. Hence, our results uncover spatially and mechanistically distinct requirements for ACT7 function during outer lateral membrane cargo trafficking and polarity establishment. They further identify an exocyst tethering complex mediator of outer lateral membrane cargo polarity. Y1 - 2016 U6 - https://doi.org/10.1104/pp.16.01252 SN - 0032-0889 SN - 1532-2548 VL - 172 SP - 2245 EP - 2260 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - VanDerVen, Peter F. M. A1 - Bartsch, Jörg A1 - Gautel, Mathias A1 - Jokusch, Harald A1 - Fürst, Dieter Oswald T1 - A functional knock-out of titin results in defective myofibril assembly Y1 - 2000 ER - TY - JOUR A1 - Schwarte, Sandra A1 - Tiedemann, Ralph T1 - A Gene Duplication/Loss Event in the Ribulose-1,5-Bisphosphate-Carboxylase/Oxygenase (Rubisco) Small Subunit Gene Family among Accessions of Arabidopsis thaliana JF - Molecular biology and evolution N2 - Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39), the most abundant protein in nature, catalyzes the assimilation of CO(2) (worldwide about 10(11) t each year) by carboxylation of ribulose-1,5-bisphosphate. It is a hexadecamer consisting of eight large and eight small subunits. Although the Rubisco large subunit (rbcL) is encoded by a single gene on the multicopy chloroplast genome, the Rubisco small subunits (rbcS) are encoded by a family of nuclear genes. In Arabidopsis thaliana, the rbcS gene family comprises four members, that is, rbcS-1a, rbcS-1b, rbcS-2b, and rbcS-3b. We sequenced all Rubisco genes in 26 worldwide distributed A. thaliana accessions. In three of these accessions, we detected a gene duplication/loss event, where rbcS-1b was lost and substituted by a duplicate of rbcS-2b (called rbcS-2b*). By screening 74 additional accessions using a specific polymerase chain reaction assay, we detected five additional accessions with this duplication/loss event. In summary, we found the gene duplication/loss in 8 of 100 A. thaliana accessions, namely, Bch, Bu, Bur, Cvi, Fei, Lm, Sha, and Sorbo. We sequenced an about 1-kb promoter region for all Rubisco genes as well. This analysis revealed that the gene duplication/loss event was associated with promoter alterations (two insertions of 450 and 850 bp, one deletion of 730 bp) in rbcS-2b and a promoter deletion (2.3 kb) in rbcS-2b* in all eight affected accessions. The substitution of rbcS-1b by a duplicate of rbcS-2b (i.e., rbcS-2b*) might be caused by gene conversion. All four Rubisco genes evolve under purifying selection, as expected for central genes of the highly conserved photosystem of green plants. We inferred a single positive selected site, a tyrosine to aspartic acid substitution at position 72 in rbcS-1b. Exactly the same substitution compromises carboxylase activity in the cyanobacterium Anacystis nidulans. In A. thaliana, this substitution is associated with an inferred recombination. Functional implications of the substitution remain to be evaluated. KW - Arabidopsis thaliana KW - Arabidopsis lyrata KW - Rubisco KW - gene duplication KW - positive selection Y1 - 2011 U6 - https://doi.org/10.1093/molbev/msr008 SN - 0737-4038 VL - 28 IS - 6 SP - 1861 EP - 1876 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Sunna, Anwar A1 - Bergquist, Peter L. T1 - A gene encoding a novel extremely thermostable 1,4-beta-xylanase isolated directly from an environmental DNA sample N2 - Small-subunit (SSU) rRNA genes (rDNA) were amplified by PCR from a hot pool environmental DNA sample using Bacteria- or Archaea-specific rDNA primers. Unique rDNA types were identified by restriction fragment length polymorphism (RFLP) analysis and representative sequences were determined. Family 10 glycoside hydrolase consensus PCR primers were used to explore the occurrence and diversity of xylanase genes in the hot pool environmental DNA sample. Partial sequences for three different xylanases were obtained and genomic walking PCR (GWPCR), in combination with nested primer pairs, was used to obtained a unique 1,741-bp nucleotide sequence. Analysis of this sequence identified a putative XynA protein encoded by the xynA open reading frame. The single module novel xylanase shared sequence similarity to the family 10 glycoside hydrolases. The purified recombinant enzyme, XynA expressed in E. coli exhibited optimum activity at 100 degrees C and pH 6.0, and was extremely thermostable at 90 degrees C. The enzyme showed high specificity toward different xylans and xylooligosaccharides. Y1 - 2003 ER - TY - JOUR A1 - Balazadeh, Salma A1 - Siddiqui, Hamad A1 - Allu, Annapurna Devi A1 - Matallana-Ramirez, Lilian Paola A1 - Caldana, Camila A1 - Mehrnia, Mohammad A1 - Zanor, Maria-Inés A1 - Koehler, Barbara A1 - Müller-Röber, Bernd T1 - A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence N2 - P>The onset and progression of senescence are under genetic and environmental control. The Arabidopsis thaliana NAC transcription factor ANAC092 (also called AtNAC2 and ORE1) has recently been shown to control age-dependent senescence, but its mode of action has not been analysed yet. To explore the regulatory network administered by ANAC092 we performed microarray-based expression profiling using estradiol-inducible ANAC092 overexpression lines. Approximately 46% of the 170 genes up-regulated upon ANAC092 induction are known senescence-associated genes, suggesting that the NAC factor exerts its role in senescence through a regulatory network that includes many of the genes previously reported to be senescence regulated. We selected 39 candidate genes and confirmed their time-dependent response to enhanced ANAC092 expression by quantitative RT-PCR. We also found that the majority of them (24 genes) are up-regulated by salt stress, a major promoter of plant senescence, in a manner similar to that of ANAC092, which itself is salt responsive. Furthermore, 24 genes like ANAC092 turned out to be stage-dependently expressed during seed growth with low expression at early and elevated expression at late stages of seed development. Disruption of ANAC092 increased the rate of seed germination under saline conditions, whereas the opposite occurred in respective overexpression plants. We also detected a delay of salinity-induced chlorophyll loss in detached anac092-1 mutant leaves. Promoter-reporter (GUS) studies revealed transcriptional control of ANAC092 expression during leaf and flower ageing and in response to salt stress. We conclude that ANAC092 exerts its functions during senescence and seed germination through partly overlapping target gene sets. Y1 - 2010 UR - http://www3.interscience.wiley.com/cgi-bin/issn?DESCRIPTOR=PRINTISSN&VALUE=0960-7412 U6 - https://doi.org/10.1111/j.1365-313X.2010.04151.x SN - 0960-7412 ER - TY - JOUR A1 - Liaimer, Anton A1 - Jensen, John B. A1 - Dittmann, Elke T1 - A Genetic and Chemical Perspective on Symbiotic Recruitment of Cyanobacteria of the Genus Nostoc into the Host Plant Blasia pusilla L. JF - Frontiers in microbiology N2 - Liverwort Blasia pusilla L. recruits soil nitrogen-fixing cyanobacteria of genus Nostoc as symbiotic partners. In this work we compared Nostoc community composition inside the plants and in the soil around them from two distant locations in Northern Norway. STRR fingerprinting and 16S rDNA phylogeny reconstruction showed a remarkable local diversity among isolates assigned to several Nostoc clades. An extensive web of negative allelopathic interactions was recorded at an agricultural site, but not at the undisturbed natural site. The cell extracts of the cyanobacteria did not show antimicrobial activities, but four isolates were shown to be cytotoxic to human cells. The secondary metabolite profiles of the isolates were mapped by MALDI-TOF MS, and the most prominent ions were further analyzed by Q-TOF for MS/MS aided identification. Symbiotic isolates produced a great variety of small peptide-like substances, most of which lack any record in the databases. Among identified compounds we found microcystin and nodularin variants toxic to eukaryotic cells. Microcystin producing chemotypes were dominating as symbiotic recruits but not in the free-living community. In addition, we were able to identify several novel aeruginosins and banyaside-like compounds, as well as nostocyclopeptides and nosperin. KW - Cyanobacteria KW - secondary metabolites KW - symbiosis KW - Blasia KW - Nostoc KW - allelopathy Y1 - 2016 U6 - https://doi.org/10.3389/fmicb.2016.01693 SN - 1664-302X VL - 7 SP - 449 EP - 474 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Lah, Ljerka A1 - Löber, Ulrike A1 - Hsiang, Tom A1 - Hartmann, Stefanie T1 - A genomic comparison of putative pathogenicity-related gene families in five members of the Ophiostomatales with different lifestyles JF - Fungal biology N2 - Ophiostomatoid fungi are vectored by their bark-beetle associates and colonize different host tree species. To survive and proliferate in the host, they have evolved mechanisms for detoxification and elimination of host defence compounds, efficient nutrient sequestration, and, in pathogenic species, virulence towards plants. Here, we assembled a draft genome of the spruce pathogen Ophiostoma bicolor. For our comparative and phylogenetic analyses, we mined the genomes of closely related species (Ophiostoma piceae, Ophiostoma ulmi, Ophiostoma novo-ulmi, and Grosmannia clavigera). Our aim was to acquire a genomic and evolutionary perspective of gene families important in host colonization. Genome comparisons showed that both the nuclear and mitochondrial genomes in our assembly were largely complete. Our O. bicolor 25.3 Mbp draft genome had 10 018 predicted genes, 6041 proteins with gene ontology (GO) annotation, 269 carbohydrate-active enzymes (CAZymes), 559 peptidases and inhibitors, and 1373 genes likely involved in pathogen-host interactions. Phylogenetic analyses of selected protein families revealed core sets of cytochrome P450 genes, ABC transporters and backbone genes involved in secondary metabolite (SM) biosynthesis (polyketide synthases (PKS) and non-ribosomal synthases), and species-specific gene losses and duplications. Phylogenetic analyses of protein families of interest provided insight into evolutionary adaptations to host biochemistry in ophiostomatoid fungi. KW - Bark beetle KW - Bluestain fungi KW - Ips typographus Y1 - 2016 U6 - https://doi.org/10.1016/j.funbio.2016.12.002 SN - 1878-6146 SN - 1878-6162 VL - 121 SP - 234 EP - 252 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Wulf, Monika A1 - Rujner, Hendrik T1 - A GIS-based method for the reconstruction of the late eighteenth century forest vegetation in the Prignitz region (NE Germany) JF - Landscape ecology N2 - Our goal was to reconstruct the late eighteenth century forest vegetation of the Prignitz region (NE Germany) at a scale of 1:50,000. We also wanted to relate the historical forest vegetation to the actual and potential natural vegetation. For these purposes, we selected 15 woody species and transferred relevant data found in historical records from various sources together with the recent localities of (very) old individuals belonging to these woody species into ArcView GIS. Following multi-step data processing including the generation of a point density layer using a moving window with kernel estimation and derivation of vegetation units applying Boolean algebra rules together with information on site conditions, we derived 17 forest communities corresponding to the potential natural vegetation. We were able to reconstruct the historical forest vegetation for 90% of the forest area ca. 1780. Only two of the 17 forest communities covered large parts of the forested area. The oak forest with Agrostis capillaris covered about 44% of the total forest area, and alder forests on fenland made up about 37%. Oak-hornbeam forests with Stellaria holostea comprised slightly less than 6% of the forest area, while all other forest communities comprised less than 1%. The historical forest vegetation is more similar to the potential forest vegetation and quite different from the actual forest vegetation because coniferous tree species currently cover approximately two-thirds of the actual forest area. The most beneficial result of this study is the map of high-resolution historical vegetation units that may serve as the basis for various further studies, e.g., modelling long-term changes in biodiversity at the landscape scale. KW - Boolean algebra KW - Historical ecology KW - Kernel estimation KW - Moving window KW - Point density KW - Potential natural vegetation KW - Schmettau map KW - Toponymy KW - Vegetation map Y1 - 2011 U6 - https://doi.org/10.1007/s10980-010-9555-1 SN - 0921-2973 VL - 26 IS - 2 SP - 153 EP - 168 PB - Springer CY - Dordrecht ER - TY - JOUR A1 - Reeg, Jette A1 - Heine, Simon A1 - Mihan, Christine A1 - McGee, Sean A1 - Preuss, Thomas G. A1 - Jeltsch, Florian T1 - A graphical user interface for the plant community model IBC-grass JF - Plos One N2 - Plants located adjacent to agricultural fields are important for maintaining biodiversity in semi-natural landscapes. To avoid undesired impacts on these plants due to herbicide application on the arable fields, regulatory risk assessments are conducted prior to registration to ensure proposed uses of plant protection products do not present an unacceptable risk. The current risk assessment approach for these non-target terrestrial plants (NTTPs) examines impacts at the individual-level as a surrogate approach for protecting the plant community due to the inherent difficulties of directly assessing population or community level impacts. However, modelling approaches are suitable higher tier tools to upscale individual-level effects to community level. IBC-grass is a sophisticated plant community model, which has already been applied in several studies. However, as it is a console application software, it was not deemed sufficiently user-friendly for risk managers and assessors to be conveniently operated without prior expertise in ecological models. Here, we present a user-friendly and open source graphical user interface (GUI) for the application of IBC-grass in regulatory herbicide risk assessment. It facilitates the use of the plant community model for predicting long-term impacts of herbicide applications on NTTP communities. The GUI offers two options to integrate herbicide impacts: (1) dose responses based on current standard experiments (acc. to testing guidelines) and (2) based on specific effect intensities. Both options represent suitable higher tier options for future risk assessments of NTTPs as well as for research on the ecological relevance of effects. Y1 - 2020 U6 - https://doi.org/10.1371/journal.pone.0230012 SN - 1932-6203 VL - 15 IS - 3 PB - Plos 1 CY - San Francisco ER - TY - JOUR A1 - Arvidsson, Samuel Janne A1 - Perez-Rodriguez, Paulino A1 - Müller-Röber, Bernd T1 - A growth phenotyping pipeline for Arabidopsis thaliana integrating image analysis and rosette area modeling for robust quantification of genotype effects JF - New phytologist : international journal of plant science N2 - To gain a deeper understanding of the mechanisms behind biomass accumulation, it is important to study plant growth behavior. Manually phenotyping large sets of plants requires important human resources and expertise and is typically not feasible for detection of weak growth phenotypes. Here, we established an automated growth phenotyping pipeline for Arabidopsis thaliana to aid researchers in comparing growth behaviors of different genotypes. The analysis pipeline includes automated image analysis of two-dimensional digital plant images and evaluation of manually annotated information of growth stages. It employs linear mixed-effects models to quantify genotype effects on total rosette area and relative leaf growth rate (RLGR) and ANOVAs to quantify effects on developmental times. Using the system, a single researcher can phenotype up to 7000 plants d(-1). Technical variance is very low (typically < 2%). We show quantitative results for the growth-impaired starch-excessmutant sex4-3 and the growth-enhancedmutant grf9. We show that recordings of environmental and developmental variables reduce noise levels in the phenotyping datasets significantly and that careful examination of predictor variables (such as d after sowing or germination) is crucial to avoid exaggerations of recorded phenotypes and thus biased conclusions. KW - development KW - growth KW - leaf area KW - modeling KW - phenotyping Y1 - 2011 U6 - https://doi.org/10.1111/j.1469-8137.2011.03756.x SN - 0028-646X VL - 191 IS - 3 SP - 895 EP - 907 PB - Wiley-Blackwell CY - Malden ER - TY - JOUR A1 - Cheng, Fuxia A1 - Hartmann, Stefanie A1 - Gupta, Mayetri A1 - Ibrahim, Joseph G. A1 - Vision, Todd J. T1 - A hierarchical model for incomplete alignments in phylogenetic inference N2 - Motivation: Full-length DNA and protein sequences that span the entire length of a gene are ideally used for multiple sequence alignments (MSAs) and the subsequent inference of their relationships. Frequently, however, MSAs contain a substantial amount of missing data. For example, expressed sequence tags (ESTs), which are partial sequences of expressed genes, are the predominant source of sequence data for many organisms. The patterns of missing data typical for EST-derived alignments greatly compromise the accuracy of estimated phylogenies. Results: We present a statistical method for inferring phylogenetic trees from EST-based incomplete MSA data. We propose a class of hierarchical models for modeling pairwise distances between the sequences, and develop a fully Bayesian approach for estimation of the model parameters. Once the distance matrix is estimated, the phylogenetic tree may be constructed by applying neighbor-joining (or any other algorithm of choice). We also show that maximizing the marginal likelihood from the Bayesian approach yields similar results to a pro. le likelihood estimation. The proposed methods are illustrated using simulated protein families, for which the true phylogeny is known, and one real protein family. Y1 - 2009 UR - http://bioinformatics.oxfordjournals.org/ U6 - https://doi.org/10.1093/bioinformatics/btp015 SN - 1367-4803 ER - TY - JOUR A1 - Zhang, Youjun A1 - Chen, Moxian A1 - Siemiatkowska, Beata A1 - Toleco, Mitchell Rey A1 - Jing, Yue A1 - Strotmann, Vivien A1 - Zhang, Jianghua A1 - Stahl, Yvonne A1 - Fernie, Alisdair T1 - A highly efficient agrobacterium-mediated method for transient gene expression and functional studies in multiple plant species JF - Plant Communications N2 - Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis. KW - transient expression KW - agro-infiltration KW - subcellular localization KW - protein-protein interaction Y1 - 2019 SN - 2590-3462 VL - 1 IS - 5 PB - Science Direct CY - New York ER - TY - JOUR A1 - Dortay, Hakan A1 - Müller-Röber, Bernd T1 - A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker N2 - Background: Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results: IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 mu L Y1 - 2010 UR - http://www.microbialcellfactories.com/home/ U6 - https://doi.org/10.1186/1475-2859-9-29 SN - 1475-2859 ER - TY - JOUR A1 - Jantzen, Friederike A1 - Wozniak, Natalia Joanna A1 - Kappel, Christian A1 - Sicard, Adrien A1 - Lenhard, Michael T1 - A high‑throughput amplicon‑based method for estimating outcrossing rates JF - Plant Methods N2 - Background: The outcrossing rate is a key determinant of the population-genetic structure of species and their long-term evolutionary trajectories. However, determining the outcrossing rate using current methods based on PCRgenotyping individual offspring of focal plants for multiple polymorphic markers is laborious and time-consuming. Results: We have developed an amplicon-based, high-throughput enabled method for estimating the outcrossing rate and have applied this to an example of scented versus non-scented Capsella (Shepherd’s Purse) genotypes. Our results show that the method is able to robustly capture differences in outcrossing rates. They also highlight potential biases in the estimates resulting from differential haplotype sharing of the focal plants with the pollen-donor population at individual amplicons. Conclusions: This novel method for estimating outcrossing rates will allow determining this key population-genetic parameter with high-throughput across many genotypes in a population, enabling studies into the genetic determinants of successful pollinator attraction and outcrossing. KW - Outcrossing KW - Mixed mating KW - Outcrossing rate KW - Capsella KW - Amplicon sequencing Y1 - 2019 U6 - https://doi.org/10.1186/s13007-019-0433-9 SN - 1746-4811 VL - 15 IS - 47 PB - BioMed Central CY - London ER - TY - JOUR A1 - Lämke, Jörn A1 - Brzezinka, Krzysztof A1 - Altmann, Simone A1 - Bäurle, Isabel T1 - A hit-and-run heat shock factor governs sustained histone methylation and transcriptional stress memory JF - The EMBO journal N2 - In nature, plants often encounter chronic or recurring stressful conditions. Recent results indicate that plants can remember a past exposure to stress to be better prepared for a future stress incident. However, the molecular basis of this is poorly understood. Here, we report the involvement of chromatin modifications in the maintenance of acquired thermotolerance (heat stress [HS] memory). HS memory is associated with the accumulation of histone H3 lysine 4 di- and trimethylation at memory-related loci. This accumulation outlasts their transcriptional activity and marks them as recently transcriptionally active. High accumulation of H3K4 methylation is associated with hyper-induction of gene expression upon a recurring HS. This transcriptional memory and the sustained accumulation of H3K4 methylation depend on HSFA2, a transcription factor that is required for HS memory, but not initial heat responses. Interestingly, HSFA2 associates with memory-related loci transiently during the early stages following HS. In summary, we show that transcriptional memory after HS is associated with sustained H3K4 hyper-methylation and depends on a hit-and-run transcription factor, thus providing a molecular framework for HS memory. KW - chromatin KW - H3K4 methylation KW - heat shock transcription factor KW - priming KW - transcriptional memory Y1 - 2016 U6 - https://doi.org/10.15252/embj.201592593 SN - 0261-4189 SN - 1460-2075 VL - 35 SP - 162 EP - 175 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Sellrie, Frank A1 - Beck, Michael A1 - Hildebrandt, Niko A1 - Micheel, Burkhard T1 - A homogeneous time-resolved fluoroimmunoassay (TR-FIA) using antibody mediated luminescence quenching N2 - The determination of low-molecular weight substances (haptens) is demonstrated with a homogeneous time-resolved immunoassay using antibody-induced luminescence quenching. Our novel assay technology uses the newly developed monoclonal antibody (G24-BA9) to quench the luminescence of europium trisbipyridine (EuTBP). We performed a competitive biotin immunoassay including an EuTBP-biotin conjugate, the anti-EuTBP antibody G24-BA9 and streptavidin as assay components. Steric hindrance allows only the binding of either G24-BA9 (to the EuTBP moiety) or streptavidin (to the biotin moiety) to the EuTBP-biotin conjugate. Addition of the analyte biotin resulted in the binding of streptavidin to biotin and a concomitant preferred binding of G24-BA9 to EuTBP-biotin. Since G24-BA9 quenches the luminescence of EuTBP within the conjugate, the luminescence signal could be used to indicate and quantify the presence of free biotin in the system. All experiments were carried out in solution in the presence of 5% serum demonstrating the possibility of using our novel assay for a very fast determination of low molecular weight substances in biological fluids. Y1 - 2010 UR - http://www.rsc.org/Publishing/Journals/AY/Index.asp U6 - https://doi.org/10.1039/C0ay00306a SN - 1759-9660 ER - TY - JOUR A1 - Reeve, Holly A. A1 - Nicholson, Jake A1 - Altaf, Farieha A1 - Lonsdale, Thomas H. A1 - Preissler, Janina A1 - Lauterbach, Lars A1 - Lenz, Oliver A1 - Leimkühler, Silke A1 - Hollmann, Frank A1 - Paul, Caroline E. A1 - Vincent, Kylie A. T1 - A hydrogen-driven biocatalytic approach to recycling synthetic analogues of NAD(P)H JF - Chemical communications : ChemComm N2 - We demonstrate a recycling system for synthetic nicotinamide cofactor analogues using a soluble hydrogenase with turnover number of >1000 for reduction of the cofactor analogues by H-2. Coupling this system to an ene reductase, we show quantitative conversion of N-ethylmaleimide to N-ethylsuccinimide. The biocatalyst system retained >50% activity after 7 h. Y1 - 2022 U6 - https://doi.org/10.1039/d2cc02411j SN - 1359-7345 SN - 1364-548X VL - 58 IS - 75 SP - 10540 EP - 10543 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Kabelitz, Tina A1 - Brzezinka, Krzysztof A1 - Friedrich, Thomas A1 - Gorka, Michal A1 - Graf, Alexander A1 - Kappel, Christian A1 - Bäurle, Isabel T1 - A JUMONJI Protein with E3 Ligase and Histone H3 Binding Activities Affects Transposon Silencing in Arabidopsis JF - Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants N2 - Transposable elements (TEs) make up a large proportion of eukaryotic genomes. As their mobilization creates genetic variation that threatens genome integrity, TEs are epigenetically silenced through several pathways, and this may spread to neighboring sequences. JUMONJI (JMJ) proteins can function as antisilencing factors and prevent silencing of genes next to TEs. Whether TE silencing is counterbalanced by the activity of antisilencing factors is still unclear. Here, we characterize JMJ24 as a regulator of TE silencing. We show that loss of JMJ24 results in increased silencing of the DNA transposon AtMu1c, while overexpression of JMJ24 reduces silencing. JMJ24 has a JumonjiC (JmjC) domain and two RING domains. JMJ24 autoubiquitinates in vitro, demonstrating E3 ligase activity of the RING domain(s). JMJ24-JmjC binds the N-terminal tail of histone H3, and full-length JMJ24 binds histone H3 in vivo. JMJ24 activity is anticorrelated with histone H3 Lys 9 dimethylation (H3K9me2) levels at AtMu1c. Double mutant analyses with epigenetic silencing mutants suggest that JMJ24 antagonizes histone H3K9me2 and requires H3K9 methyltransferases for its activity on AtMu1c. Genome-wide transcriptome analysis indicates that JMJ24 affects silencing at additional TEs. Our results suggest that the JmjC domain of JMJ24 has lost demethylase activity but has been retained as a binding domain for histone H3. This is in line with phylogenetic analyses indicating that JMJ24 (with the mutated JmjC domain) is widely conserved in angiosperms. Taken together, this study assigns a role in TE silencing to a conserved JmjC-domain protein with E3 ligase activity, but no demethylase activity. Y1 - 2016 U6 - https://doi.org/10.1104/pp.15.01688 SN - 0032-0889 SN - 1532-2548 VL - 171 SP - 344 EP - 358 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Tikhonenko, Irina A1 - Magidson, Valentin A1 - Gräf, Ralph A1 - Khodjakov, Alexey A1 - Koonce, Michael P. T1 - A kinesin-mediated mechanism that couples centrosomes to nuclei JF - Cellular and molecular life sciences N2 - The M-type kinesin isoform, Kif9, has recently been implicated in maintaining a physical connection between the centrosome and nucleus in Dictyostelium discoideum. However, the mechanism by which Kif9 functions to link these two organelles remains obscure. Here we demonstrate that the Kif9 protein is localized to the nuclear envelope and is concentrated in the region underlying the centrosome point of attachment. Nuclear anchorage appears mediated through a specialized transmembrane domain located in the carboxyl terminus. Kif9 interacts with microtubules in in vitro binding assays and effects an endwise depolymerization of the polymer. These results suggest a model whereby Kif9 is anchored to the nucleus and generates a pulling force that reels the centrosome up against the nucleus. This is a novel activity for a kinesin motor, one important for progression of cells into mitosis and to ensure centrosome-nuclear parity in a multinuclear environment. KW - Centrosome KW - Kinesin KW - Microtubule KW - Dictyostelium Y1 - 2013 U6 - https://doi.org/10.1007/s00018-012-1205-0 SN - 1420-682X VL - 70 IS - 7 SP - 1285 EP - 1296 PB - Springer CY - Basel ER - TY - JOUR A1 - Kress, H. A1 - Jarrin, A. A1 - Thuroff, E. A1 - Saunders, R. A1 - Weise, C. A1 - Schmidt am Busch, Marcel A1 - Knapp, E. W. A1 - Wedde, M. A1 - Vilcinskas, Andreas T1 - A Kunitz type protease inhibitor related protein is synthesized in Drosophila prepupal salivary glands and released into the moulting fluid during pupation N2 - From the Drosophila virilis late puff region 31C, we microcloned two neighbouring genes, Kil-1 and Kil-2, that encode putative Kunitz serine protease inhibitor like proteins. The Kil-1 gene is expressed exclusively in prepupal salivary glands. Using a size mutant of the KIL-1 protein and MALDI-TOF analysis, we demonstrate that during pupation this protein is released from the prepupal salivary glands into the pupation fluid covering the surface of the pupa. 3-D- structure predictions are consistent with the known crystal structure of the human Kunitz type protease inhibitor 2KNT. This is the first experimental proof for the extra-corporal presence of a distinct Drosophila prepupal salivary gland protein. Possible functions of KIL-1 in the context of the control of proteolytic activities in the pupation fluid are discussed. (C) 2004 Elsevier Ltd. All rights reserved Y1 - 2004 SN - 0965-1748 ER -