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 - 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 - CHAP A1 - Tikhonenko, I. A1 - Magidson, V. A1 - Gräf, Ralph A1 - Khodjakov, A. A1 - Koonce, M. T1 - A kinesin-mediated linkage between centrosomes and the nuclear envelope T2 - Molecular biology of the cell : the official publication of the American Society for Cell Biology Y1 - 2011 SN - 1059-1524 VL - 22 IS - 1 PB - American Society for Cell Biology CY - Bethesda 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 - 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 - 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 - 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 - GEN 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 T2 - Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 745 KW - Outcrossing KW - Mixed mating KW - Outcrossing rate KW - Capsella KW - Amplicon sequencing Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-435657 SN - 1866-8372 IS - 745 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 - 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 -