TY - GEN A1 - Arvidsson, Samuel Janne A1 - Kwasniewski, Miroslaw A1 - Riaño- Pachón, Diego Mauricio A1 - Mueller-Roeber, Bernd T1 - QuantPrime BT - a flexible tool for reliable high-throughput primer design for quantitative PCR T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Background Medium- to large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays. Results Here we present QuantPrime, an intuitive and user-friendly, fully automated tool for primer pair design in small- to large-scale qPCR analyses. QuantPrime can be used online through the internet http://www.quantprime.de/ or on a local computer after download; it offers design and specificity checking with highly customizable parameters and is ready to use with many publicly available transcriptomes of important higher eukaryotic model organisms and plant crops (currently 295 species in total), while benefiting from exon-intron border and alternative splice variant information in available genome annotations. Experimental results with the model plant Arabidopsis thaliana, the crop Hordeum vulgare and the model green alga Chlamydomonas reinhardtii show success rates of designed primer pairs exceeding 96%. Conclusion QuantPrime constitutes a flexible, fully automated web application for reliable primer design for use in larger qPCR experiments, as proven by experimental data. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative in situ hybridization. Future suggestions made by users can be easily implemented, thus allowing QuantPrime to be developed into a broad-range platform for the design of RNA expression assays. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 943 KW - prime pair KW - genome annotation KW - specific prime pair KW - primer pair design KW - quantification protocol Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431531 SN - 1866-8372 IS - 943 ER - TY - GEN A1 - Yang, Lei A1 - Tang, Renjie A1 - Zhu, Jinqi A1 - Liu, Hua A1 - Mueller-Roeber, Bernd A1 - Xia, Huijun A1 - Zhang, Hongxia T1 - Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Inositol phosphates (IPs) and their turnover products have been implicated to play important roles in stress signaling in eukaryotic cells. In higher plants genes encoding inositol polyphosphate kinases have been identified previously, but their physiological functions have not been fully resolved. Here we expressed Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2 beta) in two heterologous systems, i.e. the yeast Saccharomyces cerevisiae and in tobacco (Nicotiana tabacum), and tested the effect on abiotic stress tolerance. Expression of AtIpk2 beta rescued the salt-, osmotic- and temperature-sensitive growth defects of a yeast mutant strain (arg82 Delta) that lacks inositol polyphosphate multikinase activity encoded by the ARG82/IPK2 gene. Transgenic tobacco plants constitutively expressing AtIpk2 beta under the control of the Cauliflower Mosaic Virus 35S promoter were generated and found to exhibit improved tolerance to diverse abiotic stresses when compared to wild type plants. Expression patterns of various stress responsive genes were enhanced, and the activities of anti-oxidative enzymes were elevated in transgenic plants, suggesting a possible involvement of AtIpk2 beta in plant stress responses. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 954 KW - arabidopsis thaliana KW - AtIpk2 beta KW - inositol phosphate KW - IP3 KW - stress tolerance KW - transgenic tobacco Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431225 SN - 1866-8372 IS - 954 ER - TY - GEN A1 - Scarpeci, Telma E. A1 - Zanor, María I. A1 - Carrillo, Néstor A1 - Mueller-Roeber, Bernd A1 - Valle, Estela M. T1 - Generation of superoxide anion in chloroplasts of Arabidopsis thaliana during active photosynthesis BT - a focus on rapidly induced genes T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe N2 - The antioxidant defense system involves complex functional coordination of multiple components in different organelles within the plant cell. Here, we have studied the Arabidopsis thaliana early response to the generation of superoxide anion in chloroplasts during active photosynthesis. We exposed plants to methyl viologen (MV), a superoxide anion propagator in the light, and performed biochemical and expression profiling experiments using Affymetrix ATH1 GeneChip(R) microarrays under conditions in which photosynthesis and antioxidant enzymes were active. Data analysis identified superoxide-responsive genes that were compared with available microarray results. Examples include genes encoding proteins with unknown function, transcription factors and signal transduction components. A common GAAAAGTCAAAC motif containing the W-box consensus sequence of WRKY transcription factors, was found in the promoters of genes highly up-regulated by superoxide. Band shift assays showed that oxidative treatments enhanced the specific binding of leaf protein extracts to this motif. In addition, GUS reporter gene fused to WRKY30 promoter, which contains this binding motif, was induced by MV and H2O2. Overall, our study suggests that genes involved in signalling pathways and with unknown functions are rapidly activated by superoxide anion generated in photosynthetically active chloroplasts, as part of the early antioxidant response of Arabidopsis leaves. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 866 KW - antioxidant response KW - chloroplast KW - Hsp KW - oxidative stress KW - WRKY Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-434254 SN - 1866-8372 IS - 866 SP - 361 EP - 378 ER - TY - JOUR A1 - Kupsch, Andreas A1 - Mueller, Bernd R. A1 - Lange, Axel A1 - Bruno, Giovanni T1 - Microstructure characterisation of ceramics via 2D and 3D X-ray refraction techniques JF - Journal of the European Ceramic Society N2 - 3D imaging techniques are very fashionable nowadays, and allow enormous progress in understanding ceramic microstructure, its evolution, and its link to mechanical, thermal, and transport properties. In this feature article, we report the use of a powerful, yet not so wide-spread, set of X-ray techniques based on refraction effects. X-ray refraction allows determining internal specific surface (surface per unit volume) in a non-destructive fashion, position and orientation sensitive, and with a nanometric detectability. While the techniques are limited by the X-ray absorption of the material under investigation, we demonstrate showcases of ceramics and composite materials, where understanding of process parameter influence or simply of microstructural parameters could be achieved in a way unrivalled even by high-resolution techniques such as electron microscopy or computed tomography. (C) 2016 Elsevier Ltd. All rights reserved. KW - X-ray refraction KW - Porosity KW - Specific surface KW - Crack detection KW - Composites Y1 - 2017 U6 - https://doi.org/10.1016/j.jeurceramsoc.2016.12.031 SN - 0955-2219 SN - 1873-619X VL - 37 SP - 1879 EP - 1889 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Mack, Daniel Emil A1 - Laquai, Rene A1 - Mueller, Bernd A1 - Helle, Oliver A1 - Sebold, Doris A1 - Vassen, Robert A1 - Bruno, Giovanni T1 - Evolution of porosity, crack density, and CMAS penetration in thermal barrier coatings subjected to burner rig testing JF - Journal of the American Ceramic Society N2 - Degradation of thermal barrier coatings (TBCs) in gas-turbine engines due to calcium-magnesium-aluminosilicate (CMAS) glassy deposits from various sources has been a persistent issue since many years. In this study, state of the art electron microscopy was correlated with X-ray refraction techniques to elucidate the intrusion of CMAS into the porous structure of atmospheric plasma sprayed (APS) TBCs and the formation and growth of cracks under thermal cycling in a burner rig. Results indicate that the sparse nature of the infiltration as well as kinetics in the burner rig are majorly influenced by the wetting behavior of the CMAS. Despite the obvious attack of CMAS on grain boundaries, the interaction of yttria-stabilized zirconia (YSZ) with intruded CMAS has no immediate impact on structure and density of internal surfaces. At a later stage the formation of horizontal cracks is observed in a wider zone of the TBC layer. KW - characterization KW - CMAS KW - synchrotron X-ray refraction radiography KW - thermal barrier coatings Y1 - 2019 U6 - https://doi.org/10.1111/jace.16465 SN - 0002-7820 SN - 1551-2916 VL - 102 IS - 10 SP - 6163 EP - 6175 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Gisin, Jonathan A1 - Mueller, Alexandra A1 - Pfaender, Yvonne A1 - Leimkühler, Silke A1 - Narberhaus, Franz A1 - Masepohl, Bernd T1 - A Rhodobacter capsulatus member of a universal permease family imports molybdate and other oxyanions N2 - Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family. Y1 - 2010 UR - http://jb.asm.org/ U6 - https://doi.org/10.1128/Jb.00742-10 SN - 0021-9193 ER - TY - JOUR A1 - Wiethaus, Jessica A1 - Mueller, Alexandra A1 - Neumann, Meina A1 - Neumann, Sandra A1 - Leimkühler, Silke A1 - Narberhaus, Franz A1 - Masepohl, Bernd T1 - Specific interactions between four Molybdenum-binding proteins contribute to Mo-dependent gene regulation in Rhodobacter capsulatus N2 - The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA- binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop-lacZ fusion in mutant strains defective for either mopA, mopB, or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression. Y1 - 2009 UR - http://jb.asm.org/ U6 - https://doi.org/10.1128/Jb.00526-09 SN - 0021-9193 ER - TY - JOUR A1 - Messerschmidt, Katrin A1 - Hochrein, Lena A1 - Dehm, Daniel A1 - Schulz, Karina A1 - Mueller-Roeber, Bernd T1 - Characterizing seamless ligation cloning extract for synthetic biological applications JF - Analytical biochemistry : methods in the biological sciences N2 - Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular. and synthetic biology projects. (C) 2016 Elsevier Inc. All rights reserved. KW - SLiCE KW - Seamless ligation cloning KW - Homologous recombination KW - Synthetic biology Y1 - 2016 U6 - https://doi.org/10.1016/j.ab.2016.05.029 SN - 0003-2697 SN - 1096-0309 VL - 509 SP - 24 EP - 32 PB - Elsevier CY - San Diego ER - TY - JOUR A1 - Hochrein, Lena A1 - Machens, Fabian A1 - Gremmels, Juergen A1 - Schulz, Karina A1 - Messerschmidt, Katrin A1 - Mueller-Roeber, Bernd T1 - AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies JF - Nucleic acids research N2 - The assembly of large DNA constructs coding for entire pathways poses a major challenge in the field of synthetic biology. Here, we present AssemblX, a novel, user-friendly and highly efficient multi-gene assembly strategy. The software-assisted AssemblX process allows even unexperienced users to rapidly design, build and test DNA constructs with currently up to 25 functional units, from 75 or more subunits. At the gene level, AssemblX uses scar-free, overlap-based and sequence-independent methods, allowing the unrestricted design of transcriptional units without laborious parts domestication. The assembly into multi-gene modules is enabled via a standardized, highly efficient, polymerase chain reaction-free and virtually sequence-independent scheme, which relies on rare cutting restriction enzymes and optimized adapter sequences. Selection and marker switching strategies render the whole process reliable, rapid and very effective. The assembly product can be easily transferred to any desired expression host, making AssemblX useful for researchers from various fields. Y1 - 2017 U6 - https://doi.org/10.1093/nar/gkx034 SN - 0305-1048 SN - 1362-4962 VL - 45 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Stobiecki, Maciej A1 - Skirycz, Aleksandra A1 - Kerhoas, L. A1 - Kachlicki, P. A1 - Muth, D. A1 - Einhorn, J. A1 - Mueller-Roeber, Bernd T1 - Profiling of phenolic glycosidic conjugates in leaves of Arabidopsis thaliana using LC/MS JF - Metabolomics : the official journal of the Metabolomics Society N2 - Profiling of plant secondary metabolites is still a very difficult task. Liquid chromatography (LC) or capillary electrophoresis hyphenated with different kinds of detectors are methods of choice for analysis of polar, thermo labile compounds with high molecular masses. We demonstrate the applicability of LC combined with UV diode array or/and mass spectrometric detectors for the unambiguous identification and quantification of flavonoid conjugates isolated from Arahidopsis thaliana leaves of different genotypes and grown in different environmental conditions. During LC/UV/MS/MS analyses we were able to identify tetra-, tri, and di-glycosides of kaempferol, quercetin and isorhamnetin. Based on our results we can conclude that due to the co-elution of different chemical compounds in reversed phase H PLC systems the application of UV detectors does not allow to precisely profile all flavonoid conjugates existing in A. thaliana genotypes. Using MS detection it was possible to unambiguously recognize the glycosylation patterns of the aglycones. However, from the mass spectra we could not conclude neither the anomeric form of the C-1 carbon atoms of sugar moieties in glycosidic bonds between sugars or sugar and aglycone nor the position of the second carbon involved in disaccharides. The applicability of collision induced dissociation techniques (CID MS/MS) for structural analyses of the studied group of plant secondary metabolites with two types of analyzers (triple quadrupole or ion trap) was demonstrated. KW - liquid chromatography-mass spectrometry KW - metabolite profiling KW - metabolomics KW - flavonoid glycosides Y1 - 2006 U6 - https://doi.org/10.1007/s11306-006-0031-5 SN - 1573-3882 VL - 2 SP - 197 EP - 219 PB - Springer CY - New York ER -