@misc{ArvidssonKwasniewskiRianoPachonetal.2008, author = {Arvidsson, Samuel Janne and Kwasniewski, Miroslaw and Ria{\~n}o- Pach{\´o}n, Diego Mauricio and Mueller-Roeber, Bernd}, title = {QuantPrime}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {943}, issn = {1866-8372}, doi = {10.25932/publishup-43153}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-431531}, pages = {17}, year = {2008}, abstract = {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.}, language = {en} } @article{BalazadehKwasniewskiCaldanaetal.2011, author = {Balazadeh, Salma and Kwasniewski, Miroslaw and Caldana, Camila and Mehrnia, Mohammad and Zanor, Maria Ines and Xue, Gang-Ping and M{\"u}ller-R{\"o}ber, Bernd}, title = {ORS1, an H2O2-Responsive NAC Transcription Factor, Controls Senescence in Arabidopsis thaliana}, series = {Molecular plant}, volume = {4}, journal = {Molecular plant}, number = {2}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {1674-2052}, doi = {10.1093/mp/ssq080}, pages = {346 -- 360}, year = {2011}, abstract = {We report here that ORS1, a previously uncharacterized member of the NAC transcription factor family, controls leaf senescence in Arabidopsis thaliana. Overexpression of ORS1 accelerates senescence in transgenic plants, whereas its inhibition delays it. Genes acting downstream of ORS1 were identified by global expression analysis using transgenic plants producing dexamethasone-inducible ORS1-GR fusion protein. Of the 42 up-regulated genes, 30 (similar to 70\%) were previously shown to be up-regulated during age-dependent senescence. We also observed that 32 (similar to 76\%) of the ORS1-dependent genes were induced by long-term (4 d), but not short-term (6 h) salinity stress (150 mM NaCl). Furthermore, expression of 16 and 24 genes, respectively, was induced after 1 and 5 h of treatment with hydrogen peroxide (H2O2), a reactive oxygen species known to accumulate during salinity stress. ORS1 itself was found to be rapidly and strongly induced by H2O2 treatment in both leaves and roots. Using in vitro binding site selection, we determined the preferred binding motif of ORS1 and found it to be present in half of the ORS1-dependent genes. ORS1 is a paralog of ORE1/ANAC092/AtNAC2, a previously reported regulator of leaf senescence. Phylogenetic footprinting revealed evolutionary conservation of the ORS1 and ORE1 promoter sequences in different Brassicaceae species, indicating strong positive selection acting on both genes. We conclude that ORS1, similarly to ORE1, triggers expression of senescence-associated genes through a regulatory network that may involve cross-talk with salt- and H2O2-dependent signaling pathways.}, language = {en} } @article{WinckKwasniewskiWienkoopetal.2011, author = {Winck, Flavia Vischi and Kwasniewski, Miroslaw and Wienkoop, Stefanie and M{\"u}ller-R{\"o}ber, Bernd}, title = {An optimized method for the isolation of nuclei from chlamydomas Reinhardtii (Chlorophyceae)}, series = {Journal of phycology}, volume = {47}, journal = {Journal of phycology}, number = {2}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {0022-3646}, doi = {10.1111/j.1529-8817.2011.00967.x}, pages = {333 -- 340}, year = {2011}, abstract = {The cell nucleus harbors a large number of proteins involved in transcription, RNA processing, chromatin remodeling, nuclear signaling, and ribosome assembly. The nuclear genome of the model alga Chlamydomonas reinhardtii P. A. Dang. was recently sequenced, and many genes encoding nuclear proteins, including transcription factors and transcription regulators, have been identified through computational discovery tools. However, elucidating the specific biological roles of nuclear proteins will require support from biochemical and proteomics data. Cellular preparations with enriched nuclei are important to assist in such analyses. Here, we describe a simple protocol for the isolation of nuclei from Chlamydomonas, based on a commercially available kit. The modifications done in the original protocol mainly include alterations of the differential centrifugation parameters and detergent-based cell lysis. The nuclei-enriched fractions obtained with the optimized protocol show low contamination with mitochondrial and plastid proteins. The protocol can be concluded within only 3 h, and the proteins extracted can be used for gel-based and non-gel-based proteomic approaches.}, language = {en} }