TY - GEN A1 - Baumann, Tobias A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Background: DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. Results: A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions. Conclusions: The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 983 KW - cohesive ends KW - DNA cleavage KW - genetic vectors KW - modified primers KW - molecular methods KW - polymerase chain reaction KW - recombinant Escherichia coli KW - restriction enzymes Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431085 SN - 1866-8372 IS - 983 ER - TY - JOUR A1 - Baumann, Tobias A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V JF - BMC biotechnology N2 - Background: DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. Results: A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions. Conclusions: The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed. KW - Cohesive ends KW - DNA cleavage KW - Genetic vectors KW - Modified primers KW - Molecular methods KW - Polymerase chain reaction KW - Recombinant Escherichia coli KW - Restriction enzymes Y1 - 2013 U6 - https://doi.org/10.1186/1472-6750-13-81 SN - 1472-6750 VL - 13 IS - 10 PB - BioMed Central CY - London ER - TY - JOUR A1 - Speck, Janina A1 - Räuber, Christina A1 - Kükenshöner, Tim A1 - Niemöller, Christoph A1 - Mueller, Katelyn J. A1 - Schleberger, Paula A1 - Dondapati, Padmarupa A1 - Hecky, Jochen A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment complementation JF - Protein engineering design & selection N2 - The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric proteinprotein interactions enable TAT-dependent translocation of the resistance marker TEM -lactamase (L). Specifically, we demonstrate and analyze selection of (i) enhancers for folding by direct TAT translocation selection of a target protein interposed between the TorA signal sequence and L, (ii) dimeric or oligomeric proteinprotein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the L, respectively and (iii) heterotrimeric proteinprotein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split L fragments and TorA, respectively. The lactamase fragments were additionally engineered for improved activity and stability. Applicability was benchmarked with interaction partners of known affinity and multimerization whereby cellular fitness correlated well with biophysical protein properties. Ultimately, the HiT selection was employed to identify peptides, which specifically bind to leukemia- and melanoma-relevant target proteins (MITF and ETO) by coiled-coil or tetra-helix-bundle formation with high affinity. The various versions of TAT selection led to inhibiting peptides (iPEPs) of disease-promoting interactions and enabled so far difficult to achieve selections. KW - HiT selection KW - NHR2 KW - TAT selection KW - three hybrid KW - two hybrid Y1 - 2013 U6 - https://doi.org/10.1093/protein/gzs098 SN - 1741-0126 VL - 26 IS - 3 SP - 225 EP - 242 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Jedrusik-Bode, Monika A1 - Studencka, Maja A1 - Smolka, Christian A1 - Baumann, Tobias A1 - Schmidt, Henning A1 - Kampf, Jan A1 - Paap, Franziska A1 - Martin, Sophie A1 - Tazi, Jamal A1 - Müller, Kristian M. A1 - Krüger, Marcus A1 - Braun, Thomas A1 - Bober, Eva T1 - The sirtuin SIRT6 regulates stress granule formation in C. elegans and mammals JF - Journal of cell science N2 - SIRT6 is a NAD(+)-dependent deacetylase that modulates chromatin structure and safeguards genomic stability. Until now, SIRT6 has been assigned to the nucleus and only nuclear targets of SIRT6 are known. Here, we demonstrate that in response to stress, C. elegans SIR-2.4 and its mammalian orthologue SIRT6 localize to cytoplasmic stress granules, interact with various stress granule components and induce their assembly. Loss of SIRT6 or inhibition of its catalytic activity in mouse embryonic fibroblasts impairs stress granule formation and delays disassembly during recovery, whereas deficiency of SIR-2.4 diminishes maintenance of P granules and decreases survival of C. elegans under stress conditions. Our findings uncover a novel, evolutionary conserved function of SIRT6 in the maintenance of stress granules in response to stress. KW - C. elegans KW - G3BP KW - SIRT6 KW - Sirtuins KW - Stress KW - Stress granules Y1 - 2013 U6 - https://doi.org/10.1242/jcs.130708 SN - 0021-9533 SN - 1477-9137 VL - 126 IS - 22 SP - 5166 EP - + PB - Company of Biologists Limited CY - Cambridge ER - TY - JOUR A1 - Klingstrom, Tomas A1 - Soldatova, Larissa A1 - Stevens, Robert A1 - Roos, T. Erik A1 - Swertz, Morris A. A1 - Müller, Kristian M. A1 - Kalas, Matus A1 - Lambrix, Patrick A1 - Taussig, Michael J. A1 - Litton, Jan-Eric A1 - Landegren, Ulf A1 - Bongcam-Rudloff, Erik T1 - Workshop on laboratory protocol standards for the molecular methods database JF - New biotechnology N2 - Management of data to produce scientific knowledge is a key challenge for biological research in the 21st century. Emerging high-throughput technologies allow life science researchers to produce big data at speeds and in amounts that were unthinkable just a few years ago. This places high demands on all aspects of the workflow: from data capture (including the experimental constraints of the experiment), analysis and preservation, to peer-reviewed publication of results. Failure to recognise the issues at each level can lead to serious conflicts and mistakes; research may then be compromised as a result of the publication of non-coherent protocols, or the misinterpretation of published data. In this report, we present the results from a workshop that was organised to create an ontological data-modelling framework for Laboratory Protocol Standards for the Molecular Methods Database (MolMeth). The workshop provided a set of short- and long-term goals for the MolMeth database, the most important being the decision to use the established EXACT description of biomedical ontologies as a starting point. Y1 - 2013 U6 - https://doi.org/10.1016/j.nbt.2012.05.019 SN - 1871-6784 VL - 30 IS - 2 SP - 109 EP - 113 PB - Elsevier CY - Amsterdam ER -