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Publications - Cell and Metabolic Biology

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Displaying results 251 to 260 of 478.

Publications

Engler, C.; Gruetzner, R.; Kandzia, R.; Marillonnet, S.; Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes PLOS ONE 4, e5553, (2009) DOI: 10.1371/journal.pone.0005553

We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.
Publications

Ehrlich, H.; Hanke, T.; Frolov, A.; Langrock, T.; Hoffmann, R.; Fischer, C.; Schwarzenbolz, U.; Henle, T.; Born, R.; Worch, H.; Modification of collagen in vitro with respect to formation of Nɛ-carboxymethyllysine Int. J. Biol. Macromol. 44, 51-56, (2009) DOI: 10.1016/j.ijbiomac.2008.10.001

Developing new biopolymer-based materials with bio-identical properties is a significant challenge in modern science. One interesting route to this goal involves the biomineralization of collagen, a pre-structured and widely available protein, into a material with interesting properties. A prerequisite for biomineralization is the ability of cations (e.g., calcium) to bind to the protein and to result in concert with appropriate anions (e.g., phosphate) in composite material with e.g., bone-like properties. In order to increase the number of binding sites it is necessary to modify the protein prior to mineralization. For this glucuronic acid (GA) was used due to its carbonyl and carboxyl groups to derivatize proteinogenic amino groups transferring them into negatively charged carboxyl groups. Our experiments showed for the first time, that Nɛ-carboxymethyllysine is the major product of in vitro non-enzymatic glycosylation of collagen by glucuronic acid. For an unequivocal determination of the reaction products, the lysine residues of collagen and of the model peptide were carboxymethylated through a reductive alkylation with glyoxalic acid and compared to the glucuronic acid derivatives. Beside their identical mass spectra the common structure elements could be confirmed with FTIR. Thus, in the context of matrix engineering, by producing Nɛ-carboxymethyllysine, glucuronic acid offers a convenient way of introducing additional stable acidic groups into protein matrices.
Publications

Ehrlich, H.; Hanke, T.; Born, R.; Fischer, C.; Frolov, A.; Langrock, T.; Hoffmann, R.; Schwarzenbolz, U.; Henle, T.; Simon, P.; Geiger, D.; Bazhenov, V. V.; Worch, H.; Mineralization of biomimetically carboxymethylated collagen fibrils in a model dual membrane diffusion system J. Membr. Sci. 326, 254-259, (2009) DOI: 10.1016/j.memsci.2008.10.003

In the present work, we show for the first time, that Nɛ-carboxymethyllysine is the major product of the in vitro non-enzymatic glycation reaction between fibrillar collagen and glucuronic acid. Dual diffusion membrane system was effectively used for oriented crystal growth of octacalcium phosphate/hydroxyapatite on the biomimetically carboxymethylated collagen fibrils. We hypothesize that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions in such a way that a critical nucleus of ions can be formed, leading to the formation of the mineral under specific micro-environment conditions achieved by using diffusion membrane system.
Publications

Lukačin, R.; Matern, U.; Hehmann, M.; Specker, S.; Vogt, T.; Corrigendum to “Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus” [FEBS Lett. 577 (2004) 367-370] FEBS Lett. 583, 855-855, (2009) DOI: 10.1016/j.febslet.2009.01.050

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Publications

Hohnjec, N.; Lenz, F.; Fehlberg, V.; Vieweg, M. F.; Baier, M. C.; Hause, B.; Küster, H.; The Signal Peptide of the Medicago truncatula Modular Nodulin MtNOD25 Operates as an Address Label for the Specific Targeting of Proteins to Nitrogen-Fixing Symbiosomes Mol. Plant Microbe Interact. 22, 63-72, (2009) DOI: 10.1094/MPMI-22-1-0063

The nodule-specific MtNOD25 gene of the model legume Medicago truncatula encodes a modular nodulin composed of different repetitive modules flanked by distinct N- and C-termini. Although similarities are low with respect to all repetitive modules, both the N-terminal signal peptide (SP) and the C-terminus are highly conserved in modular nodulins from different legumes. On the cellular level, MtNOD25 is only transcribed in the infected cells of root nodules, and this activation is mediated by a 299-bp minimal promoter containing an organ-specific element. By expressing mGFP6 translational fusions in transgenic nodules, we show that MtNOD25 proteins are exclusively translocated to the symbiosomes of infected cells. This specific targeting only requires an N-terminal MtNOD25 SP that is highly conserved across a family of legume-specific symbiosome proteins. Our finding sheds light on one possible mechanism for the delivery of host proteins to the symbiosomes of infected root nodule cells and, in addition, defines a short molecular address label of only 24 amino acids whose N-terminal presence is sufficient to translocate proteins across the peribacteroid membrane.
Books and chapters

Frolov, A.; Singer, D.; Zauner, T.; Hoffmann, R.; Solid Phase Synthesis and Analysis of Amadori Peptides Adv. Exp. Med. Biol. 611, 423-424, (2009) ISBN: 978-0-387-73657-0 DOI: 10.1007/978-0-387-73657-0_182

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Books and chapters

Dorka, R.; Miersch, O.; Hause, B.; Weik, P.; Wasternack, C.; Chronobiologische Phänomene und Jasmonatgehalt bei Viscum album L. 49-66, (2009)

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Publications

Weier, D.; Mittasch, J.; Strack, D.; Milkowski, C.; The genes BnSCT1 and BnSCT2 from Brassica napus encoding the final enzyme of sinapine biosynthesis: molecular characterization and suppression Planta 227, 375-385, (2008) DOI: 10.1007/s00425-007-0624-x

This study describes the molecular characterization of the genes BnSCT1 and BnSCT2 from oilseed rape (Brassica napus) encoding the enzyme 1-O-sinapoyl-β-glucose:choline sinapoyltransferase (SCT; EC 2.3.1.91). SCT catalyzes the 1-O-β-acetal ester-dependent biosynthesis of sinapoylcholine (sinapine), the most abundant phenolic compound in seeds of B. napus. GUS fusion experiments indicated that seed specificity of BnSCT1 expression is caused by an inducible promoter confining transcription to embryo tissues and the aleurone layer. A dsRNAi construct designed to silence seed-specifically the BnSCT1 gene was effective in reducing the sinapine content of Arabidopsis seeds thus defining SCT genes as targets for molecular breeding of low sinapine cultivars of B. napus. Sequence analyses revealed that in the allotetraploid genome of B. napus the gene BnSCT1 represents the C genome homologue from the B. oleracea progenitor whereas BnSCT2 was derived from the Brassica A genome of B. rapa. The BnSCT1 and BnSCT2 loci showed colinearity with the homologous Arabidopsis SNG2 gene locus although the genomic microstructure revealed the deletion of a cluster of three genes and several coding regions in the B. napus genome.
Publications

Wadenbäck, J.; von Arnold, S.; Egertsdotter, U.; Walter, M. H.; Grima-Pettenati, J.; Goffner, D.; Gellerstedt, G.; Gullion, T.; Clapham, D.; Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR) Transgenic Res. 17, 379-392, (2008) DOI: 10.1007/s11248-007-9113-z

An attractive objective in tree breeding is to reduce the content of lignin or alter its composition, in order to facilitate delignification in pulping. This has been achieved in transgenic angiosperm tree species. In this study we show for the first time that changes in lignin content and composition can be achieved in a conifer by taking a transgenic approach. Lignin content and composition have been altered in five-year-old transgenic plants of Norway spruce (Picea abies [L.] Karst) expressing the Norway spruce gene encoding cinnamoyl CoA reductase (CCR) in antisense orientation. The asCCR plants had a normal phenotype but smaller stem widths compared to the transformed control plants. The transcript abundance of the sense CCR gene was reduced up to 35% relative to the transformed control. The corresponding reduction in lignin content was up to 8%, which is at the lower limit of the 90–99% confidence intervals reported for natural variation. The contribution of H-lignin to the non-condensed fraction of lignin, as judged by thioacidolysis, was reduced up to 34%. The H-lignin content was strongly correlated with the total lignin content. Furthermore, the kappa number of small-scale Kraft pulps from one of the most down-regulated lines was reduced 3.5%. The transcript abundances of the various lignin biosynthetic genes were down-regulated indicating co-regulation of the biosynthetic pathway.
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