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Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
Tobias Fromme, Martin Klingenspor
Journal of Biological Engineering , 2007, DOI: 10.1186/1754-1611-1-7
Abstract: We here present a novel benchtop procedure to achieve rapid recombination into any destination vector of choice with the sole requirement of an endonuclease recognition site. The method relies on a specifically designed entry vector and the combined action of type II and type IIs endonucleases with ligase. The formulation leads to accumulation of a single stable cloning product representing the desired insert carrying destination vector.The described method provides a fast single step procedure for routine subcloning from an entry vector into a series of destination vectors with the same restriction enzyme recognition site.One of the most routinely performed tasks in molecular biology labs around the world undoubtedly is the subcloning of a given DNA fragment from one plasmid vector into a different one. The reasons to do so are as numerous and diverse as are the applied methods. We here describe a further such technique involving the orchestrated action of a typeII and a typeIIs endonuclease ("outside cutter") with ligase. This procedure achieves the speed of recombinase based methods without the need for unusual recognition elements in the target vector.In an example experiment we subcloned a 1166 bp long DNA fragment* from an entry vector (modified pGEM-T easy, Promega) into a NheI site of a destination vector (pEGFP-N1, Clontech). The method relies on a specifically engineered entry vector which comprises two key elements flanking the insert to be subcloned (Figure 1A). To test our method we accordingly modified the plasmid pGEM-T easy and included a BglII site to be able to insert a DNA fragment between these elements. A blunt end cutting enzyme to Taq-generate T overhangs for TA cloning of PCR products or any other means to insert a fragment of choice are of course feasible as well. Both of the two identical key elements comprise a recognition sequence for a type IIs restriction endonuclease – Esp3I in our case – and a restriction site (Figure 1B). The latter
A Functional Nexus between Photoperiod Acclimation, Torpor Expression and Somatic Fatty Acid Composition in a Heterothermic Mammal
Fritz Geiser, Martin Klingenspor, Bronwyn M. McAllan
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0063803
Abstract: The seasonal changes in thermal physiology and torpor expression of many heterothermic mammals are controlled by photoperiod. As function at low body temperatures during torpor requires changes of tissue lipid composition, we tested for the first time whether and how fatty acids are affected by photoperiod acclimation in hamsters, Phodopus sungorus, a strongly photoperiodic species. We also examined changes in fatty acid composition in relation to those in morphology and thermal biology. Hamsters in short photoperiod had smaller reproductive organs and most had a reduced body mass in comparison to those in long photoperiod. Pelage colour of hamsters under short photoperiod was almost white while that of long photoperiod hamsters was grey-brown and black. Short photoperiod acclimation resulted in regular (28% of days) torpor use, whereas all hamsters in long photoperiod remained normothermic. The composition of total fatty acids differed between acclimation groups for brown adipose tissue (5 of 8 fatty acids), heart muscle (4 of 7 fatty acids) and leg muscle (3 of 11 fatty acids). Importantly, 54% of all fatty acids detected were correlated (r2 = 0.60 to 0.87) with the minimum surface temperature of individuals, but the responses of tissues differed. While some of the compositional changes of fatty acids were consistent with a ‘homeoviscous’ response, this was not the case for all, including the sums of saturated and unsaturated fatty acids, which did not differ between acclimation groups. Our data identify a possible nexus between photoperiod acclimation, morphology, reproductive biology, thermal biology and fatty acid composition. They suggest that some of the changes in thermal physiology are linked to the composition of tissue and organ fatty acids.
Molecular evolution of UCP1 and the evolutionary history of mammalian non-shivering thermogenesis
David A Hughes, Martin Jastroch, Mark Stoneking, Martin Klingenspor
BMC Evolutionary Biology , 2009, DOI: 10.1186/1471-2148-9-4
Abstract: Models of adaptive evolution through phylogenetic analysis of amino acid sequences by maximum likelihood were implemented to determine the mode of UCP1 protein evolution in Eutherians. An increase in the rate of amino acid substitutions on the branch leading to Eutherians is observed, but is best explained by relaxed constraints, not positive selection. Further, evidence for branch and site heterogeneity in selection pressures, as well as divergent selection pressures between UCP1 and its paralogs (UCP2-3) is observed.We propose that the unique thermogenic function of UCP1 in Eutherians may be best explained by neutral processes. Along with other evidence, this suggests that the primary biochemical properties of UCP1 may not differ between Eutherians and non-Eutherians.Uncoupling protein 1 (UCP1) is a mitochondrial protein carrier which, until recently, was thought to be found only in endothermic placental (Eutherian) mammals [1,2]. In Eutherians, UCP1 is the only gene known to be exclusively expressed in brown adipose tissue (BAT), accounting for up to 5% of the total mitochondrial protein in BAT [3]; UCP1 (also known as thermogenin) provides Eutherians, particularly small mammals, hibernators and newborns, with a unique mechanism of non-shivering thermogenesis (NST) [4]. UCP1-dependent NST is probably a feature of most Eutherian mammals, as it has been found recently in the rock elephant shrew, a member of the Afrotherian mammalian lineage which separated early during the evolution of the Eutherians [5]. NST is produced by increasing the proton conductance in the inner membrane of brown adipocyte mitochondria. This increased proton conductance uncouples mitochondrial respiration from ATP synthesis and thereby dissipates the proton motive force as heat [6-9]. It is the high oxidative capacity of mitochondria in BAT and the cellular composition of BAT that allows heat dissipation rates at a power of 300 – 400 W/kg [10-12]. It is these properties of BAT, their mitoch
Chicken ovalbumin upstream promoter transcription factor II regulates uncoupling protein 3 gene transcription in Phodopus sungorus
Tobias Fromme, Kathrin Reichwald, Matthias Platzer, Xing-Sheng Li, Martin Klingenspor
BMC Molecular Biology , 2007, DOI: 10.1186/1471-2199-8-1
Abstract: By quantitative PCR we demonstrated a positive correlation of Coup-TFII and Ucp3 mRNA expression in skeletal muscle and brown adipose tissue in response to food deprivation and cold exposure, respectively. In reporter gene assays Coup-TFII enhanced transactivation of the Ucp3 promoter conveyed by MyoD, PPARalpha, RXRalpha and/or p300. Using deletions and mutated constructs, we identified a Coup-TFII enhancer element 816–840 bp upstream of the transcriptional start site. Binding of Coup-TFII to this upstream enhancer was confirmed in electrophoretic mobility shift and supershift assays.Transcriptional regulation of the Coup-TFII gene in response to starvation and cold exposure seems to be the regulatory mechanism of Ucp3 mRNA expression in brown adipose and skeletal muscle tissue determining the final appropriate rate of transcript synthesis. These findings add a crucial component to the complex transcriptional machinery controlling expression of Ucp3. Given the substantial evidence for a function of Ucp3 in lipid metabolism, Coup-TFII may not only be a negative regulator of glucose responsive genes but also transactivate genes involved in lipid metabolism.Uncoupling protein 3 (Ucp3) is a member of the family of uncoupling proteins, which are located in the inner mitochondrial membrane and uncouple the respiratory chain from ATP synthesis by dissipating the proton motive force [1,2]. The physiological function of Ucp3 is subject to an ongoing debate [3]. Regulation of Ucp3 expression suggests a role in lipid metabolism. Skeletal muscle Ucp3 transcription is increased in response to food deprivation, a robust mechanism consistently observable in man, rodents and even fish [4]. Further physiological conditions positively regulating Ucp3 include cold exposure [5,6], acute exercise [7] and streptozotocin-induced diabetes [8]. Increased levels of circulating free fatty acids (FFA) are common to all these physiological states; infusion experiments imply that these are the
A Novel SP1/SP3 Dependent Intronic Enhancer Governing Transcription of the UCP3 Gene in Brown Adipocytes
Christoph Hoffmann, Anika Zimmermann, Anke Hinney, Anna-Lena Volckmar, Harry W. Jarrett, Tobias Fromme, Martin Klingenspor
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0083426
Abstract: Uncoupling protein (UCP) 3 is a mitochondrial inner membrane protein implicated in lipid handling and metabolism of reactive oxygen species. Its transcription is mainly regulated by peroxisome proliferator-activated receptors (PPAR), a family of nuclear hormone receptors. Employing bandshift assays, RNA interference and reporter gene assays we examine an intronic region in the UCP3 gene harboring a cis-element essential for expression in brown adipocytes. We demonstrate binding of SP1 and SP3 to this element which is adjacent to a direct repeat 1 element mediating activation of UCP3 expression by PPARγ agonists. Transactivation mediated by these elements is interdependent and indispensable for UCP3 expression. Systematic deletion uncovered a third binding element, a putative NF1 site, in close proximity to the SP1/3 and PPARγ binding elements. Data mining demonstrated binding of MyoD and Myogenin to this third element in C2C12 cells, and, furthermore, revealed recruitment of p300. Taken together, this intronic region is the main enhancer driving UCP3 expression with SP1/3 and PPARγ as the core factors required for expression.
Comparison of particle-exposure triggered pulmonary and systemic inflammation in mice fed with three different diets
Alexander A G?tz, Jan Rozman, Heiko G R?del, Helmut Fuchs, Valérie Gailus-Durner, Martin Hrabě de Angelis, Martin Klingenspor, Tobias Stoeger
Particle and Fibre Toxicology , 2011, DOI: 10.1186/1743-8977-8-30
Abstract: In this study we addressed the question, whether a diet challenge increases the inflammatory response in the alveolar and the blood compartment in response to carbon nanoparticles (CNP), as a surrogate for ambient/urban particulate air pollutants.Mice were fed a high caloric carbohydrate-rich (CA) or a fat-rich (HF) diet for six weeks and were compared to mice kept on a purified low fat (LF) diet, respectively. Bronchoalveolar lavage (BAL) and blood samples were taken 24 h after intratracheal CNP instillation and checked for cellular and molecular markers of inflammation.The high caloric diets resulted in distinct effects when compared with LF mice, respectively: CA resulted in increased body and fat mass without affecting blood cellular immunity. Conversely, HF activated the blood system, increasing lymphocyte and neutrophil counts, and resulted in slightly increased body fat content. In contrast to higher pro-inflammatory BAL Leptin in CA and HF mice, on a cellular level, both diets did not lead to an increased pro-inflammatory basal status in the alveolar compartment per se, nor did result in differences in the particle-triggered response. However both diets resulted in a disturbance of the alveolar capillary barrier as indicated by enhanced BAL protein and lactate-dehydrogenase concentrations. Systemically, reduced serum Adiponectin in HF mice might be related to the observed white blood cell increase.The increase in BAL pro-inflammatory factors in high caloric groups and reductions in serum concentrations of anti-inflammatory factors in HF mice, clearly show diet-specific effects, pointing towards augmented systemic inflammatory conditions. Our data suggest that extended feeding periods, leading to manifest obesity, are necessary to generate an increased susceptibility to particle-induced lung inflammation; although the diet-challenge already was efficient in driving pro-inflammatory systemic events.Obesity and its common sequelae (e.g. type II diabetes and car
High Fat Diet Accelerates Pathogenesis of Murine Crohn’s Disease-Like Ileitis Independently of Obesity
Lisa Gruber, Sigrid Kisling, Pia Lichti, Fran?ois-Pierre Martin, Stephanie May, Martin Klingenspor, Martina Lichtenegger, Michael Rychlik, Dirk Haller
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0071661
Abstract: Background Obesity has been associated with a more severe disease course in inflammatory bowel disease (IBD) and epidemiological data identified dietary fats but not obesity as risk factors for the development of IBD. Crohn’s disease is one of the two major IBD phenotypes and mostly affects the terminal ileum. Despite recent observations that high fat diets (HFD) impair intestinal barrier functions and drive pathobiont selection relevant for chronic inflammation in the colon, mechanisms of high fat diets in the pathogenesis of Crohn’s disease are not known. The aim of this study was to characterize the effect of HFD on the development of chronic ileal inflammation in a murine model of Crohn’s disease-like ileitis. Methods TNFΔARE/WT mice and wildtype C57BL/6 littermates were fed a HFD compared to control diet for different durations. Intestinal pathology and metabolic parameters (glucose tolerance, mesenteric tissue characteristics) were assessed. Intestinal barrier integrity was characterized at different levels including polyethylene glycol (PEG) translocation, endotoxin in portal vein plasma and cellular markers of barrier function. Inflammatory activation of epithelial cells as well as immune cell infiltration into ileal tissue were determined and related to luminal factors. Results HFD aggravated ileal inflammation but did not induce significant overweight or typical metabolic disorders in TNFΔARE/WT. Expression of the tight junction protein Occludin was markedly reduced in the ileal epithelium of HFD mice independently of inflammation, and translocation of endotoxin was increased. Epithelial cells showed enhanced expression of inflammation-related activation markers, along with enhanced luminal factors-driven recruitment of dendritic cells and Th17-biased lymphocyte infiltration into the lamina propria. Conclusions HFD feeding, independently of obesity, accelerated disease onset of small intestinal inflammation in Crohn’s disease-relevant mouse model through mechanisms that involve increased intestinal permeability and altered luminal factors, leading to enhanced dendritic cell recruitment and promoted Th17 immune responses.
Gene Set of Nuclear-Encoded Mitochondrial Regulators Is Enriched for Common Inherited Variation in Obesity
Nadja Knoll, Ivonne Jarick, Anna-Lena Volckmar, Martin Klingenspor, Thomas Illig, Harald Grallert, Christian Gieger, Heinz-Erich Wichmann, Annette Peters, Johannes Hebebrand, André Scherag, Anke Hinney
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0055884
Abstract: There are hints of an altered mitochondrial function in obesity. Nuclear-encoded genes are relevant for mitochondrial function (3 gene sets of known relevant pathways: (1) 16 nuclear regulators of mitochondrial genes, (2) 91 genes for oxidative phosphorylation and (3) 966 nuclear-encoded mitochondrial genes). Gene set enrichment analysis (GSEA) showed no association with type 2 diabetes mellitus in these gene sets. Here we performed a GSEA for the same gene sets for obesity. Genome wide association study (GWAS) data from a case-control approach on 453 extremely obese children and adolescents and 435 lean adult controls were used for GSEA. For independent confirmation, we analyzed 705 obesity GWAS trios (extremely obese child and both biological parents) and a population-based GWAS sample (KORA F4, n = 1,743). A meta-analysis was performed on all three samples. In each sample, the distribution of significance levels between the respective gene set and those of all genes was compared using the leading-edge-fraction-comparison test (cut-offs between the 50th and 95th percentile of the set of all gene-wise corrected p-values) as implemented in the MAGENTA software. In the case-control sample, significant enrichment of associations with obesity was observed above the 50th percentile for the set of the 16 nuclear regulators of mitochondrial genes (pGSEA,50 = 0.0103). This finding was not confirmed in the trios (pGSEA,50 = 0.5991), but in KORA (pGSEA,50 = 0.0398). The meta-analysis again indicated a trend for enrichment (pMAGENTA,50 = 0.1052, pMAGENTA,75 = 0.0251). The GSEA revealed that weak association signals for obesity might be enriched in the gene set of 16 nuclear regulators of mitochondrial genes.
Loss of the Actin Remodeler Eps8 Causes Intestinal Defects and Improved Metabolic Status in Mice
Arianna Tocchetti,Charlotte Blanche Ekalle Soppo,Fabio Zani,Fabrizio Bianchi,Maria Cristina Gagliani,Benedetta Pozzi,Jan Rozman,Ralf Elvert,Nicole Ehrhardt,Birgit Rathkolb,Corinna Moerth,Marion Horsch,Helmut Fuchs,Valérie Gailus-Durner,Johannes Beckers,Martin Klingenspor,Eckhard Wolf,Martin Hrabé de Angelis,Eugenio Scanziani,Carlo Tacchetti,Giorgio Scita,Pier Paolo Di Fiore,Nina Offenh?user
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0009468
Abstract: In a variety of organisms, including mammals, caloric restriction improves metabolic status and lowers the incidence of chronic-degenerative diseases, ultimately leading to increased lifespan.
Dll1 Haploinsufficiency in Adult Mice Leads to a Complex Phenotype Affecting Metabolic and Immunological Processes
Isabel Rubio-Aliaga, Gerhard K. H. Przemeck, Helmut Fuchs, Valérie Gailus-Durner, Thure Adler, Wolfgang Hans, Marion Horsch, Birgit Rathkolb, Jan Rozman, Anja Schrewe, Sibylle Wagner, Sabine M. Hoelter, Lore Becker, Thomas Klopstock, Wolfgang Wurst, Eckhard Wolf, Martin Klingenspor, Boris T. Ivandic, Dirk H. Busch, Johannes Beckers, Martin Hrabé de Angelis
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0006054
Abstract: Background The Notch signaling pathway is an evolutionary conserved signal transduction pathway involved in embryonic patterning and regulation of cell fates during development and self-renewal. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, involving as well other signal transduction pathways, and implicated in distinct human diseases. Delta-like 1 (Dll1) is one of the known ligands of the Notch receptors. The role of the Notch ligands is less well understood. Loss-of-function of Dll1 leads to embryonic lethality, but reduction of Delta-like 1 protein levels has not been studied in adult stage. Methodology/Principal Findings Here we present the haploinsufficient phenotype of Dll1 and a missense mutant Dll1 allele (Dll1C413Y). Haploinsufficiency leads to a complex phenotype with several biological processes altered. These alterations reveal the importance of Dll1 mainly in metabolism, energy balance and in immunology. The animals are smaller, lighter, with altered fat to lean ratio and have increased blood pressure and a slight bradycardia. The animals have reduced cholesterol and triglyceride levels in blood. At the immunological level a subtle phenotype is observed due to the effect and fine-tuning of the signaling network at the different levels of differentiation, proliferation and function of lymphocytes. Moreover, the importance of the proteolytic regulation of the Notch signaling network emphasized. Conclusions/Significance In conclusion, slight alterations in one player of Notch signaling alter the entire organism, emphasizing the fine-tuning character of this pathway in a high number of processes.
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