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Strong spatiotemporal variation in population size often leads to reduced genetic diversity limiting the adaptive potential of individual populations. Key genes of adaptive variation are encoded by the immune genes of the major histocompatibility complex (MHC) playing an essential role in parasite resistance. How MHC variation persists in rodent populations that regularly experience population bottlenecks remains an important topic in evolutionary genetics. We analysed the consequences of strong population fluctuations on MHC class II DRB exon 2 diversity in two distant common vole (Microtus arvalis) populations in three consecutive years using a high-throughput sequencing approach. In 143 individuals, we detected 25 nucleotide alleles translating into 14 unique amino acid MHC alleles belonging to at least three loci. Thus, the overall allelic diversity and amino acid distance among the remaining MHC alleles, used as a surrogate for the range of pathogenic antigens that can be presented to T-cells, are still remarkably high. Both study populations did not show significant population differentiation between years, but significant differences were found between sites. We concluded that selection processes seem to be strong enough to maintain moderate levels of MHC diversity in our study populations outcompeting genetic drift, as the same MHC alleles were conserved between years. Differences in allele frequencies between populations might be the outcome of different local parasite pressures and/or genetic drift. Further understanding of how pathogens vary across space and time will be crucial to further elucidate the mechanisms maintaining MHC diversity in cyclic populations.
Voltage-gated sodium channels, Nav1, play a crucial role in the generation and propagation of action potentials and substantially contribute to the shape of their rising phase. The electric organ discharge (EOD) of African weakly electric fish (Mormyroidea) is the sum of action potentials fired from all electrocytes of the electric organ at the same time and hence voltage-gated sodium channels are one factor—together with the electrocyte’s morphology and innervation pattern—that determines the properties of these EODs. Due to the fish-specific genome duplication, teleost fish possess eight copies of sodium channel genes (SCN), which encode for Nav1 channels. In mormyroids, SCN4aa is solely expressed in the electrocytes of the adult electric organ. In this study, we compared entire SCN4aa sequences of six species of the genus Campylomormyrus and identified nonsynonymous substitutions among them. SCN4aa in Campylomormyrus exhibits a much higher evolutionary rate compared to its paralog SCN4ab, whose expression is not restricted to the electric organ. We also found evidence for strong positive selection on the SCN4aa gene within Mormyridae and along the lineage ancestral to the Mormyridae. We have identified sites at which all nonelectric teleosts are monomorphic in their amino acid, but mormyrids have different amino acids. Our findings confirm the crucial role of SCN4aa in EOD evolution among mormyrid weakly electric fish. The inferred positive selection within Mormyridae makes this gene a prime candidate for further investigation of the divergent evolution of pulse-type EODs among closely related species.
Ice-rich permafrost coasts in the Arctic are highly sensitive to climate warming and erode at a pace that exceeds the global average. Permafrost coasts deliver vast amounts of organic carbon into the nearshore zone of the Arctic Ocean. Numbers on flux exist for particulate organic carbon (POC) and total or soil organic carbon (TOC, SOC). However, they do not exist for dissolved organic carbon (DOC), which is known to be highly bioavailable. This study aims to estimate DOC stocks in coastal permafrost as well as the annual flux into the ocean. DOC concentrations in ground ice were analyzed along the ice-rich Yukon coast (YC) in the western Canadian Arctic. The annual DOC flux was estimated using available numbers for coast length, cliff height, annual erosion rate, and volumetric ice content in different stratigraphic horizons. Our results showed that DOC concentrations in ground ice range between 0.3 and 347.0mgL(-1) with an estimated stock of 13.63.0gm(-3) along the YC. An annual DOC flux of 54.90.9Mgyr(-1) was computed. These DOC fluxes are low compared to POC and SOC fluxes from coastal erosion or POC and DOC fluxes from Arctic rivers. We conclude that DOC fluxes from permafrost coasts play a secondary role in the Arctic carbon budget. However, this DOC is assumed to be highly bioavailable. We hypothesize that DOC from coastal erosion is important for ecosystems in the Arctic nearshore zones, particularly in summer when river discharge is low, and in areas where rivers are absent.
Bank voles can harbour Puumala virus (PUUV) and vole populations usually peak in years after beech mast. A beech mast occurred in 2014 and a predictive model indicates high vole abundance in 2015. This pattern is similar to the years 2009/2011 when beech mast occurred, bank voles multiplied and human PUUV infections increased a year later. Given similar environmental conditions in 2014/2015, increased risk of human PUUV infections in 2015 is likely. Risk management measures are recommended.
Molecularly imprinted polymers (MIPs) for the recognition of proteins are expected to possess high affinity through the establishment of multiple interactions between the polymer matrix and the large number of functional groups of the target. However, while highly affine recognition sites need building blocks rich in complementary functionalities to their target, such units are likely to generate high levels of nonspecific binding. This paradox, that nature solved by evolution for biological receptors, needs to be addressed by the implementation of new concepts in molecular imprinting of proteins. Additionally, the structural variability, large size and incompatibility with a range of monomers made the development of protein MIPs to take a slow start. While the majority of MIP preparation methods are variants of chemical polymerization, the polymerization of electroactive functional monomers emerged as a particularly advantageous approach for chemical sensing application. Electropolymerization can be performed from aqueous solutions to preserve the natural conformation of the protein templates, with high spatial resolution and electrochemical control of the polymerization process. This review compiles the latest results, identifying major trends and providing an outlook on the perspectives of electrosynthesised protein-imprinted MIPs for chemical sensing. (C) 2016 Elsevier B.V. All rights reserved.
Microelectrodes modified with electropolymerized plumbagin (PLG) were used for the generation of superoxide radical (O-2(center dot-)) and hydrogen peroxide (H2O2) during oxygen reduction reaction (ORR) in an aqueous medium, specifically in serum-free cell culture media. This is enabled by the specific design of a polymer film on the microelectrode. The generation and diffusion of O-2(center dot-) during electrocatalytic ORR at a positionable PLG polymer-modified microelectrode was followed by fluorescence microscopy with the selective dye 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and by amperometric detection using a cytochrome c-modified electrode at + 0.13 V. H2O2 production, either by direct oxygen reduction or as product of O-2(center dot-) disproportionation, was monitored by the reaction with Amplex UltraRed. The PLG polymer-modified microelectrodes were used to expose mammalian B6-RPE07 retinal cells to defined local fluxes of reactive oxygen species (ROS), and cellular responses and morphological alterations were observed. The use of a controllable source of ROS opens many possibilities to study how living cells respond to the presence of a certain flux of specific ROS.
In this dissertation, an electric field-assisted method was developed and applied to achieve immobilization and alignment of biomolecules on metal electrodes in a simple one-step experiment. Neither modifications of the biomolecule nor of the electrodes were needed. The two major electrokinetic effects that lead to molecule motion in the chosen electrode configurations used were identified as dielectrophoresis and AC electroosmotic flow. To minimize AC electroosmotic flow, a new 3D electrode configuration was designed. Thus, the influence of experimental parameters on the dielectrophoretic force and the associated molecule movement could be studied. Permanent immobilization of proteins was examined and quantified absolutely using an atomic force microscope. By measuring the volumes of the immobilized protein deposits, a maximal number of proteins contained therein was calculated. This was possible since the proteins adhered to the tungsten electrodes even after switching off the electric field. The permanent immobilization of functional proteins on surfaces or electrodes is one crucial prerequisite for the fabrication of biosensors.
Furthermore, the biofunctionality of the proteins must be retained after immobilization. Due to the chemical or physical modifications on the proteins caused by immobilization, their biofunctionality is sometimes hampered. The activity of dielectrophoretically immobilized proteins, however, was proven here for an enzyme for the first time. The enzyme horseradish peroxidase was used exemplarily, and its activity was demonstrated with the oxidation of dihydrorhodamine 123, a non-fluorescent precursor of the fluorescence dye rhodamine 123.
Molecular alignment and immobilization - reversible and permanent - was achieved under the influence of inhomogeneous AC electric fields. For orientational investigations, a fluorescence microscope setup, a reliable experimental procedure and an evaluation protocol were developed and validated using self-made control samples of aligned acridine orange molecules in a liquid crystal.
Lambda-DNA strands were stretched and aligned temporarily between adjacent interdigitated electrodes, and the orientation of PicoGreen molecules, which intercalate into the DNA strands, was determined. Similarly, the aligned immobilization of enhanced Green Fluorescent Protein was demonstrated exploiting the protein's fluorescence and structural properties. For this protein, the angle of the chromophore with respect to the protein's geometrical axis was determined in good agreement with X-ray crystallographic data. Permanent immobilization with simultaneous alignment of the proteins was achieved along the edges, tips and on the surface of interdigitated electrodes. This was the first demonstration of aligned immobilization of proteins by electric fields.
Thus, the presented electric field-assisted immobilization method is promising with regard to enhanced antibody binding capacities and enzymatic activities, which is a requirement for industrial biosensor production, as well as for general interaction studies of proteins.
Micro-RNAs are cellular components regulating gene expression at the post-transcription level. In the present study, artificial micro-RNAs were used to decrease the transcript level of two genes, AtExpA8 (encoding an expansin) and AHL25 (encoding an AT-hook motif nuclear localized protein) in Arabidopsis thaliana. The backbone of the Arabidopsis endogenous MIR319a micro-RNA was used in a site-directed mutagenesis approach for the generation of artificial micro-RNAs targeting two genes. The recombinant cassettes were expressed under the control of the CaMV 35S promoter in individual A. thaliana plants. Transgenic lines of the third generation were tested by isolating total RNA and by subsequent cDNA synthesis using oligo-dT18 primers and mRNAs as templates. The expression of the two target genes was checked through quantitative realtime polymerase chain reaction to confirm reduced transcript levels for AtExpA8 and AHL25. Downregulation of AtExpA8 resulted in the formation of short hypocotyls compared with those of the wild-type control in response to low pH and high salt concentration. This technology could be used to prevent the expression of exogenous and invading genes posing a threat to the normal cellular physiology of the host plant.
Background: The efficiency of multiplex editing in plants by the RNA-guided Cas9 system is limited by efficient introduction of its components into the genome and by their activity. The possibility of introducing large fragment deletions by RNA-guided Cas9 tool provides the potential to study the function of any DNA region of interest in its
‘endogenous’ environment.
Results: Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable largefragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression.
Conclusions: Our modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.
Background
The efficiency of multiplex editing in plants by the RNA-guided Cas9 system is limited by efficient introduction of its components into the genome and by their activity. The possibility of introducing large fragment deletions by RNA-guided Cas9 tool provides the potential to study the function of any DNA region of interest in its ‘endogenous’ environment.
Results
Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression.
Conclusions
Our modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.
Results: Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression. Conclusions: Our modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.
Effects of plant community diversity and composition on fungal pathogens in experimental grasslands
(2016)
About a quarter of anthropogenic CO2 emissions are currently taken up by the oceans, decreasing seawater pH. We performed a mesocosm experiment in the Baltic Sea in order to investigate the consequences of increasing CO2 levels on pelagic carbon fluxes. A gradient of different CO2 scenarios, ranging from ambient (similar to 370 mu atm) to high (similar to 1200 mu atm), were set up in mesocosm bags (similar to 55m(3)). We determined standing stocks and temporal changes of total particulate carbon (TPC), dissolved organic carbon (DOC), dissolved inorganic carbon (DIC), and particulate organic carbon (POC) of specific plankton groups. We also measured carbon flux via CO2 exchange with the atmosphere and sedimentation (export), and biological rate measurements of primary production, bacterial production, and total respiration. The experiment lasted for 44 days and was divided into three different phases (I: t0-t16; II: t17-t30; III: t31-t43). Pools of TPC, DOC, and DIC were approximately 420, 7200, and 25 200 mmol Cm-2 at the start of the experiment, and the initial CO2 additions increased the DIC pool by similar to 7% in the highest CO2 treatment. Overall, there was a decrease in TPC and increase of DOC over the course of the experiment. The decrease in TPC was lower, and increase in DOC higher, in treatments with added CO2. During phase I the estimated gross primary production (GPP) was similar to 100 mmol C m(-2) day(-1), from which 75-95% was respired, similar to 1% ended up in the TPC (including export), and 5-25% was added to the DOC pool. During phase II, the respiration loss increased to similar to 100% of GPP at the ambient CO2 concentration, whereas respiration was lower (85-95% of GPP) in the highest CO2 treatment. Bacterial production was similar to 30% lower, on average, at the highest CO2 concentration than in the controls during phases II and III. This resulted in a higher accumulation of DOC and lower reduction in the TPC pool in the elevated CO2 treatments at the end of phase II extending throughout phase III. The "extra" organic carbon at high CO2 remained fixed in an increasing biomass of small-sized plankton and in the DOC pool, and did not transfer into large, sinking aggregates. Our results revealed a clear effect of increasing CO2 on the carbon budget and mineralization, in particular under nutrient limited conditions. Lower carbon loss processes (respiration and bacterial remineralization) at elevated CO2 levels resulted in higher TPC and DOC pools than ambient CO2 concentration. These results highlight the importance of addressing not only net changes in carbon standing stocks but also carbon fluxes and budgets to better disentangle the effects of ocean acidification.
Studies investigating the effect of increasing CO2 levels on the phosphorus cycle in natural waters are lacking although phosphorus often controls phytoplankton development in many aquatic systems. The aim of our study was to analyse effects of elevated CO2 levels on phosphorus pool sizes and uptake. The phosphorus dynamic was followed in a CO2-manipulation mesocosm experiment in the Storfjarden (western Gulf of Finland, Baltic Sea) in summer 2012 and was also studied in the surrounding fjord water. In all mesocosms as well as in surface waters of Storfjarden, dissolved organic phosphorus (DOP) concentrations of 0.26aEuro-+/- aEuro-0.03 and 0.23aEuro-+/- aEuro-0.04aEuro-A mu molaEuro-L-1, respectively, formed the main fraction of the total P-pool (TP), whereas phosphate (PO4) constituted the lowest fraction with mean concentration of 0.15aEuro-A +/- aEuro-0.02 in the mesocosms and 0.17aEuro-A +/- aEuro-0.07aEuro-A mu molaEuro-L-1 in the fjord. Transformation of PO4 into DOP appeared to be the main pathway of PO4 turnover. About 82aEuro-% of PO4 was converted into DOP whereby only 18aEuro-% of PO4 was transformed into particulate phosphorus (PP). PO4 uptake rates measured in the mesocosms ranged between 0.6 and 3.9aEuro-nmolaEuro-L(-1)aEuro-h(-1). About 86aEuro-% of them was realized by the size fraction < aEuro-3aEuro-A mu m. Adenosine triphosphate (ATP) uptake revealed that additional P was supplied from organic compounds accounting for 25-27aEuro-% of P provided by PO4 only. CO2 additions did not cause significant changes in phosphorus (P) pool sizes, DOP composition, and uptake of PO4 and ATP when the whole study period was taken into account. However, significant short-term effects were observed for PO4 and PP pool sizes in CO2 treatments > aEuro-1000aEuro-A mu atm during periods when phytoplankton biomass increased. In addition, we found significant relationships (e.g., between PP and Chl a) in the untreated mesocosms which were not observed under high fCO(2) conditions. Consequently, it can be hypothesized that the relationship between PP formation and phytoplankton growth changed with CO2 elevation. It can be deduced from the results, that visible effects of CO2 on P pools are coupled to phytoplankton growth when the transformation of PO4 into POP was stimulated. The transformation of PO4 into DOP on the other hand does not seem to be affected. Additionally, there were some indications that cellular mechanisms of P regulation might be modified under CO2 elevation changing the relationship between cellular constituents.
The effect of CO2 supply is likely to play an important role in algal ecology. Since inorganic carbon (C-i) acquisition strategies are very diverse among microalgae and C-i availability varies greatly within and among habitats, we hypothesized that C-i acquisition depends on the pH of their preferred natural environment (adaptation) and that the efficiency of C-i uptake is affected by CO2 availability (acclimation). To test this, four species of green algae originating from different habitats were studied. The pH-drift and C-i uptake kinetic experiments were used to characterize C-i acquisition strategies and their ability to acclimate to high and low CO2 conditions and high and low pH was evaluated. Results from pH drift experiments revealed that the acidophile and acidotolerant Chlamydomonas species were mainly restricted to CO2, whereas the two neutrophiles were efficient bicarbonate users. CO2 compensation points in low CO2-acclimated cultures ranged between 0.6 and 1.4 mu M CO2 and acclimation to different culture pH and CO2 conditions suggested that CO2 concentrating mechanisms were present in most species. High CO2 acclimated cultures adapted rapidly to low CO2 condition during pH-drifts. C-i uptake kinetics at different pH values showed that the affinity for C-i was largely influenced by external pH, being highest under conditions where CO2 dominated the C-i pool. In conclusion, C-i acquisition was highly variable among four species of green algae and linked to growth pH preference, suggesting that there is a connection between C-i acquisition and ecological distribution.
Eco-evolutionary dynamics have been shown to be important for understanding population and community stability and their adaptive potential. However, coevolution in the framework of eco-evolutionary theory has not been addressed directly. Combining experiments with an algal host and its viral parasite, and mathematical model analyses we show eco-evolutionary dynamics in antagonistic coevolving populations. The interaction between antagonists initially resulted in arms race dynamics (ARD) with selective sweeps, causing oscillating host-virus population dynamics. However, ARD ended and populations stabilised after the evolution of a general resistant host, whereas a trade-off between host resistance and growth then maintained host diversity over time (trade-off driven dynamics). Most importantly, our study shows that the interaction between ecology and evolution had important consequences for the predictability of the mode and tempo of adaptive change and for the stability and adaptive potential of populations.
Recruitment of European eels (Anguilla anguilla) has declined to the extent that they have been added to the IUCN Red List of Threatened Species. Therefore, it is critical to ensure that eels complete their outward river migration in order to contribute to the available spawning stock. We conducted a 4-year (2007-2011) telemetry study to understand the migratory behaviour and potential impact of environmental factors on the eel during this critical life stage. Out of 399 female eels tagged with acoustic transmitters, only 28% demonstrated clear downstream migratory behaviour. Fifty-five percent were detected exhibiting no downstream migration behaviour and 17% were not detected at any monitoring station. Movement patterns of downstream-migrating (silver) eels were characterized by nocturnal activity and seasonal migration, with distinct peaks in autumn and spring. Migration was often discontinuous and exhibited phases of active locomotion and expanded stopovers. The most important determinants of movement activity were water temperature, cumulative precipitation and moonlight, although the significance varied by season and location in the river basin. Our results evidence a discontinuous, stepwise migration over an extended period. Furthermore, our findings indicate that migration success depends on holding duration prior to tagging and environmental predictors with varying importance depending on the season, as well as the locations of capture, tagging and release. Copyright (c) 2015 John Wiley & Sons, Ltd.
Eusociality is one of the most complex forms of social organization, characterized by cooperative and reproductive units termed colonies. Altruistic behavior of workers within colonies is explained by inclusive fitness, with indirect fitness benefits accrued by helping kin. Members of a social insect colony are expected to be more closely related to one another than they are to other conspecifics. In many social insects, the colony can extend to multiple socially connected but spatially separate nests (polydomy). Social connections, such as trails between nests, promote cooperation and resource exchange, and we predict that workers from socially connected nests will have higher internest relatedness than those from socially unconnected, and noncooperating, nests. We measure social connections, resource exchange, and internest genetic relatedness in the polydomous wood ant Formica lugubris to test whether (1) socially connected but spatially separate nests cooperate, and (2) high internest relatedness is the underlying driver of this cooperation. Our results show that socially connected nests exhibit movement of workers and resources, which suggests they do cooperate, whereas unconnected nests do not. However, we find no difference in internest genetic relatedness between socially connected and unconnected nest pairs, both show high kinship. Our results suggest that neighboring pairs of connected nests show a social and cooperative distinction, but no genetic distinction. We hypothesize that the loss of a social connection may initiate ecological divergence within colonies. Genetic divergence between neighboring nests may build up only later, as a consequence rather than a cause of colony separation.