570 Biowissenschaften; Biologie
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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.
The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5-10 codons of protein-coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate.
TRAPID
(2013)
Transcriptome analysis through next-generation sequencing technologies allows the generation of detailed gene catalogs for non-model species, at the cost of new challenges with regards to computational requirements and bioinformatics expertise. Here, we present TRAPID, an online tool for the fast and efficient processing of assembled RNA-Seq transcriptome data, developed to mitigate these challenges. TRAPID offers high-throughput open reading frame detection, frameshift correction and includes a functional, comparative and phylogenetic toolbox, making use of 175 reference proteomes. Benchmarking and comparison against state-of-the-art transcript analysis tools reveals the efficiency and unique features of the TRAPID system. TRAPID is freely available at http://bioinformatics.psb.ugent.be/webtools/trapid/.
Background: The linear noise approximation (LNA) is commonly used to predict how noise is regulated and exploited at the cellular level. These predictions are exact for reaction networks composed exclusively of first order reactions or for networks involving bimolecular reactions and large numbers of molecules. It is however well known that gene regulation involves bimolecular interactions with molecule numbers as small as a single copy of a particular gene. It is therefore questionable how reliable are the LNA predictions for these systems.
Results: We implement in the software package intrinsic Noise Analyzer (iNA), a system size expansion based method which calculates the mean concentrations and the variances of the fluctuations to an order of accuracy higher than the LNA. We then use iNA to explore the parametric dependence of the Fano factors and of the coefficients of variation of the mRNA and protein fluctuations in models of genetic networks involving nonlinear protein degradation, post-transcriptional, post-translational and negative feedback regulation. We find that the LNA can significantly underestimate the amplitude and period of noise-induced oscillations in genetic oscillators. We also identify cases where the LNA predicts that noise levels can be optimized by tuning a bimolecular rate constant whereas our method shows that no such regulation is possible. All our results are confirmed by stochastic simulations.
Conclusion: The software iNA allows the investigation of parameter regimes where the LNA fares well and where it does not. We have shown that the parametric dependence of the coefficients of variation and Fano factors for common gene regulatory networks is better described by including terms of higher order than LNA in the system size expansion. This analysis is considerably faster than stochastic simulations due to the extensive ensemble averaging needed to obtain statistically meaningful results. Hence iNA is well suited for performing computationally efficient and quantitative studies of intrinsic noise in gene regulatory networks.
Background: Short lived, iteroparous animals in seasonal environments experience variable social and environmental conditions over their lifetime. Animals can be divided into those with a "young-of-the-year" life history (YY, reproducing and dying in the summer of birth) and an "overwinter" life history (OW, overwintering in a subadult state before reproducing next spring).
We investigated how behavioural patterns across the population were affected by season and sex, and whether variation in behaviour reflects the variation in life history patterns of each season. Applications of pace-of-life (POL) theory would suggest that long-lived OW animals are shyer in order to increase survival, and YY are bolder in order to increase reproduction. Therefore, we expected that in winter and spring samples, when only OW can be sampled, the animals should be shyer than in summer and autumn, when both OW and YY animals can be sampled. We studied common vole (Microtus arvalis) populations, which express typical, intra-annual density fluctuation. We captured a total of 492 voles at different months over 3 years and examined boldness and activity level with two standardised behavioural experiments.
Results: Behavioural variables of the two tests were correlated with each other. Boldness, measured as short latencies in both tests, was extremely high in spring compared to other seasons. Activity level was highest in spring and summer, and higher in males than in females.
Conclusion: Being bold in laboratory tests may translate into higher risk-taking in nature by being more mobile while seeking out partners or valuable territories. Possible explanations include asset-protection, with OW animals being rather old with low residual reproductive value in spring. Therefore, OW may take higher risks during this season. Offspring born in spring encounter a lower population density and may have higher reproductive value than offspring of later cohorts. A constant connection between life history and animal personality, as suggested by the POL theory, however, was not found. Nevertheless, correlations of traits suggest the existence of animal personalities. In conclusion, complex patterns of population dynamics, seasonal variation in life histories, and variability of behaviour due to asset-protection may cause complex seasonal behavioural dynamics in a population.
Background: Adaptive behavioural strategies promoting co-occurrence of competing species are known to result from a sympatric evolutionary past. Strategies should be different for indirect resource competition (exploitation, e.g., foraging and avoidance behaviour) than for direct interspecific interference (e.g., aggression, vigilance, and nest guarding). We studied the effects of resource competition and nest predation in sympatric small mammal species using semi-fossorial voles and shrews, which prey on vole offspring during their sensitive nestling phase. Experiments were conducted in caged outdoor enclosures. Focus common vole mothers (Microtus arvalis) were either caged with a greater white-toothed shrew (Crocidura russula) as a potential nest predator, with an herbivorous field vole (Microtus agrestis) as a heterospecific resource competitor, or with a conspecific resource competitor.
Results: We studied behavioural adaptations of vole mothers during pregnancy, parturition, and early lactation, specifically modifications of the burrow architecture and activity at burrow entrances. Further, we measured pre- and postpartum faecal corticosterone metabolites (FCMs) of mothers to test for elevated stress hormone levels. Only in the presence of the nest predator were prepartum FCMs elevated, but we found no loss of vole nestlings and no differences in nestling body weight in the presence of the nest predator or the heterospecific resource competitor. Although the presence of both the shrew and the field vole induced prepartum modifications to the burrow architecture, only nest predators caused an increase in vigilance time at burrow entrances during the sensitive nestling phase.
Conclusion: Voles displayed an adequate behavioural response for both resource competitors and nest predators. They modified burrow architecture to improve nest guarding and increased their vigilance at burrow entrances to enhance offspring survival chances. Our study revealed differential behavioural adaptations to resource competitors and nest predators.
Movement of organisms is one of the key mechanisms shaping biodiversity, e.g. the distribution of genes, individuals and species in space and time. Recent technological and conceptual advances have improved our ability to assess the causes and consequences of individual movement, and led to the emergence of the new field of ‘movement ecology’. Here, we outline how movement ecology can contribute to the broad field of biodiversity research, i.e. the study of processes and patterns of life among and across different scales, from genes to ecosystems, and we propose a conceptual framework linking these hitherto largely separated fields of research. Our framework builds on the concept of movement ecology for individuals, and demonstrates its importance for linking individual organismal movement with biodiversity. First, organismal movements can provide ‘mobile links’ between habitats or ecosystems, thereby connecting resources, genes, and processes among otherwise separate locations. Understanding these mobile links and their impact on biodiversity will be facilitated by movement ecology, because mobile links can be created by different modes of movement (i.e., foraging, dispersal, migration) that relate to different spatiotemporal scales and have differential effects on biodiversity. Second, organismal movements can also mediate coexistence in communities, through ‘equalizing’ and ‘stabilizing’ mechanisms. This novel integrated framework provides a conceptual starting point for a better understanding of biodiversity dynamics in light of individual movement and space-use behavior across spatiotemporal scales. By illustrating this framework with examples, we argue that the integration of movement ecology and biodiversity research will also enhance our ability to conserve diversity at the genetic, species, and ecosystem levels.
Introduction: We examined patterns of genetic divergence in 26 Mediterranean populations of the semi-terrestrial beachflea Orchestia montagui using mitochondrial (cytochrome oxidase subunit I), microsatellite (eight loci) and allozymic data. The species typically forms large populations within heaps of dead seagrass leaves stranded on beaches at the waterfront. We adopted a hierarchical geographic sampling to unravel population structure in a species living at the sea-land transition and, hence, likely subjected to dramatically contrasting forces.
Results: Mitochondrial DNA showed historical phylogeographic breaks among Adriatic, Ionian and the remaining basins (Tyrrhenian, Western and Eastern Mediterranean Sea) likely caused by the geological and climatic changes of the Pleistocene. Microsatellites (and to a lesser extent allozymes) detected a further subdivision between and within the Western Mediterranean and the Tyrrhenian Sea due to present-day processes. A pattern of isolation by distance was not detected in any of the analyzed data set.
Conclusions: We conclude that the population structure of O. montagui is the result of the interplay of two contrasting forces that act on the species population genetic structure. On one hand, the species semi-terrestrial life style would tend to determine the onset of local differences. On the other hand, these differences are partially counter-balanced by passive movements of migrants via rafting on heaps of dead seagrass leaves across sites by sea surface currents. Approximate Bayesian Computations support dispersal at sea as prevalent over terrestrial regionalism.
Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells
(2013)
Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system.