Genome-wide investigation reveals high evolutionary rates in annual model plants

Background: ;Rates of molecular evolution vary widely among species. While significant deviations from molecular clock have been found in many taxa, effects of life histories on molecular evolution are not fully understood. In plants, annual/perennial life history traits have long been suspected to influence the evolutionary rates at the molecular level. To date, however, the number of genes investigated on this subject is limited and the conclusions are mixed. To evaluate the possible heterogeneity in evolutionary rates between annual and perennial plants at the genomic level, we investigated 85 nuclear housekeeping genes, 10 non-housekeeping families, and 34 chloroplast;genes using the genomic data from model plants including Arabidopsis thaliana and Medicago truncatula for annuals and grape (Vitis vinifera) and popular (Populus trichocarpa) for perennials.;Results: ;According to the cross-comparisons among the four species, 74-82% of the nuclear genes and 71-97% of the chloroplast genes suggested higher rates of molecular evolution in the two annuals than those in the two perennials. The significant heterogeneity in evolutionary rate between annuals and perennials was consistently found both in nonsynonymous sites and synonymous sites. While a linear correlation of evolutionary rates in orthologous genes between species was observed in nonsynonymous sites, the correlation was weak or invisible in synonymous sites. This tendency was clearer in nuclear genes than in chloroplast genes, in which the overall;evolutionary rate was small. The slope of the regression line was consistently lower than unity, further confirming the higher evolutionary rate in annuals at the genomic level.;Conclusions: ;The higher evolutionary rate in annuals than in perennials appears to be a universal phenomenon both in nuclear and chloroplast genomes in the four dicot model plants we investigated. Therefore, such heterogeneity in evolutionary rate should result from factors that have genome-wide influence, most likely those associated with annual/perennial life history. Although we acknowledge current limitations of this kind of study, mainly due to a small sample size available and a distant taxonomic relationship of the model organisms, our results indicate that the genome-wide survey is a promising approach toward further understanding of the;mechanism determining the molecular evolutionary rate at the genomic level.

Eukaryotic translation elongation factor 1A (eEF1A) domain I from S. cerevisiae is required but not sufficient for inter-species complementation

Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisiae.

Concentration- and time-dependent effects of the synthetic estrogen, 17alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio

Partial or full life-cycle tests are needed to assess the potential of endocrine-disrupting compounds (EDCs) to adversely affect development and reproduction of fish. Small fish species such as zebrafish, Danio rerio, are under consideration as model organisms for appropriate test protocols. The present study examines how reproductive effects resulting from exposure of zebrafish to the synthetic estrogen 17alpha-ethinylestradiol (EE2) vary with concentration (0.05 to 10 ng EE2 L(-1), nominal), and with timing/duration of exposure (partial life-cycle, full life-cycle, and two-generation exposure). Partial life-cycle exposure of the parental (F1) generation until completion of gonad differentiation (0-75 d postfertilization, dpf) impaired juvenile growth, time to sexual maturity, adult fecundity (egg production/female/day), and adult fertilization success at 1.1 ng EE2 L(-1) and higher. Lifelong exposure of the F1 generation until 177 dpf resulted in lowest observed effect concentrations (LOECs) for time to sexual maturity, fecundity, and fertilization success identical to those of the developmental test (0-75 dpf), but the slope of the concentration-response curve was steeper. Reproduction of zebrafish was completely inhibited at 9.3 ng EE2 L(-1), and this was essentially irreversible as a 3-mo depuration restored fertilization success to only a very low rate. Accordingly, elevated endogenous vitellogenin (VTG) synthesis and degenerative changes in gonad morphology persisted in depurated zebrafish. Full life-cycle exposure of the filial (F2) generation until 162 dpf impaired growth, delayed onset of spawning and reduced fecundity and fertilization success at 2.0 ng EE2 L(-1). In conclusion, results show that the impact of estrogenic agents on zebrafish sexual development and reproductive functions as well as the reversibility of effects, varies with exposure concentration (reversibility at < or = 1.1 ng EE2 L(-1) and irreversibility at 9.3 ng EE2 L(-1)), and between partial and full life-cycle exposure (exposure to 10 ng EE2 L(-1) during critical period exerted no permanent effect on sexual differentiation, but life-cycle exposure did).

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

The ribosome as novel target for small regulatory RNAs

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. Furthermore it is strongly assumed that the primordial translation system in the ‘RNA world’ most likely received direct regulatory input from ncRNA-like cofactors. The fundamental question that we would like to ask is: Does the ‘RNA world still communicate’ with the ribosome? To address this question, we have analyzed the small ncRNA interactomes of ribosomes of prokaryotic (H. volcanii, S. aureus) and unicellular eukaryotic model organisms. Deep-sequencing and subsequent bioinformatic analyses revealed thousands of putative ribosome-associated ncRNAs. For a subset of these ncRNA candidates we have gathered experimental evidence that they are expressed in a stress-dependent manner and indeed directly target the ribosome. In the archaeon H. volcanii a tRNA-derived fragment was identified to target the small ribosomal subunit upon alkaline stress in vitro and in vivo. As a consequence of ribosome binding, this tRNA-fragment reduces protein synthesis by interfering with the peptidyl transferase activity. Our data reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life. Ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory sRNAs.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. To investigate whether such a class of regulatory ncRNAs does exist we performed genomic screens for small ribosome-associated RNAs in various model organisms of all three domains [1,2]. Here we focus on the functional characterisation of an 18 nucleotide long ncRNA candidate derived from an open reading frame (ORF) of an annotated S. cerevisiae gene, which encodes a tRNA methyltransferase. Yeast cells lacking this tRNA methyltransferase showed clear growth defects in high salt containing media. Genetic analysis showed that the absence of the mRNA-derived ncRNA rather than the absence of the tRNA methyltransferase activity is responsible for the observed phenotype. Since we performed a screen for small ribosome-associated RNAs we examined the regulatory potential of the synthetic 18mer during translation in vitro and in vivo. Metabolic labeling experiments in the presence of the synthetic 18mer RNA revealed an inhibitory potential on the global protein biosynthesis rate. In vitro translation and northern blot analysis further strengthen the hypothesis, that this RNA is a ribosome-associated regulatory ncRNA. Our studies in pro- and eukaryotic model organisms reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life. Ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory ncRNAs.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNA (ncRNA) molecules are key players in controlling gene expression at multiple steps in all domains of life. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily (such as micro RNAs and small-interfering RNAs), not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). This is unexpected, given the central position the ribosome plays during gene expression. To investigate whether such a class of regulatory ncRNAs does exist we performed genomic screens for small ribosome-associated RNAs in various model organisms of all three domains [1,2]. Here we show that an mRNA-derived 18 nucleotide long ncRNA is capable of down-regulating translation in Saccharomyces cerevisiae by directly targeting the ribosome [3]. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence, rather than the mRNA-encoded enzyme, as the translation regulator under these stress conditions. Our data reveal the ribosome as a target for small regulatory ncRNAs and unveil the existence of a novel mechanism of translation regulation. Analogous genomic screens in organisms spanning all three domains of life demonstrate the existence of thousands of ncRNA candidates putatively regulating the ribosome. We therefore anticipate that ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory ncRNAs.

Temperate Mountain Forest Biodiversity under Climate Change: Compensating Negative Effects by Increasing Structural Complexity

Species adapted to cold-climatic mountain environments are expected to face a high risk of range contractions, if not local extinctions under climate change. Yet, the populations of many endothermic species may not be primarily affected by physiological constraints, but indirectly by climate-induced changes of habitat characteristics. In mountain forests, where vertebrate species largely depend on vegetation composition and structure, deteriorating habitat suitability may thus be mitigated or even compensated by habitat management aiming at compositional and structural enhancement. We tested this possibility using four cold-adapted bird species with complementary habitat requirements as model organisms. Based on species data and environmental information collected in 300 1-km2 grid cells distributed across four mountain ranges in central Europe, we investigated (1) how species’ occurrence is explained by climate, landscape, and vegetation, (2) to what extent climate change and climate-induced vegetation changes will affect habitat suitability, and (3) whether these changes could be compensated by adaptive habitat management. Species presence was modelled as a function of climate, landscape and vegetation variables under current climate; moreover, vegetation-climate relationships were assessed. The models were extrapolated to the climatic conditions of 2050, assuming the moderate IPCC-scenario A1B, and changes in species’ occurrence probability were quantified. Finally, we assessed the maximum increase in occurrence probability that could be achieved by modifying one or multiple vegetation variables under altered climate conditions. Climate variables contributed significantly to explaining species occurrence, and expected climatic changes, as well as climate-induced vegetation trends, decreased the occurrence probability of all four species, particularly at the low-altitudinal margins of their distribution. These effects could be partly compensated by modifying single vegetation factors, but full compensation would only be achieved if several factors were changed in concert. The results illustrate the possibilities and limitations of adaptive species conservation management under climate change.

The transcriptome of equine peripheral blood mononuclear cells.

Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To improve the horse genome annotation we performed RNA-seq on 561 samples of peripheral blood mononuclear cells (PBMCs) derived from 85 Warmblood horses. The mapped sequencing reads were used to build a new transcriptome assembly. The new assembly revealed many alternative isoforms associated to known genes or to those predicted by the Ensembl and/or Gnomon pipelines. We also identified 7,531 transcripts not associated with any horse gene annotated in public databases. Of these, 3,280 transcripts did not have a homologous match to any sequence deposited in the NCBI EST database suggesting horse specificity. The unknown transcripts were categorized as coding and noncoding based on predicted coding potential scores. Among them 230 transcripts had high coding potential score, at least 2 exons, and an open reading frame of at least 300 nt. We experimentally validated 9 new equine coding transcripts using RT-PCR and Sanger sequencing. Our results provide valuable detailed information on many transcripts yet to be annotated in the horse genome.

The ribosome as novel target for stress-induced small regulatory RNAs

Small non-protein-coding RNA (ncRNA) molecules represent major contributors to regulatory networks in controlling gene expression in a highly efficient manner. All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. To address the question, whether small ncRNA regulators exist that are capable of modulating the rate of protein production by directly interacting with the ribosome, we have analyzed the small ncRNA interactomes of ribosomes Deep-sequencing and subsequent bioinformatic analyses revealed thousands of putative ribosome-associated ncRNAs in various model organisms (1,2). For a subset of these ncRNA candidates we have gathered experimental evidence that they associate with ribosomes in a stress-dependent manner and are capable of regulating gene expression by fine-tuning the rate of protein biosynthesis (3,4). Many of the investigated ribosome-bound small ncRNA appear to be processing products from larger functional RNAs, such as tRNAs (2,3) or mRNAs (3). Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. Our data reveal the ribosome as a target for small regulatory ncRNAs and demonstrate the existence of a yet unknown mechanism of translation regulation. Ribosome-associated ncRNAs (rancRNAs) are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules (5). Future work on the small ncRNA interactomes of ribosomes in a variety of model systems will allow deeper insight into the conservation and functional repertoire of this emerging class of regulatory ncRNA molecules.

Ribosome-associated ncRNAs (rancRNAs) An emerging class of translation regulators

Small non-protein-coding RNA (ncRNA) molecules represent major contributors to regulatory networks in controlling gene expression in a highly efficient manner. Most of the recently discovered regulatory ncRNAs acting on translation target the mRNA rather than the ribosome (e.g.: miRNAs, siRNAs, antisense RNAs). To address the question, whether ncRNA regulators exist that are capable of modulating the rate of protein production by directly interacting with the ribosome, we have analyzed the small ncRNA interactomes of ribosomes. Deep-sequencing analyses revealed thousands of putative rancRNAs in various model organisms (1,2). For a subset of these ncRNA candidates we have gathered experimental evidence that they associate with ribosomes in a stress-dependent manner and fine-tune the rate of protein biosynthesis (3,4). Many of the investigated rancRNAs appear to be processing products of larger functional RNAs, such as tRNAs (2,3), mRNAs (3), or snoRNAs (2). Post-transcriptional cleavage of RNA to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. Our data disclose the ribosome as target for small regulatory RNAs. rancRNAs are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules (5). Ongoing work in our lab revealed first insight into rancRNA processing and mechanism of this emerging class of translation regulators.

2C-Cas9: a versatile tool for clonal analysis of gene function

CRISPR/Cas9-mediated targeted mutagenesis allows efficient generation of loss-of-function alleles in zebrafish. To date this technology has been primarily used to generate genetic knockout animals. Nevertheless, the study of the function of certain loci might require tight spatiotemporal control of gene inactivation. Here, we show that tissue-specific gene disruption can be achieved by driving Cas9 expression with the Gal4/UAS system. Furthermore, by combining the Gal4/UAS and Cre/loxP systems, we establish a versatile tool to genetically label mutant cell clones, enabling their phenotypic analysis. Our technique has the potential to be applied to diverse model organisms, enabling tissue-specific loss-of-function and phenotypic characterization of live and fixed tissues.

Citizen science reveals unexpected continental-scale evolutionary change in a model organism

Organisms provide some of the most sensitive indicators of climate change and evolutionary responses are becoming apparent in species with short generation times. Large datasets on genetic polymorphism that can provide an historical benchmark against which to test for recent evolutionary responses are very rare, but an exception is found in the brown-lipped banded snail (Cepaea nemoralis). This species is sensitive to its thermal environment and exhibits several polymorphisms of shell colour and banding pattern affecting shell albedo in the majority of populations within its native range in Europe. We tested for evolutionary changes in shell albedo that might have been driven by the warming of the climate in Europe over the last half century by compiling an historical dataset for 6,515 native populations of C. nemoralis and comparing this with new data on nearly 3,000 populations. The new data were sampled mainly in 2009 through the Evolution MegaLab, a citizen science project that engaged thousands of volunteers in 15 countries throughout Europe in the biggest such exercise ever undertaken. A known geographic cline in the frequency of the colour phenotype with the highest albedo (yellow) was shown to have persisted and a difference in colour frequency between woodland and more open habitats was confirmed, but there was no general increase in the frequency of yellow shells. This may have been because snails adapted to a warming climate through behavioural thermoregulation. By contrast, we detected an unexpected decrease in the frequency of Unbanded shells and an increase in the Mid-banded morph. Neither of these evolutionary changes appears to be a direct response to climate change, indicating that the influence of other selective agents, possibly related to changing predation pressure and habitat change with effects on micro-climate.

Pharmacodynamics of antibiotics in the therapy of meningitis: infection model observations

Detailed studies of pharmacodynamic principles relevant to the therapy of bacterial meningitis are difficult to perform in man, while the rabbit model of bacterial meningitis has proved to be extremely valuable and has led to insights that appear relevant for the treatment of humans. Most importantly in the light of the restricted penetration of antibiotics into the CSF, animal studies have shown that in meningitis there is a dose-response curve between the CSF concentrations achieved by antibiotics and their bactericidal activity. This appears to be true for all classes of antibiotics thus far examined, including the beta-lactams, which do not show such a dose-response behaviour in other infections. Only CSF concentrations that exceed the MBC of the infecting organism by at least 10-30-fold achieve consistent and rapid bactericidal activity. Such rapid bactericidal activity is a requirement for successful therapy with beta-lactams and can be impaired with certain antibiotics by the specific conditions in infected CSF (protein content; acidic pH; slow-growing bacteria). However, rapid antibiotic killing of the infecting organisms may not be without adverse effects either. Some antibiotics, particularly beta-lactams lead to the brisk liberation of bacterial cell wall components (e.g. endotoxin, in the case of Gram-negative organisms) which have an inflammatory effect on the host and can lead to a temporary deterioration of the disease. Dexamethasone, when administered with the antibiotic, can prevent some of the adverse effects of rapid bacterial lysis.