Although technical advances in genomics and proteomics research have yielded a better understanding of the coding capacity of a genome, one major challenge remaining is the identification of all expressed proteins, especially those less than 100 amino acids in length. Such information can be particularly relevant to human pathogens, such as Trypanosoma brucei, the causative agent of African trypanosomiasis, since it will provide further insight into the parasite biology and life cycle.
Starting with 993 T. brucei transcripts, previously shown by RNA-Sequencing not to coincide with annotated coding sequences (CDS), homology searches revealed that 173 predicted short open reading frames in these transcripts are conserved across kinetoplastids with 13 also conserved in representative eukaryotes. Mining mass spectrometry data sets revealed 42 transcripts encoding at least one matching peptide. RNAi-induced down-regulation of these 42 transcripts revealed seven to be essential in insect-form trypanosomes with two also required for the bloodstream life cycle stage. To validate the specificity of the RNAi results, each lethal phenotype was rescued by co-expressing an RNAi-resistant construct of each corresponding CDS. These previously non-annotated essential small proteins localized to a variety of cell compartments, including the cell surface, mitochondria, nucleus and cytoplasm, inferring the diverse biological roles they are likely to play in T. brucei. We also provide evidence that one of these small proteins is required for replicating the kinetoplast (mitochondrial) DNA.
Our studies highlight the presence and significance of small proteins in a protist and expose potential new targets to block the survival of trypanosomes in the insect vector and/or the mammalian host.
Genomics; Proteomics; Mass spectrometry data; Non-coding RNA; Mitochondria
A study in BMC Evolutionary Biology represents the most comprehensive effort to clarify the phylogeny of green plants using sequences from the plastid genome. This study highlights the strengths and limitations of plastome data for resolving the green plant phylogeny, and points toward an exciting future for plant phylogenetics, during which the vast and largely untapped territory of nuclear genomes will be explored.
The Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. The cellular function of the mammalian members of this family, the Nm23 proteins, has not yet been clearly defined. Previous awd genetic analyses unraveled its endocytic role that is required for proper internalization of receptors controlling different signaling pathways. In this study, we analyzed the role of Awd in controlling Notch signaling during development.
To study the awd gene function we used genetic mosaic approaches to obtain cells homozygous for a loss of function allele. In awd mutant follicle cells and wing disc cells, Notch accumulates in enlarged early endosomes, resulting in defective Notch signaling. Our results demonstrate that awd function is required before γ-secretase mediated cleavage since over-expression of the constitutively active form of the Notch receptor in awd mutant follicle cells allows rescue of the signaling. By using markers of different endosomal compartments we show that Notch receptor accumulates in early endosomes in awd mutant follicle cells. A trafficking assay in living wing discs also shows that Notch accumulates in early endosomes. Importantly, constitutively active Rab5 cannot rescue the awd phenotype, suggesting that awd is required for Rab5 function in early endosome maturation.
In this report we demonstrate that awd is essential for Notch signaling via its endocytic role. In addition, we identify the endocytic step at which Awd function is required for Notch signaling and we obtain evidence indicating that Awd is necessary for Rab5 function. These findings provide new insights into the developmental and pathophysiological function of this important gene family.
Awd; Notch signaling; Endocytosis
Among the most common pollutants that enter the environment after passing municipal wastewater treatment are estrogens, especially the synthetic 17α-ethinylestradiol that is used in oral contraceptives. Estrogens are potent endocrine disruptors at concentrations frequently observed in surface waters. However, new genetic analyses suggest that some fish populations can be self-sustaining even in heavily polluted waters. We now need to understand the basis of this tolerance.
See research article: http://www.biomedcentral.com/1741-7007/12/1
Planktonic ciliated larvae are characteristic for the life cycle of marine invertebrates. Their most prominent feature is the apical organ harboring sensory cells and neurons of largely undetermined function. An elucidation of the relationships between various forms of primary larvae and apical organs is key to understanding the evolution of animal life cycles. These relationships have remained enigmatic due to the scarcity of comparative molecular data.
To compare apical organs and larval body patterning, we have studied regionalization of the episphere, the upper hemisphere of the trochophore larva of the marine annelid Platynereis dumerilii. We examined the spatial distribution of transcription factors and of Wnt signaling components previously implicated in anterior neural development. Pharmacological activation of Wnt signaling with Gsk3β antagonists abolishes expression of apical markers, consistent with a repressive role of Wnt signaling in the specification of apical tissue. We refer to this Wnt-sensitive, six3- and foxq2-expressing part of the episphere as the ‘apical plate’. We also unraveled a molecular signature of the apical organ - devoid of six3 but expressing foxj, irx, nkx3 and hox - that is shared with other marine phyla including cnidarians. Finally, we characterized the cell types that form part of the apical organ by profiling by image registration, which allows parallel expression profiling of multiple cells. Besides the hox-expressing apical tuft cells, this revealed the presence of putative light- and mechanosensory as well as multiple peptidergic cell types that we compared to apical organ cell types of other animal phyla.
The similar formation of a six3+, foxq2+ apical plate, sensitive to Wnt activity and with an apical tuft in its six3-free center, is most parsimoniously explained by evolutionary conservation. We propose that a simple apical organ - comprising an apical tuft and a basal plexus innervated by sensory-neurosecretory apical plate cells - was present in the last common ancestors of cnidarians and bilaterians. One of its ancient functions would have been the control of metamorphosis. Various types of apical plate cells would then have subsequently been added to the apical organ in the divergent bilaterian lineages. Our findings support an ancient and common origin of primary ciliated larvae.
Apical-blastoporal axis; Apical organ; Body plan; Larval evolution
The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host’s liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood.
Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite’s glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin.
Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs.
Cestode; Tapeworm; Echinococcus; Echinococcosis; Insulin; Receptor kinase; Kinase inhibitor; Host-parasite interaction
Atomic Force Microscopy (AFM) is a polyvalent tool that allows biological and mechanical studies of full living microorganisms, and therefore the comprehension of molecular mechanisms at the nanoscale level. By combining AFM with genetical and biochemical methods, we explored the biophysical response of the yeast Saccharomyces cerevisiae to a temperature stress from 30°C to 42°C during 1 h.
We report for the first time the formation of an unprecedented circular structure at the cell surface that takes its origin at a single punctuate source and propagates in a concentric manner to reach a diameter of 2–3 μm at least, thus significantly greater than a bud scar. Concomitantly, the cell wall stiffness determined by the Young’s Modulus of heat stressed cells increased two fold with a concurrent increase of chitin. This heat-induced circular structure was not found either in wsc1Δ or bck1Δ mutants that are defective in the CWI signaling pathway, nor in chs1Δ, chs3Δ and bni1Δ mutant cells, reported to be deficient in the proper budding process. It was also abolished in the presence of latrunculin A, a toxin known to destabilize actin cytoskeleton.
Our results suggest that this singular morphological event occurring at the cell surface is due to a dysfunction in the budding machinery caused by the heat shock and that this phenomenon is under the control of the CWI pathway.
Atomic Force Microscopy (AFM); Saccharomyces cerevisiae; Heat-shock; Cell wall; Chitin; Budding
In the first weeks of vertebrate postnatal life, neural networks in the visual thalamus undergo activity-dependent refinement thought to be important for the development of functional vision. This process involves pruning of synaptic connections between retinal ganglion cells and excitatory thalamic neurons that relay signals on to visual areas of the cortex. A recent report in Neural Development shows that this does not occur in inhibitory neurons, questioning our current understanding of the development of mature neural circuits.
See research article: http://www.neuraldevelopment.com/content/8/1/24
At the beginning of the transcription process, the RNA polymerase (RNAP) core enzyme requires a σ-factor to recognize the genomic location at which the process initiates. Although the crucial role of σ-factors has long been appreciated and characterized for many individual promoters, we do not yet have a genome-scale assessment of their function.
Using multiple genome-scale measurements, we elucidated the network of σ-factor and promoter interactions in Escherichia coli. The reconstructed network includes 4,724 σ-factor-specific promoters corresponding to transcription units (TUs), representing an increase of more than 300% over what has been previously reported. The reconstructed network was used to investigate competition between alternative σ-factors (the σ70 and σ38 regulons), confirming the competition model of σ substitution and negative regulation by alternative σ-factors. Comparison with σ-factor binding in Klebsiella pneumoniae showed that transcriptional regulation of conserved genes in closely related species is unexpectedly divergent.
The reconstructed network reveals the regulatory complexity of the promoter architecture in prokaryotic genomes, and opens a path to the direct determination of the systems biology of their transcriptional regulatory networks.
Escherichia coli; Sigma factor; Network reconstruction; Comparative analysis; Klebsiella pneumoniae; Omics data; Systems biology
Treated effluents from wastewater treatment works can comprise a large proportion of the flow of rivers in the developed world. Exposure to these effluents, or the steroidal estrogens they contain, feminizes wild male fish and can reduce their reproductive fitness. Long-term experimental exposures have resulted in skewed sex ratios, reproductive failures in breeding colonies, and population collapse. This suggests that environmental estrogens could threaten the sustainability of wild fish populations.
Here we tested this hypothesis by examining population genetic structures and effective population sizes (Ne) of wild roach (Rutilus rutilus L.) living in English rivers contaminated with estrogenic effluents. Ne was estimated from DNA microsatellite genotypes using approximate Bayesian computation and sibling assignment methods. We found no significant negative correlation between Ne and the predicted estrogen exposure at 28 sample sites. Furthermore, examination of the population genetic structure of roach in the region showed that some populations have been confined to stretches of river with a high proportion of estrogenic effluent for multiple generations and have survived, apparently without reliance on immigration of fish from less polluted sites.
These results demonstrate that roach populations living in some effluent-contaminated river stretches, where feminization is widespread, are self-sustaining. Although we found no evidence to suggest that exposure to estrogenic effluents is a significant driving factor in determining the size of roach breeding populations, a reduction in Ne of up to 65% is still possible for the most contaminated sites because of the wide confidence intervals associated with the statistical model.
Fishery; Genetic diversity; DNA microsatellites; Waste water treatment work; Ecotoxicology
The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown.
Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An ipsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Further studies revealed the absence of inositol-derived lipids in the cell wall and a complete loss of mycothiol biosynthesis. The phenotype of the C. glutamicum ipsA deletion mutant was complemented to different extend by homologs from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. Myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro.
This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development.
Transcriptional regulator; Corynebacterium glutamicum; Corynebacterium diphtheriae; Mycobacterium tuberculosis; Cell wall synthesis; Mycolic acids; Mycothiol; Inositol; LM; LAM
Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mRNAs to examine the defects in translation in a poly(A) binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1.
The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins. Detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mRNAs. First, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. Second, a group of mRNAs with incomplete coding sequences appeared to be uncoupled from the regulon, since their dependence on RPL24B differed from regular mRNAs. Third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild-type. Some sister paralogs also differed in their response to the rpl24b mutation. In contrast to rpl24b, the pab2 pab8 mutant revealed few gene specific translational defects, but a group of seed storage protein mRNAs were stimulated in their ribosome occupancy. In the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected 12 biological replicates of translation states from wild-type seedlings. We defined 20% of mRNAs as having a high variance in their translation state. Many of these mRNAs were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mRNAs.
These data represent the first genome-wide analysis of translation in a eukaryote defective in the large ribosomal subunit. RPL24 and eIF3h play similar but non-identical roles in eukaryotic translation. The data also shed light on the fine structure of the regulon of ribosomal protein mRNAs.
Translation state; Ribosome occupancy; RPL24; PABP; Regulon; Arabidopsis
ICE (Inducer of CBF Expression) family genes play an important role in the regulation of cold tolerance pathways. In an earlier study, we isolated the gene CdICE1 from Chrysanthemum dichrum and demonstrated that freezing tolerance was enhanced by CdICE1 overexpression. Therefore, we sought to determine the mechanism by which ICE1 family genes participate in freezing tolerance.
Using EMSA (Electrophoretic Mobility Shift Assay) and yeast one-hybrid assays, we confirmed that CdICE1 binds specifically to the MYC element in the CdDREBa promoter and activates transcription. In addition, overexpression of CdICE1 enhanced Arabidopsis freezing tolerance after transition from 23°C to 4°C or 16°C. We found that after acclimation to 4°C, CdICE1, like Arabidopsis AtICE1, promoted expression of CBFs (CRT/DRE Binding Factor) and their genes downstream involved in freezing tolerance, including COR15a (Cold-Regulated 15a), COR6.6, and RD29a (Responsive to Dessication 29a). Interestingly, we observed that CdICE1-overexpressing plants experienced significant reduction in miR398. In addition, its target genes CSD1 (Copper/zinc Superoxide Dismutase 1) and CSD2 showed inducible expression under acclimation at 16°C, indicating that the miR398-CSD pathway was involved in the induction of freezing tolerance.
Our data indicate that CdICE1-mediated freezing tolerance occurs via different pathways, involving either CBF or miR398, under acclimation at two different temperatures.
Chrysanthemum dichrum; CdICE1; Freezing tolerance; miR398; CSD
The lag phase of bacterial growth is important from a medical and food safety perspective, but difficult to study due to the low density and metabolic rate of cells. A new study by Alon and colleagues reveals that the gene expression program during early lag phase prioritizes carbon source utilization enzymes over genes responsible for biomass accumulation. This cellular strategy ultimately maximizes growth, making the best long-term use of the new nutrient-rich environment.
See research article: http://www.biomedcentral.com/1752-0509/7/136
Most segmented animals add segments sequentially as the animal grows. In vertebrates, segment patterning depends on oscillations of gene expression coordinated as travelling waves in the posterior, unsegmented mesoderm. Recently, waves of segmentation gene expression have been clearly documented in insects. However, it remains unclear whether cyclic gene activity is widespread across arthropods, and possibly ancestral among segmented animals. Previous studies have suggested that a segmentation oscillator may exist in Strigamia, an arthropod only distantly related to insects, but further evidence is needed to document this.
Using the genes even skipped and Delta as representative of genes involved in segment patterning in insects and in vertebrates, respectively, we have carried out a detailed analysis of the spatio-temporal dynamics of gene expression throughout the process of segment patterning in Strigamia. We show that a segmentation clock is involved in segment formation: most segments are generated by cycles of dynamic gene activity that generate a pattern of double segment periodicity, which is only later resolved to the definitive single segment pattern. However, not all segments are generated by this process. The most posterior segments are added individually from a localized sub-terminal area of the embryo, without prior pair-rule patterning.
Our data suggest that dynamic patterning of gene expression may be widespread among the arthropods, but that a single network of segmentation genes can generate either oscillatory behavior at pair-rule periodicity or direct single segment patterning, at different stages of embryogenesis.
Segmentation clock; Oscillation; Pair-rule genes; Single segment patterning; Arthropod; Evolution
The tumor suppressor protein p53 is activated by cellular stress. DNA double strand breaks (DSBs) induce the activation of the kinase ATM, which stabilizes p53 and activates its transcriptional activity. Single cell analysis revealed that DSBs induced by gamma irradiation trigger p53 accumulation in a series of pulses that vary in number from cell to cell. Higher levels of irradiation increase the number of p53 pulses suggesting that they arise from periodic examination of the damage by ATM. If damage persists, additional pulses of p53 are triggered. The threshold of damage required for activating a p53 pulse is unclear. Previous studies that averaged the response across cell populations suggested that one or two DNA breaks are sufficient for activating ATM and p53. However, it is possible that by averaging over a population of cells important features of the dependency between DNA breaks and p53 dynamics are missed.
Using fluorescent reporters we developed a system for following in individual cells the number of DSBs, the kinetics of repair and the p53 response. We found a large variation in the initial number of DSBs and the rate of repair between individual cells. Cells with higher number of DSBs had higher probability of showing a p53 pulse. However, there was no distinct threshold number of breaks for inducing a p53 pulse. We present evidence that the decision to activate p53 given a specific number of breaks is not entirely stochastic, but instead is influenced by both cell-intrinsic factors and previous exposure to DNA damage. We also show that the natural variations in the initial amount of p53, rate of DSB repair and cell cycle phase do not affect the probability of activating p53 in response to DNA damage.
The use of fluorescent reporters to quantify DNA damage and p53 levels in live cells provided a quantitative analysis of the complex interrelationships between both processes. Our study shows that p53 activation differs even between cells that have a similar number of DNA breaks. Understanding the origin and consequences of such variability in normal and cancerous cells is crucial for developing efficient and selective therapeutic interventions.
Double strand breaks; p53; Live imaging; Single cells; Pulses
Reciprocal interactions between lung mesenchymal and epithelial cells play essential roles in lung organogenesis and homeostasis. Although the molecular markers and related animal models that target lung epithelial cells are relatively well studied, molecular markers of lung mesenchymal cells and the genetic tools to target and/or manipulate gene expression in a lung mesenchyme-specific manner are not available, which becomes a critical barrier to the study of lung mesenchymal biology and the related pulmonary diseases.
We have identified a mouse Tbx4 gene enhancer that contains conserved DNA sequences across many vertebrate species with lung or lung-like gas exchange organ. We then generate a mouse line to express rtTA/LacZ under the control of the Tbx4 lung enhancer, and therefore a Tet-On inducible transgenic system to target lung mesenchymal cells at different developmental stages. By combining a Tbx4-rtTA driven Tet-On inducible Cre expression mouse line with a Cre reporter mouse line, the spatial-temporal patterns of Tbx4 lung enhancer targeted lung mesenchymal cells were defined. Pulmonary endothelial cells and vascular smooth muscle cells were targeted by the Tbx4-rtTA driver line prior to E11.5 and E15.5, respectively, while other subtypes of lung mesenchymal cells including airway smooth muscle cells, fibroblasts, pericytes could be targeted during the entire developmental stage.
Developmental lung mesenchymal cells can be specifically marked by Tbx4 lung enhancer activity. With our newly created Tbx4 lung enhancer-driven Tet-On inducible system, lung mesenchymal cells can be specifically and differentially targeted in vivo for the first time by controlling the doxycycline induction time window. This novel system provides a unique tool to study lung mesenchymal cell lineages and gene functions in lung mesenchymal development, injury repair, and regeneration in mice.
Lung mesenchyme; Tbx4 lung enhancer; Tet-On system
Neural crest defects lead to congenital heart disease involving outflow tract malformation. Integrin-linked-kinase (ILK) plays important roles in multiple cellular processes and embryogenesis. ILK is expressed in the neural crest, but its role in neural crest and outflow tract morphogenesis remains unknown.
We ablated ILK specifically in the neural crest using the Wnt1-Cre transgene. ILK ablation resulted in abnormal migration and overpopulation of neural crest cells in the pharyngeal arches and outflow tract and a significant reduction in the expression of neural cell adhesion molecule (NCAM) and extracellular matrix components. ILK mutant embryos exhibited an enlarged common arterial trunk and ventricular septal defect. Reduced smooth muscle differentiation, but increased ossification and neurogenesis/innervation were observed in ILK mutant outflow tract that may partly be due to reduced transforming growth factor β2 (TGFβ2) but increased bone morphogenetic protein (BMP) signaling. Consistent with these observations, microarray analysis of fluorescence-activated cell sorting (FACS)-sorted neural crest cells revealed reduced expression of genes associated with muscle differentiation, but increased expression of genes of neurogenesis and osteogenesis.
Our results demonstrate that ILK plays essential roles in neural crest and outflow tract development by mediating complex crosstalk between cell matrix and multiple signaling pathways. Changes in these pathways may collectively result in the unique neural crest and outflow tract phenotypes observed in ILK mutants.
ILK; Cardiac neural crest; Outflow tract; Migration; Congenital heart disease