RESUMO
Mortality rates of marine fish larvae are incredibly high and can determine year-class strength. The major causes of larval mortality are predation and starvation, and the performance of larvae in survival skills that can mitigate this mortality (predator evasion, foraging) varies among individuals and cohorts, but the causes of the variation are not known. Transcriptomics can link gene expression variation to phenotypic variation at the whole-system level to investigate the molecular basis of behavioural variation. We used tag-based RNA-sequencing to examine the molecular basis of variation in predator evasion and routine swimming (trait related to foraging efficiency) in the larval red drum, Sciaenops ocellatus. We looked for functional gene networks in which interindividual variation would explain variation in larval behavioural performance. We identified co-expressed gene groups ("modules") associated with predator evasion traits and found enrichment of motor, neural and energy metabolism pathways. These functional associations and pattern of correlations between modules and traits suggest that energy availability and allocation were responsible for the magnitude of startle responses, while differential neural and motor activation were associated with differences in response latency.
Assuntos
Perciformes , Animais , Larva/fisiologia , Perciformes/genética , Perciformes/metabolismo , Peixes , Natação , Expressão GênicaRESUMO
The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable.
Assuntos
Cisteína , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Cisteína/genética , Cisteína/metabolismo , Regulação da Expressão Gênica , DNA/genética , DNA/metabolismo , Oxirredução , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
The transcription factor ΔFosB accumulates in response to chronic insults such as drugs of abuse, L-3,4-dihydroxyphenylalanine (l-DOPA) or stress in specific regions of the brain, triggering long lasting neural and behavioral changes that underlie aspects of drug addiction, dyskinesia, and depression. Thus, small molecule chemical probes are urgently needed to investigate biological functions of ΔFosB. Herein we describe the identification of a novel phenanthridine analogue ZL0220 (27) as an active and promising ΔFosB chemical probe with micromolar inhibitory activities against ΔFosB homodimers and ΔFosB/JunD heterodimers.
Assuntos
DNA/efeitos dos fármacos , Descoberta de Drogas , Fenantridinas/farmacologia , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , DNA/química , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fenantridinas/química , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-jun/química , Relação Estrutura-AtividadeRESUMO
ΔFosB is a highly stable transcription factor that accumulates in specific brain regions upon chronic exposure to drugs of abuse, stress, or seizures, and mediates lasting behavioral responses. ΔFosB reportedly heterodimerizes with JunD forming a canonical bZIP leucine zipper coiled coil that clamps onto DNA. However, the striking accumulation of ΔFosB protein in brain upon chronic insult has brought its molecular status into question. Here, we demonstrate through a series of crystal structures that the ΔFosB bZIP domain self-assembles into stable oligomeric assemblies that defy the canonical arrangement. The ΔFosB bZIP domain also self-assembles in solution, and in neuron-like Neuro 2a cells it is trapped into molecular arrangements that are consistent with our structures. Our data suggest that, as ΔFosB accumulates in brain in response to chronic insult, it forms non-canonical assemblies. These species may be at the root of ΔFosB's striking protein stability, and its unique transcriptional and behavioral consequences.
RESUMO
Telomeres cap the physical ends of eukaryotic chromosomes to ensure complete DNA replication and genome stability. Heritable natural variation in telomere length exists in yeast, mice, plants and humans at birth; however, major effect loci underlying such polymorphism remain elusive. Here, we employ quantitative trait locus (QTL) mapping and transgenic manipulations to identify genes controlling telomere length set point in a multi-parent Arabidopsis thaliana mapping population. We detect several QTL explaining 63.7% of the total telomere length variation in the Arabidopsis MAGIC population. Loss-of-function mutants of the NOP2A candidate gene located inside the largest effect QTL and of two other ribosomal genes RPL5A and RPL5B establish a shorter telomere length set point than wild type. These findings indicate that evolutionarily conserved components of ribosome biogenesis and cell proliferation pathways promote telomere elongation.
Assuntos
Arabidopsis/genética , Ribossomos/genética , Telômero/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mapeamento Cromossômico , Polimorfismo Genético , Locos de Características Quantitativas , Ribossomos/metabolismo , Telômero/genéticaRESUMO
Reef-building corals can increase their resistance to heat-induced bleaching through adaptation and acclimatization and/or by associating with a more thermo-tolerant strain of algal symbiont (Symbiodinium sp.). Here, we show that these two adaptive pathways interact. We collected Acropora millepora corals from two contrasting thermal environments on the Great Barrier Reef: cooler, mid-latitude Orpheus Island, where all corals hosted a heat-sensitive clade C Symbiodinium, and warmer, low-latitude Wilkie Island, where corals hosted either a clade C or a more thermo-tolerant clade D. Corals were kept in a benign common garden to reveal differences in baseline gene expression, reflecting prior adaptation/long-term acclimatization. Model-based analysis identified gene expression differences between Wilkie and Orpheus corals that were negatively correlated with previously described transcriptome-wide signatures of heat stress, signifying generally elevated thermotolerance of Wilkie corals. Yet, model-free analyses of gene expression revealed that Wilkie corals hosting clade C were distinct from Wilkie corals hosting clade D, whereas Orpheus corals were more variable. Wilkie corals hosting clade C symbionts exhibited unique functional signatures, including downregulation of histone proteins and ion channels and upregulation of chaperones and RNA processing genes, putatively representing constitutive "frontloading" of stress response genes. Furthermore, clade C Symbiodinium exhibited constitutive expression differences between Wilkie and Orpheus, indicative of contrasting life history strategies. Our results demonstrate that hosting alternative Symbiodinium types is associated with different pathways of local adaptation for the coral host. These interactions could play a significant role in setting the direction of genetic adaptation to global warming in the two symbiotic partners.
Assuntos
Antozoários/genética , Aclimatação , Animais , Mudança Climática , Recifes de Corais , Simbiose/genética , Simbiose/fisiologiaRESUMO
Can genetic adaptation in reef-building corals keep pace with the current rate of sea surface warming? Here we combine population genomics, biophysical modeling, and evolutionary simulations to predict future adaptation of the common coral Acropora millepora on the Great Barrier Reef (GBR). Genomics-derived migration rates were high (0.1-1% of immigrants per generation across half the latitudinal range of the GBR) and closely matched the biophysical model of larval dispersal. Both genetic and biophysical models indicated the prevalence of southward migration along the GBR that would facilitate the spread of heat-tolerant alleles to higher latitudes as the climate warms. We developed an individual-based metapopulation model of polygenic adaptation and parameterized it with population sizes and migration rates derived from the genomic analysis. We find that high migration rates do not disrupt local thermal adaptation, and that the resulting standing genetic variation should be sufficient to fuel rapid region-wide adaptation of A. millepora populations to gradual warming over the next 20-50 coral generations (100-250 years). Further adaptation based on novel mutations might also be possible, but this depends on the currently unknown genetic parameters underlying coral thermal tolerance and the rate of warming realized. Despite this capacity for adaptation, our model predicts that coral populations would become increasingly sensitive to random thermal fluctuations such as ENSO cycles or heat waves, which corresponds well with the recent increase in frequency of catastrophic coral bleaching events.
Assuntos
Adaptação Fisiológica/genética , Antozoários/genética , Mudança Climática , Recifes de Corais , Animais , Antozoários/fisiologia , Austrália , Conservação dos Recursos Naturais , Variação Genética , Genótipo , Temperatura , IncertezaRESUMO
BACKGROUND: Molecular mechanisms underlying coral larval competence, the ability of larvae to respond to settlement cues, determine their dispersal potential and are potential targets of natural selection. Here, we profiled competence, fluorescence and genome-wide gene expression in embryos and larvae of the reef-building coral Acropora millepora daily throughout 12 days post-fertilization. RESULTS: Gene expression associated with competence was positively correlated with transcriptomic response to the natural settlement cue, confirming that mature coral larvae are "primed" for settlement. Rise of competence through development was accompanied by up-regulation of sensory and signal transduction genes such as ion channels, genes involved in neuropeptide signaling, and G-protein coupled receptor (GPCRs). A drug screen targeting components of GPCR signaling pathways confirmed a role in larval settlement behavior and metamorphosis. CONCLUSIONS: These results gives insight into the molecular complexity underlying these transitions and reveals receptors and pathways that, if altered by changing environments, could affect dispersal capabilities of reef-building corals. In addition, this dataset provides a toolkit for asking broad questions about sensory capacity in multicellular animals and the evolution of development.
Assuntos
Antozoários/crescimento & desenvolvimento , Antozoários/genética , Animais , Antozoários/anatomia & histologia , Antozoários/embriologia , Comportamento Animal/efeitos dos fármacos , Fertilização , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Proteínas Luminescentes/metabolismo , Metamorfose Biológica/genética , TranscriptomaRESUMO
Adaptive variation in social behaviour depends upon standing genetic variation, but we know little about how evolutionary forces shape genetic diversity relevant to brain and behaviour. In prairie voles (Microtus ochrogaster), variants at the Avpr1a locus predict expression of the vasopressin 1a receptor in the retrosplenial cortex (RSC), a brain region that mediates spatial and contextual memory; cortical V1aR abundance in turn predicts diversity in space use and sexual fidelity in the field. To examine the potential contributions of adaptive and neutral forces to variation at the Avpr1a locus, we explore sequence diversity at the Avpr1a locus and throughout the genome in two populations of wild prairie voles. First, we refine results demonstrating balancing selection at the locus by comparing the frequency spectrum of variants at the locus to a random sample of the genome. Next, we find that the four single nucleotide polymorphisms that predict high V1aR expression in the RSC are in stronger linkage disequilibrium than expected by chance despite high recombination among intervening variants, suggesting that epistatic selection maintains their association. Analysis of population structure and a haplotype network for two populations revealed that this excessive LD was unlikely to be due to admixture alone. Furthermore, the two populations differed considerably in the region shown to be a regulator of V1aR expression despite the extremely low levels of genomewide genetic differentiation. Together, our data suggest that complex selection on Avpr1a locus favours specific combinations of regulatory polymorphisms, maintains the resulting alleles at population-specific frequencies, and may contribute to unique patterns of spatial cognition and sexual fidelity among populations.
Assuntos
Arvicolinae/genética , Cognição/fisiologia , Receptores de Vasopressinas/genética , Comportamento Sexual Animal/fisiologia , Alelos , Animais , Arvicolinae/fisiologia , Encéfalo/fisiologia , Variação Genética , Repetições de Microssatélites/genética , Comportamento SocialRESUMO
The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions.
Assuntos
Antozoários/fisiologia , Evolução Biológica , Células Germinativas , Animais , Linhagem da Célula , Análise Mutacional de DNA , Variação Genética , Genótipo , Reprodução/genética , Reprodução/fisiologia , Células-Tronco/citologiaRESUMO
Effective dispersal across environmental gradients is the key to species resilience to environmental perturbation, including climate change. Coral reefs are among the most sensitive ecosystems to global warming, but factors predicting coral dispersal potential remain unknown. In a reef-building coral Acropora millepora, larval fluorescence emerged as a possible indicator of dispersal potential since it correlates with responsiveness to a settlement cue. Here, we show that gene expression in red fluorescent larvae of A. millepora is correlated with diapause-like characteristics highly likely to be associated with extended dispersal. We compared gene expression among three larval fluorescent morphs under three coloured light treatments. While colour morphs did not differ in their gene expression responses to light colour, red larvae demonstrated gene expression signatures of cell cycle arrest and decreased transcription accompanied by elevated ribosome production and heightened defenses against oxidative stress. A meta-analysis revealed that this profile was highly similar to the signatures of elevated thermal tolerance in the same coral species, and moreover, functionally resembled diapause states in an insect and a nematode. Our results support a connection between red fluorescence and long-range dispersal, which offers a new perspective on the molecular underpinnings of coral larval dispersal and the biological function of GFP-like fluorescent proteins.
Assuntos
Antozoários/fisiologia , Fluorescência , Transcriptoma , Distribuição Animal , Animais , Antozoários/genética , Mudança Climática , Cor , Genética Populacional , Larva/genética , Larva/fisiologia , Metamorfose Biológica , Fenótipo , Análise de Sequência de RNA , Austrália OcidentalRESUMO
As global warming continues, reef-building corals could avoid local population declines through "genetic rescue" involving exchange of heat-tolerant genotypes across latitudes, but only if latitudinal variation in thermal tolerance is heritable. Here, we show an up-to-10-fold increase in odds of survival of coral larvae under heat stress when their parents come from a warmer lower-latitude location. Elevated thermal tolerance was associated with heritable differences in expression of oxidative, extracellular, transport, and mitochondrial functions that indicated a lack of prior stress. Moreover, two genomic regions strongly responded to selection for thermal tolerance in interlatitudinal crosses. These results demonstrate that variation in coral thermal tolerance across latitudes has a strong genetic basis and could serve as raw material for natural selection.
Assuntos
Aclimatação/genética , Antozoários/genética , Antozoários/fisiologia , Recifes de Corais , Aquecimento Global , Temperatura Alta , Animais , Extinção Biológica , Expressão Gênica , Frequência do Gene , Marcadores Genéticos , Larva/genética , Larva/fisiologia , Seleção Genética , Estresse Fisiológico/genéticaRESUMO
BACKGROUND: Corals are capable of launching diverse immune defenses at the site of direct contact with pathogens, but the molecular mechanisms of this activity and the colony-wide effects of such stressors remain poorly understood. Here we compared gene expression profiles in eight healthy Acropora hyacinthus colonies against eight colonies exhibiting tissue loss commonly associated with white syndromes, all collected from a natural reef environment near Palau. Two types of tissues were sampled from diseased corals: visibly affected and apparently healthy. RESULTS: Tag-based RNA-Seq followed by weighted gene co-expression network analysis identified groups of co-regulated differentially expressed genes between all health states (disease lesion, apparently healthy tissues of diseased colonies, and fully healthy). Differences between healthy and diseased tissues indicate activation of several innate immunity and tissue repair pathways accompanied by reduced calcification and the switch towards metabolic reliance on stored lipids. Unaffected parts of diseased colonies, although displaying a trend towards these changes, were not significantly different from fully healthy samples. Still, network analysis identified a group of genes, suggestive of altered immunity state, that were specifically up-regulated in unaffected parts of diseased colonies. CONCLUSIONS: Similarity of fully healthy samples to apparently healthy parts of diseased colonies indicates that systemic effects of white syndromes on A. hyacinthus are weak, which implies that the coral colony is largely able to sustain its physiological performance despite disease. The genes specifically up-regulated in unaffected parts of diseased colonies, instead of being the consequence of disease, might be related to the originally higher susceptibility of these colonies to naturally occurring white syndromes.
Assuntos
Antozoários/genética , Recifes de Corais , Transcriptoma , Animais , Ontologia Genética , Redes Reguladoras de Genes , Análise de Sequência de RNARESUMO
Rhizaria is one of the six supergroups of eukaryotes, which comprise the majority of amoeboid and skeleton-building protists living in freshwater and marine ecosystems. There is an overall lack of molecular data for the group and therefore the deep phylogeny of rhizarians is unresolved. Molecular data are particularly scarce for the clade of Retaria, which include two prominent groups of microfossils: foraminiferans and radiolarians. To fill this gap, we have produced and sequenced EST libraries for 14 rhizarian species including seven foraminiferans, Gromia and six taxa belonging to traditional Haeckel's Radiolaria: Acantharea, Polycystinea, and Phaeodarea. A matrix was constructed for phylogenetic analysis based on 109 genes and a total of 56 species, of which 22 are rhizarians. Our analyses provide the first multigene evidence for branching of Phaeodarea within Cercozoa, confirming the polyphyly of Haeckel's Radiolaria. It confirms the monophyly of Retaria, a clade grouping Foraminifera with other lineages of Radiolaria. However, contrary to what could be expected from morphological observations, Foraminifera do not form a sister group to radiolarians, but branch within them as sister to either Acantharea or Polycystinea depending on the multigene data set. While the monophyly of Foraminifera and Acantharea is well supported, that of Polycystinea, represented in our data by Spumellaria and Collodaria is questionable. In view of our study, Haeckel's Radiolaria appears as both, a polyphyletic and paraphyletic assemblage of independent groups that should be considered as separate lineages in protist classification.
Assuntos
Filogenia , Rhizaria/classificação , Teorema de Bayes , DNA de Protozoário/genética , Etiquetas de Sequências Expressas , Biblioteca Gênica , Genes de Protozoários , Funções Verossimilhança , Modelos Genéticos , Rhizaria/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Proteins homologous to Green Fluorescent Protein (GFP) are widely used as genetically encoded fluorescent labels. Many developments of this technology were spurred by discoveries of novel types of GFP-like proteins (FPs) in nature. Here we report two proteins displaying primary structures never before encountered in natural FPs: they consist of multiple GFP-like domains repeated within the same polypeptide chain. A two-domain green FP (abeGFP) and a four-domain orange-fluorescent FP (Ember) were isolated from the siphonophore Abylopsis eschscholtzii and an unidentified juvenile jellyfish (order Anthoathecata), respectively. Only the most evolutionary ancient domain of Ember is able to synthesize an orange-emitting chromophore (emission at 571 nm), while the other three are purely green (emission at 520 nm) and putatively serve to maintain the stability and solubility of the multidomain protein. When expressed individually, two of the green Ember domains form dimers and the third one exists as a monomer. The low propensity for oligomerization of these domains would simplify their adoption as in vivo labels. Our results reveal a previously unrecognized direction in which natural FPs have diversified, suggesting new avenues to look for FPs with novel and potentially useful features.
Assuntos
Hidrozoários/metabolismo , Proteínas Luminescentes/química , Sequência de Aminoácidos , Animais , Dimerização , Proteínas Luminescentes/classificação , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de FluorescênciaRESUMO
Coral reefs are declining worldwide due to increased incidence of climate-induced coral bleaching, which will have widespread biodiversity and economic impacts. A simple method to measure the sub-bleaching level of heat-light stress experienced by corals would greatly inform reef management practices by making it possible to assess the distribution of bleaching risks among individual reef sites. Gene expression analysis based on quantitative PCR (qPCR) can be used as a diagnostic tool to determine coral condition in situ. We evaluated the expression of 13 candidate genes during heat-light stress in a common Caribbean coral Porites astreoides, and observed strong and consistent changes in gene expression in two independent experiments. Furthermore, we found that the apparent return to baseline expression levels during a recovery phase was rapid, despite visible signs of colony bleaching. We show that the response to acute heat-light stress in P. astreoides can be monitored by measuring the difference in expression of only two genes: Hsp16 and actin. We demonstrate that this assay discriminates between corals sampled from two field sites experiencing different temperatures. We also show that the assay is applicable to an Indo-Pacific congener, P. lobata, and therefore could potentially be used to diagnose acute heat-light stress on coral reefs worldwide.
Assuntos
Recifes de Corais , Perfilação da Expressão Gênica , Temperatura Alta/efeitos adversos , Luz/efeitos adversos , Estresse Fisiológico/genética , Actinas/genética , Animais , Biomarcadores , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genéticaRESUMO
The presence of green fluorescent protein (GFP) within the bioluminescent system of Obelia (Cnidaria, Hydrozoa, Campanulariidae) was inferred shortly after the discovery of GFP in Aequorea. Despite the enormous success of Aequorea GFP as a genetically encoded fluorescent label, Obelia GFP thus far has been defeating attempts to clone it from the hydroid life cycle stage. Here, we report cloning of three GFP-like fluorescent proteins (FPs) from Obelia medusa, representing cyan, green, and yellow spectral types. Such color diversity has never been detected outside class Anthozoa, suggesting a more general function for multi-colored fluorescence in cnidarians than has been previously hypothesized. An unusual property of the new FPs is the formation of large soluble complexes of well-defined sizes and molecular weights, corresponding to up to 128 individual polypeptides. This aligns well with the earlier observation that luminescence in Obelia, unlike in Aequorea, is localized within subcellular granules, which prompts further inquiry into the self-assembly properties of the new FPs and their interactions with the photoprotein. The discovery of Obelia FPs fills the four-decade-old gap in the knowledge of cnidarian bioluminescence and provides experimental material to further investigate the details of its molecular mechanism.
Assuntos
Hidrozoários/metabolismo , Proteínas Luminescentes/química , Sequência de Aminoácidos , Animais , Cor , Proteínas de Fluorescência Verde/química , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Espectrofotometria UltravioletaRESUMO
BACKGROUND: Recent phylogenomic analyses have revolutionized our view of eukaryote evolution by revealing unexpected relationships between and within the eukaryotic supergroups. However, for several groups of uncultivable protists, only the ribosomal RNA genes and a handful of proteins are available, often leading to unresolved evolutionary relationships. A striking example concerns the supergroup Rhizaria, which comprises several groups of uncultivable free-living protists such as radiolarians, foraminiferans and gromiids, as well as the parasitic plasmodiophorids and haplosporids. Thus far, the relationships within this supergroup have been inferred almost exclusively from rRNA, actin, and polyubiquitin genes, and remain poorly resolved. To address this, we have generated large Expressed Sequence Tag (EST) datasets for 5 species of Rhizaria belonging to 3 important groups: Acantharea (Astrolonche sp., Phyllostaurus sp.), Phytomyxea (Spongospora subterranea, Plasmodiophora brassicae) and Gromiida (Gromia sphaerica). RESULTS: 167 genes were selected for phylogenetic analyses based on the representation of at least one rhizarian species for each gene. Concatenation of these genes produced a supermatrix composed of 36,735 amino acid positions, including 10 rhizarians, 9 stramenopiles, and 9 alveolates. Phylogenomic analyses of this large dataset revealed a strongly supported clade grouping Foraminifera and Acantharea. The position of this clade within Rhizaria was sensitive to the method employed and the taxon sampling: Maximum Likelihood (ML) and Bayesian analyses using empirical model of evolution favoured an early divergence, whereas the CAT model and ML analyses with fast-evolving sites or the foraminiferan species Reticulomyxa filosa removed suggested a derived position, closely related to Gromia and Phytomyxea. In contrast to what has been previously reported, our analyses also uncovered the presence of the rhizarian-specific polyubiquitin insertion in Acantharea. Finally, this work reveals another possible rhizarian signature in the 60S ribosomal protein L10a. CONCLUSIONS: Our study provides new insights into the evolution of Rhizaria based on phylogenomic analyses of ESTs from three groups of previously under-sampled protists. It was enabled through the application of a recently developed method of transcriptome analysis, requiring very small amount of starting material. Our study illustrates the potential of this method to elucidate the early evolution of eukaryotes by providing large amount of data for uncultivable free-living and parasitic protists.
Assuntos
Evolução Biológica , Etiquetas de Sequências Expressas , Filogenia , Rhizaria/genética , Actinas/genética , Teorema de Bayes , Mapeamento de Sequências Contíguas , Biblioteca Gênica , Funções Verossimilhança , Dados de Sequência Molecular , Poliubiquitina/genética , Rhizaria/classificação , Proteínas Ribossômicas/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
BACKGROUND: New methods are needed for genomic-scale analysis of emerging model organisms that exemplify important biological questions but lack fully sequenced genomes. For example, there is an urgent need to understand the potential for corals to adapt to climate change, but few molecular resources are available for studying these processes in reef-building corals. To facilitate genomics studies in corals and other non-model systems, we describe methods for transcriptome sequencing using 454, as well as strategies for assembling a useful catalog of genes from the output. We have applied these methods to sequence the transcriptome of planulae larvae from the coral Acropora millepora. RESULTS: More than 600,000 reads produced in a single 454 sequencing run were assembled into approximately 40,000 contigs with five-fold average sequencing coverage. Based on sequence similarity with known proteins, these analyses identified approximately 11,000 different genes expressed in a range of conditions including thermal stress and settlement induction. Assembled sequences were annotated with gene names, conserved domains, and Gene Ontology terms. Targeted searches using these annotations identified the majority of genes associated with essential metabolic pathways and conserved signaling pathways, as well as novel candidate genes for stress-related processes. Comparisons with the genome of the anemone Nematostella vectensis revealed approximately 8,500 pairs of orthologs and approximately 100 candidate coral-specific genes. More than 30,000 SNPs were detected in the coral sequences, and a subset of these validated by re-sequencing. CONCLUSION: The methods described here for deep sequencing of the transcriptome should be widely applicable to generate catalogs of genes and genetic markers in emerging model organisms. Our data provide the most comprehensive sequence resource currently available for reef-building corals, and include an extensive collection of potential genetic markers for association and population connectivity studies. The characterization of the larval transcriptome for this widely-studied coral will enable research into the biological processes underlying stress responses in corals and evolutionary adaptation to global climate change.
Assuntos
Antozoários/genética , Perfilação da Expressão Gênica , Análise de Sequência de DNA/métodos , Animais , Análise por Conglomerados , Biologia Computacional , Mapeamento de Sequências Contíguas , Biblioteca Gênica , Genômica/métodos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
In the developing ventral midbrain, the signaling molecule sonic hedgehog (SHH) is sufficient to specify a striped pattern of cell fates (midbrain arcs). Here, we asked whether and precisely how hedgehog (HH) signaling might be necessary for ventral midbrain patterning. By blocking HH signaling by in ovo misexpression of Ptc1(Delta)(loop2), we show that HH signaling is necessary and can act directly at a distance to specify midbrain cell fates. Ventral midbrain progenitors extinguish their dependence upon HH in a spatiotemporally complex manner, completing cell-fate specification at the periphery by Hamburger and Hamilton stage 13. Thus, patterning at the lateral periphery of the ventral midbrain is accomplished early, when the midbrain is small and the HH signal needs to travel relatively short distances (approximately 30 cell diameters). Interestingly, single-cell injections demonstrate that patterning in the midbrain occurs within the context of cortex-like radial columns of cells that can share HH blockade and are cytoplasmically connected by gap junctions. HH blockade results in increased cell scatter, disrupting the spatial coherence of the midbrain arc pattern. Finally, HH signaling is required for the integrity and the signaling properties of the boundaries of the midbrain (e.g. the midbrain-hindbrain boundary, the dorsoventral boundary), its perturbations resulting in abnormal cell mixing across 'leaky' borders.