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Helitron-like elements (HLEs) are widespread eukaryotic DNA transposons employing a rolling-circle transposition mechanism. Despite their prevalence in fungi, animals, and plant genomes, identifying Helitrons remains a formidable challenge. We introduce HELIANO, a software for annotating and classifying autonomous and non-autonomous HLE sequences from whole genomes. HELIANO overcomes several limitations of existing tools in speed and accuracy, demonstrated through benchmarking and its application to the complex genomes of frogs (Xenopus tropicalis and Xenopus laevis) and rice (Oryza sativa), where it uncovered numerous previously unidentified HLEs. In an extensive analysis of 404 eukaryote genomes, we found HLEs widely distributed across phyla, with exceptions in specific taxa. HELIANO's application led to the discovery of numerous new HLEs in land plants and identified 20 protein domains captured by certain autonomous HLE families. A comprehensive phylogenetic analysis further classified HLEs into two primary clades, HLE1 and HLE2, and revealed nine subgroups, some of which are enriched within specific taxa. The future use of HELIANO promises to improve the global analysis of HLEs across genomes, significantly advancing our understanding of this fascinating transposon superfamily.
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BACKGROUND: Variation in locomotor capacity among animals often reflects adaptations to different environments. Despite evidence that physical performance is heritable, the molecular basis of locomotor performance and performance trade-offs remains poorly understood. In this study we identify the genes, signaling pathways, and regulatory processes possibly responsible for the trade-off between burst performance and endurance observed in Xenopus allofraseri, using a transcriptomic approach. RESULTS: We obtained a total of about 121 million paired-end reads from Illumina RNA sequencing and analyzed 218,541 transcripts obtained from a de novo assembly. We identified 109 transcripts with a significant differential expression between endurant and burst performant individuals (FDR ≤ 0.05 and logFC ≥2), and blast searches resulted in 103 protein-coding genes. We found major differences between endurant and burst-performant individuals in the expression of genes involved in the polymerization and ATPase activity of actin filaments, cellular trafficking, proteoglycans and extracellular proteins secreted, lipid metabolism, mitochondrial activity and regulators of signaling cascades. Remarkably, we revealed transcript isoforms of key genes with functions in metabolism, apoptosis, nuclear export and as a transcriptional corepressor, expressed in either burst-performant or endurant individuals. Lastly, we find two up-regulated transcripts in burst-performant individuals that correspond to the expression of myosin-binding protein C fast-type (mybpc2). This suggests the presence of mybpc2 homoeologs and may have been favored by selection to permit fast and powerful locomotion. CONCLUSION: These results suggest that the differential expression of genes belonging to the pathways of calcium signaling, endoplasmic reticulum stress responses and striated muscle contraction, in addition to the use of alternative splicing and effectors of cellular activity underlie locomotor performance trade-offs. Ultimately, our transcriptomic analysis offers new perspectives for future analyses of the role of single nucleotide variants, homoeology and alternative splicing in the evolution of locomotor performance trade-offs.
Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Animais , Anuros , Xenopus , Xenopus laevisRESUMO
Lateral gene transfer is a very common process in bacterial and archaeal evolution, playing an important role in the adaptation to new environments. In eukaryotes, its role and frequency remain highly debated, although recent research supports that gene transfer from bacteria to diverse eukaryotes may be much more common than previously appreciated. However, most of this research focused on animals and the true phylogenetic and functional impact of bacterial genes in less-studied microbial eukaryotic groups remains largely unknown. Here, we have analyzed transcriptome data from the deep-branching stramenopile Opalinidae, common members of frog gut microbiomes, and distantly related to the well-known genus Blastocystis. Phylogenetic analyses suggest the early acquisition of several bacterial genes in a common ancestor of both lineages. Those lateral gene transfers most likely facilitated the adaptation of the free-living ancestor of the Opalinidae-Blastocystis symbiotic group to new niches in the oxygen-depleted animal gut environment.
Assuntos
Proteínas de Algas/genética , Bactérias/genética , Blastocystis/genética , Estramenópilas/genética , Animais , Blastocystis/classificação , Evolução Molecular , Perfilação da Expressão Gênica , Transferência Genética Horizontal , Genes Bacterianos , Filogenia , Ranidae/parasitologia , Estramenópilas/classificação , Xenopus/parasitologiaRESUMO
BACKGROUND: Mitochondrial DNA is remarkably polymorphic. This is why animal geneticists survey mitochondrial genomes variations for fundamental and applied purposes. We present here an approach to sequence whole mitochondrial genomes using nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA using an exonuclease treatment and on the amplification of circular mitochondrial DNA using a multiple displacement amplification step. RESULTS: We optimized each preparative step to obtain a 100 million-fold enrichment of horse mitochondrial DNA relative to nuclear DNA. We sequenced these amplified mitochondrial DNA using nanopore sequencing technology and obtained mitochondrial DNA reads that represented up to half of the sequencing output. The sequence reads were 2.3 kb of mean length and provided an even coverage of the mitochondrial genome. Long-reads spanning half or more of the whole mtDNA provided a coverage that varied between 118X and 488X. We evaluated SNPs identified using these long-reads by Sanger sequencing as ground truth and found a precision of 100.0%; a recall of 93.1% and a F1-score of 0.964 using the Twilight horse mtDNA reference. The choice of the mtDNA reference impacted variant calling efficiency with F1-scores varying between 0.947 and 0.964. CONCLUSIONS: Our method to amplify mtDNA and to sequence it using the nanopore technology is usable for mitochondrial DNA variant analysis. With minor modifications, this approach could easily be applied to other large circular DNA molecules.
Assuntos
Genoma Mitocondrial , Nanoporos , Animais , DNA Mitocondrial/genética , Genoma Mitocondrial/genética , Sequenciamento de Nucleotídeos em Larga Escala , Cavalos/genética , Análise de Sequência de DNARESUMO
Acoustic communication is widespread in animals. According to the sensory drive hypothesis [Endler JA (1993) Philos Trans R Soc Lond B Biol Sci 340(1292):215-225], communication signals and perceptual systems have coevolved. A clear illustration of this is the evolution of the tetrapod middle ear, adapted to life on land. Here we report the discovery of a bone conduction-mediated stimulation of the ear by wave propagation in Sechellophryne gardineri, one of the world's smallest terrestrial tetrapods, which lacks a middle ear yet produces acoustic signals. Based on X-ray synchrotron holotomography, we measured the biomechanical properties of the otic tissues and modeled the acoustic propagation. Our models show how bone conduction enhanced by the resonating role of the mouth allows these seemingly deaf frogs to communicate effectively without a middle ear.
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Anuros/fisiologia , Condução Óssea/fisiologia , Orelha Interna/anatomia & histologia , Audição/fisiologia , Modelos Biológicos , Animais , Fenômenos Biomecânicos , Boca/anatomia & histologia , Boca/fisiologia , Síncrotrons , Vocalização Animal/fisiologiaRESUMO
Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Crista Neural/embriologia , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , Hibridização In Situ , Análise em Microsséries , Fator de Transcrição PAX3 , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xenopus laevis/genéticaRESUMO
The genome size in eukaryotes does not correlate well with the number of genes they contain. We can observe this so-called C-value paradox in amphibian species. By analyzing an amphibian genome we asked how repetitive DNA can impact genome size and architecture. We describe here our discovery of a Tc1/mariner miniature inverted-repeat transposon family present in Xenopus frogs. These transposons named miDNA4 are unique since they contain a satellite DNA motif. We found that miDNA4 measured 331 bp, contained 25 bp long inverted terminal repeat sequences and a sequence motif of 119 bp present as a unique copy or as an array of 2-47 copies. We characterized the structure, dynamics, impact and evolution of the miDNA4 family and its satellite DNA in Xenopus frog genomes. This led us to propose a model for the evolution of these two repeated sequences and how they can synergize to increase genome size.
Assuntos
Elementos de DNA Transponíveis , DNA Satélite/genética , Evolução Molecular , Tamanho do Genoma , Xenopus/genética , Animais , Sequência de Bases , Sequência Conservada , Filogenia , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
BACKGROUND: Accurate interpretation of transcriptome profiling by quantitative PCR requires the establishment of species-specific standards. However, the selection of reference genes for assessing RNA expression profiles in Xenopus laevis and Xenopus tropicalis was mostly based on historical reasons and they often only reflect the traditions of a laboratory. RESULTS: We investigated the expression stability of 10 genes (dicer1, drosha, eef1a1, elavl3, gsc, h4, odc1, rpl8, smn2, tbp), 8 of which are commonly used as internal controls in published RT-qPCR experiments. We defined specific primer pairs and evaluated their suitability as reference genes by performing RT-qPCR expression profiling in Xenopus tropicalis. Gene expression stability was assayed in a set of 15 developmental stages from the egg to the froglet, and in dissected embryos. CONCLUSIONS: Overall, we determined a set of qualified reference genes for distinct developmental periods. We recommend the use of dicer1, drosha, eef1a1, and smn2 from early embryonic development up to the end of metamorphosis. During early embryogenesis drosha, eef1a1, smn2 are suitable. For the whole post-embryonic development and for metamorphic stages including pro-metamorphosis and metamorphic climax, we recommend the use of drosha and smn2. These reference genes should prove their usefulness for data comparison across studies.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Xenopus/genética , Animais , Primers do DNA/genética , DNA Complementar/metabolismo , Biologia do Desenvolvimento/métodos , Perfilação da Expressão Gênica/métodos , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Xenopus/embriologia , Xenopus laevisRESUMO
In humans and mice, the early development of αß T cells is controlled by the pre-T-cell receptor α chain (pTα) that is covalently associated with the T-cell receptor ß (TCRß) chain to form the pre-T-cell receptor (pre-TCR) at the thymocyte surface. Pre-TCR functions in a ligand-independent manner through self-oligomerization mediated by pTα. Using in silico and gene synteny-based approaches, we identified the pTα gene (PTCRA) in four sauropsid (three birds and one reptile) genomes. We also identified 25 mammalian PTCRA sequences now covering all mammalian lineages. Gene synteny around PTCRA is remarkably conserved in mammals but differences upstream of PTCRA in sauropsids suggest chromosomal rearrangements. PTCRA organization is highly similar in sauropsids and mammals. However, comparative analyses of the pTα functional domains indicate that sauropsids, monotremes, marsupials, and lagomorphs display a short pTα cytoplasmic tail and lack most residues shown to be critical for human and murine pre-TCR self-oligomerization. Chicken PTCRA transcripts similar to those in mammals were detected in immature double-negative and double-positive thymocytes. These findings give clues about the evolution of this key molecule in amniotes and suggest that the ancestral function of pTα was exclusively to enable expression of the TCRß chain at the thymocyte surface and to allow binding of pre-TCR to the CD3 complex. Together, our data provide arguments for revisiting the current model of pTα signaling.
Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Vertebrados/imunologia , Sequência de Aminoácidos , Animais , Anuros/imunologia , Aves/imunologia , Peixes/imunologia , Regulação da Expressão Gênica , Humanos , Mamíferos/imunologia , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Répteis/imunologia , Alinhamento de Sequência , Relação Estrutura-Atividade , Vertebrados/genéticaRESUMO
Cell lines are useful tools to facilitate in vitro studies of many biological and molecular processes. We describe a new permanent fibroblast-type cell line obtained from disaggregated Xenopus tropicalis limb bud. The cell line population doubling time was ~24 h. Its karyotype was genetically stable with a chromosome number of 2n = 21 and a chromosome 10 trisomy. These cells could be readily transfected and expressed transgenes faithfully. We obtained stable transformants using transposon-based gene transfer technology. These cells responded to thyroid hormone and thus can provide a complementary research tool to study thyroid hormone signaling events. In conclusion, this cell line baptized "Speedy" should prove useful to couple in vitro and in vivo biological studies in the X. tropicalis frog model.
Assuntos
Linhagem Celular , Xenopus/genética , Animais , Elementos de DNA Transponíveis , Ordem dos Genes , Técnicas de Transferência de Genes , Genes Reporter , Vetores Genéticos/genética , Cariótipo , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Hormônios Tireóideos/farmacologia , Transfecção , Transgenes , Xenopus/metabolismoRESUMO
BACKGROUND: Inverted repeat genes encode precursor RNAs characterized by hairpin structures. These RNA hairpins are then metabolized by biosynthetic pathways to produce functional small RNAs. In eukaryotic genomes, short non-autonomous transposable elements can have similar size and hairpin structures as non-coding precursor RNAs. This resemblance leads to problems annotating small RNAs. RESULTS: We mapped all microRNA precursors from miRBASE to several genomes and studied the repetition and dispersion of the corresponding loci. We then searched for repetitive elements overlapping these loci. We developed an automatic method called ncRNAclassifier to classify pre-ncRNAs according to their relationship with transposable elements (TEs). We showed that there is a correlation between the number of scattered occurrences of ncRNA precursor candidates and the presence of TEs. We applied ncRNAclassifier on six chordate genomes and report our findings. Among the 1,426 human and 721 mouse pre-miRNAs of miRBase, we identified 235 and 68 mis-annotated pre-miRNAs respectively corresponding completely to TEs. CONCLUSIONS: We provide a tool enabling the identification of repetitive elements in precursor ncRNA sequences. ncRNAclassifier is available at http://EvryRNA.ibisc.univ-evry.fr.
Assuntos
Sequências Repetitivas Dispersas , Sequências Repetidas Invertidas , MicroRNAs/genética , Precursores de RNA/química , Software , Animais , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , MicroRNAs/química , MicroRNAs/classificação , Precursores de RNA/classificação , Precursores de RNA/genética , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/classificação , Pequeno RNA não Traduzido/genética , RatosRESUMO
Synthetic biology and metabolic engineering are used to develop new strategies for producing valuable compounds ranging from therapeutics to biofuels in engineered microorganisms. When developing methods for high-titer production cells, toxicity is an important element to consider. Indeed the production rate can be limited due to toxic intermediates or accumulation of byproducts of the heterologous biosynthetic pathway of interest. Conversely, highly toxic molecules are desired when designing antimicrobials. Compound toxicity in bacteria plays a major role in metabolic engineering as well as in the development of new antibacterial agents. Here, we screened a diversified chemical library of 166 compounds for toxicity in Escherichia coli. The dataset was built using a clustering algorithm maximizing the chemical diversity in the library. The resulting assay data was used to develop a toxicity predictor that we used to assess the toxicity of metabolites throughout the metabolome. This new tool for predicting toxicity can thus be used for fine-tuning heterologous expression and can be integrated in a computational-framework for metabolic pathway design. Many structure-activity relationship tools have been developed for toxicology studies in eukaryotes [Valerio (2009), Toxicol Appl Pharmacol, 241(3): 356-370], however, to the best of our knowledge we present here the first E. coli toxicity prediction web server based on QSAR models (EcoliTox server: http://www.issb.genopole.fr/â¼faulon/EcoliTox.php).
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Escherichia coli/efeitos dos fármacos , Preparações Farmacêuticas/química , Relação Estrutura-Atividade , Biotecnologia/métodos , Internet , Engenharia Metabólica/métodosRESUMO
We gathered a collection of termite mutualistic strains from French Guiana to explore the metabolites of symbiotic microorganisms. Molecular networks reconstructed from a metabolomic analysis using LC-ESI-MS/MS methodology led us to identify two families of chlorinated polyketides, i.e., azaphilones from Penicillium sclerotiorum and ilicicolins from Neonectria discophora. To define the biosynthetic pathways related to these two types of scaffolds, we used a whole genome sequencing approach followed by hybrid assembly from short and long reads. We found two biosynthetic gene clusters, including two FAD-dependent halogenases. To exploit the enzymatic promiscuity of the two identified FAD halogenases, we sought to biosynthesize novel halogenated metabolites. An OSMAC strategy was used and resulted in the production of brominated analogs of ilicicolins and azaphilones as well as iodinated analogs of azaphilones.
Assuntos
Isópteros , Policetídeos , Animais , Benzopiranos , Flavina-Adenina Dinucleotídeo , Genômica , Isópteros/genética , Isópteros/metabolismo , Pigmentos Biológicos , Policetídeos/metabolismo , Espectrometria de Massas em TandemRESUMO
Fibroblast growth factors (FGFs) signal through high-affinity tyrosine kinase receptors to regulate a diverse range of cellular processes, including cell growth, differentiation and migration, as well as cell death. Here we identify XFLRT3, a member of a leucine-rich-repeat transmembrane protein family, as a novel modulator of FGF signalling. XFLRT3 is co-expressed with FGFs, and its expression is both induced after activation and downregulated after inhibition of FGF signalling. In gain- and loss-of function experiments, FLRT3 and FLRT2 phenocopy FGF signalling in Xenopus laevis. XFLRT3 signalling results in phosphorylation of ERK and is blocked by MAPK phosphatase 1, but not by expression of a dominant-negative phosphatidyl inositol 3-OH kinase (PI(3)K) mutant. XFLRT3 interacts with FGF receptors (FGFRs) in co-immunoprecipitation experiments in vitro and in bioluminescence resonance energy transfer assays in vivo. The results indicate that XFLRT3 is a transmembrane modulator of FGF-MAP kinase signalling in vertebrates.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Sequência de Bases/genética , Diferenciação Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , DNA Complementar/análise , DNA Complementar/genética , Regulação para Baixo/genética , Fosfatase 1 de Especificidade Dupla , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Indução Embrionária/genética , Retroalimentação Fisiológica/genética , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/farmacologia , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Xenopus laevis/metabolismoRESUMO
We describe a protocol to prepare a multiplexed mtDNA library from a blood sample for performing a long read sequencing of the mitochondrial genome. All steps are carefully described to get a high enrichment of mtDNA relative to total DNA extracted from the blood sample. The obtained mutiplexed library allows the production of long sequence mtDNA reads up to 16.5 kbp with a quality enabling variant-calling by using a portable sequencer (MinION, Oxford Nanopore Technologies).
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DNA Mitocondrial/genética , Genoma Mitocondrial , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Coleta de Amostras Sanguíneas , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , HumanosRESUMO
The Mediterranean corn borer (Sesamia nonagrioides, Noctuidae, Lepidoptera) is a major pest of maize in Europe and Africa. Here, we report an assembly of the nuclear and mitochondrial genome of a pool of inbred males and females third-instar larvae, based on short- and long-read sequencing. The complete mitochondrial genome is 15,330 bp and contains all expected 13 and 24 protein-coding and RNA genes, respectively. The nuclear assembly is 1021 Mb, composed of 2553 scaffolds and it has an N50 of 1105 kb. It is more than twice larger than that of all Noctuidae species sequenced to date, mainly due to a higher repeat content. A total of 17,230 protein-coding genes were predicted, including 15,776 with InterPro domains. We provide detailed annotation of genes involved in sex determination (doublesex, insulin-like growth factor 2 mRNA-binding protein, and P-element somatic inhibitor) and of alpha-amylase genes possibly involved in interaction with parasitoid wasps. We found no evidence of recent horizontal transfer of bracovirus genes from parasitoid wasps. These genome assemblies provide a solid molecular basis to study insect genome evolution and to further develop biocontrol strategies against S. nonagrioides.
Assuntos
Genoma Mitocondrial , Mariposas , Vespas , Animais , Zea mays/genética , Mariposas/genética , Vespas/genética , LarvaRESUMO
Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by motor neuron loss and skeletal muscle atrophy. The loss of function of the smn1 gene, the main supplier of survival motor neuron protein (SMN) protein in human, leads to reduced levels of SMN and eventually to SMA. Here, we ask if the amphibian Xenopus tropicalis can be a good model system to study SMA. Inhibition of the production of SMN using antisense morpholinos leads to caudal muscular atrophy in tadpoles. Of note, early developmental patterning of muscles and motor neurons is unaffected in this system as well as acetylcholine receptors clustering. Muscular atrophy seems to rather result from aberrant pathfinding and growth arrest and/or shortening of motor axons. This event occurs in the absence of neuronal cell bodies apoptosis, a process comparable to that of amyotrophic lateral sclerosis. Xenopus tropicalis is revealed as a complementary animal model for the study of SMA.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/biossíntese , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Modelos Animais de Doenças , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Receptores Colinérgicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , XenopusRESUMO
The early development of many animals relies on the posttranscriptional regulations of maternally stored mRNAs. In particular, the translation of maternal mRNAs is tightly controlled during oocyte maturation and early mitotic cycles in Xenopus. The Embryonic Deadenylation ElemeNt (EDEN) and its associated protein EDEN-BP are known to trigger deadenylation and translational silencing to several mRNAs bearing an EDEN. This Xenopus RNA-binding protein is an ortholog of the human protein CUG-BP1/CELF1. Five mRNAs, encoding cell cycle regulators and a protein involved in the notch pathway, have been identified as being deadenylated by EDEN/EDEN-BP. To identify new EDEN-BP targets, we immunoprecipitated EDEN-BP/mRNA complexes from Xenopus tropicalis egg extracts. We identified 153 mRNAs as new binding targets for EDEN-BP using microarrays. Sequence analyses of the 3' untranslated regions of the newly identified EDEN-BP targets reveal an enrichment in putative EDEN sequences. EDEN-BP binding to a subset of the targets was confirmed both in vitro and in vivo. Among the newly identified targets, Cdk1, a key player of oocyte maturation and cell cycle progression, is specifically targeted by its 3' UTR for an EDEN-BP-dependent deadenylation after fertilization.
Assuntos
RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/genética , Regiões 3' não Traduzidas/química , Regiões 3' não Traduzidas/metabolismo , Animais , Sítios de Ligação , Proteína Quinase CDC2/metabolismo , Imunoprecipitação , Análise de Sequência com Séries de Oligonucleotídeos , Óvulo/metabolismo , Proteínas de Ligação a RNA/imunologia , Xenopus/crescimento & desenvolvimento , Proteínas de Xenopus/imunologiaRESUMO
Xenbase (www.xenbase.org) is a model organism database integrating a diverse array of biological and genomic data on the frogs, Xenopus laevis and Xenopus (Silurana) tropicalis. Data is collected from other databases, high-throughput screens and the scientific literature and integrated into a number of database modules covering subjects such as community, literature, gene and genomic analysis. Gene pages are automatically assembled from data piped from the Entrez Gene, Gurdon Institute, JGI, Metazome, MGI, OMIM, PubMed, Unigene, Zfin, commercial suppliers and others. These data are then supplemented with in-house annotation. Xenbase has implemented the Gbrowse genome browser and also provides a BLAST service that allows users to specifically search either laevis or tropicalis DNA or protein targets. A table of Xenopus gene synonyms has been implemented and allows the genome, genes, publications and high-throughput gene expression data to be seamlessly integrated with other Xenopus data and to external database resources, making the wealth of developmental and functional data from the frog available to the broader research community.
Assuntos
Bases de Dados Genéticas , Genômica , Xenopus laevis/genética , Xenopus/genética , Animais , Expressão Gênica , Genes , Internet , PubMed , Terminologia como Assunto , Interface Usuário-Computador , Xenopus/anatomia & histologiaRESUMO
The large amphibian genomes contain numerous repetitive DNA components that have played an important role in the karyotypic diversification of this vertebrate group. Hypotheses based on the presumable primitive karyotype (2n = 20) of the anurans of the family Pipidae suggest that they have evolved principally through intrachromosomal rearrangements. Pipa is the only South American pipid, while all the other genera are found in Africa. The divergence of the South American lineages from the African ones occurred at least 136 million years ago and is thought to have had a strong biogeographic component. Here, we tested the potential of the repetitive DNA to enable a better understanding of the differentiation of the karyotype among the family Pipidae and to expand our capacity to interpret the chromosomal evolution in this frog family. Our results indicate a long history of conservation in the chromosome bearing the H3 histone locus, corroborating inferences on the chromosomal homologies between the species in pairs 6, 8, and 9. The chromosomal distribution of the microsatellite motifs also provides useful markers for comparative genomics at the chromosome level between Pipa carvalhoi and Xenopus tropicalis, contributing new insights into the evolution of the karyotypes of these species. We detected similar patterns in the distribution and abundance of the microsatellite arrangements, which reflect the shared organization in the terminal/subterminal region of the chromosomes between these two species. By contrast, the microsatellite probes detected a differential arrangement of the repetitive DNA among the chromosomes of the two species, allowing longitudinal differentiation of pairs that are identical in size and morphology, such as pairs 1, 2, 4, and 5. We also found evidence of the distinctive composition of the repetitive motifs of the centromeric region between the species analyzed in the present study, with a clear enrichment of the (CA) and (GA) microsatellite motifs in P. carvalhoi. Finally, microsatellite enrichment in the pericentromeric region of chromosome pairs 6, 8, and 9 in the P. carvalhoi karyotype, together with interstitial telomeric sequences (ITS), validate the hypothesis that pericentromeric inversions occurred during the chromosomal evolution of P. carvalhoi and reinforce the role of the repetitive DNA in the remodeling of the karyotype architecture of the Pipidae.