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1.
Mol Biol Evol ; 37(1): 149-166, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31553476

RESUMO

Small nucleolar RNAs (snoRNAs) function primarily as guide RNAs for posttranscriptional modification of rRNAs and spliceosomal snRNAs, both of which are functionally important and evolutionarily conserved molecules. It is commonly believed that snoRNAs and the modifications they mediate are highly conserved across species. However, most relevant data on snoRNA annotation and RNA modification are limited to studies on human and yeast. Here, we used RNA-sequencing data from the giant oocyte nucleus of the frog Xenopus tropicalis to annotate a nearly complete set of snoRNAs. We compared the frog data with snoRNA sets from human and other vertebrate genomes, including mammals, birds, reptiles, and fish. We identified many Xenopus-specific (or nonhuman) snoRNAs and Xenopus-specific domains in snoRNAs from conserved RNA families. We predicted that some of these nonhuman snoRNAs and domains mediate modifications at unexpected positions in rRNAs and snRNAs. These modifications were mapped as predicted when RNA modification assays were applied to RNA from nine vertebrate species: frogs X. tropicalis and X. laevis, newt Notophthalmus viridescens, axolotl Ambystoma mexicanum, whiptail lizard Aspidoscelis neomexicana, zebrafish Danio rerio, chicken, mouse, and human. This analysis revealed that only a subset of RNA modifications is evolutionarily conserved and that modification patterns may vary even between closely related species. We speculate that each functional domain in snoRNAs (half of an snoRNA) may evolve independently and shuffle between different snoRNAs.


Assuntos
RNA Nucleolar Pequeno/genética , Xenopus/genética , Animais , Genoma , Humanos , Anotação de Sequência Molecular , Mutação Puntual
2.
Rev Environ Contam Toxicol ; 252: 131-171, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463624

RESUMO

Anurans from the genus Xenopus have long been used as standard testing organisms and occur naturally in tropical and sub-tropical areas where malaria vector control pesticides are actively used. However, literature on the toxic effects of these pesticides is limited. This review analyses the available data pertaining to both Xenopus and the pesticides used for malaria vector control in order to determine the pesticides that have the greatest potential to influence amphibian health while also identifying gaps in literature that need to be addressed. Amphibian diversity has shown the fastest decline of any group, yet there are still voids in our understanding of how this is happening. The lack of basic toxicity data on amphibians with regard to pesticides is an issue that needs to be addressed in order to improve effectiveness of amphibian conservation strategies. Meta-analyses performed in this review show that, at current usage, with the available acute toxicity literature, the pyrethroid pesticide group could hold the highest potential to cause acute toxicity to Xenopus sp. in relation to the other MVCPs discussed, but the lack of data cripples the efficacy with which meta-analyses can be performed and conclusions made from such analyses. Several studies have shown that DDT accumulates in Xenopus sp. from malaria vector control areas, but accumulation of other MVCPs in frogs is still largely unknown. Through this review we hope to encourage future research into the field of amphibian ecotoxicology and to promote the use of the Xenopus standard model in order to build comprehensive datasets that may be used in amphibian conservation.


Assuntos
Ecotoxicologia , Poluentes Ambientais/toxicidade , Malária , Controle de Mosquitos , Mosquitos Vetores , Praguicidas/toxicidade , Animais , Anopheles , Xenopus , Xenopus laevis
3.
Elife ; 82019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31566562

RESUMO

Base excision repair (BER) functions not only in the maintenance of genomic integrity but also in active DNA demethylation and epigenetic gene regulation. This dual role raises the question if phenotypic abnormalities resulting from deficiency of BER factors are due to DNA damage or impaired DNA demethylation. Here we investigate the bifunctional DNA glycosylases/lyases NEIL1 and NEIL2, which act in repair of oxidative lesions and in epigenetic demethylation. Neil-deficiency in Xenopus embryos and differentiating mouse embryonic stem cells (mESCs) leads to a surprisingly restricted defect in cranial neural crest cell (cNCC) development. Neil-deficiency elicits an oxidative stress-induced TP53-dependent DNA damage response, which impairs early cNCC specification. Epistasis experiments with Tdg-deficient mESCs show no involvement of epigenetic DNA demethylation. Instead, Neil-deficiency results in oxidative damage specific to mitochondrial DNA, which triggers a TP53-mediated intrinsic apoptosis. Thus, NEIL1 and NEIL2 DNA glycosylases protect mitochondrial DNA against oxidative damage during neural crest differentiation.


Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , Células-Tronco Embrionárias/fisiologia , Mitocôndrias/metabolismo , Crista Neural/embriologia , Estresse Oxidativo , Animais , Diferenciação Celular , Linhagem Celular , Reparo do DNA , Camundongos , Xenopus
4.
Nat Chem Biol ; 15(12): 1156-1164, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31591563

RESUMO

Phospholipids are key components of cellular membranes and are emerging as important functional regulators of different membrane proteins, including pentameric ligand-gated ion channels (pLGICs). Here, we take advantage of the prokaryote channel ELIC (Erwinia ligand-gated ion channel) as a model to understand the determinants of phospholipid interactions in this family of receptors. A high-resolution structure of ELIC in a lipid-bound state reveals a phospholipid site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics. This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABAA/C and glycine receptors. A combined approach reveals that M4 is intrinsically flexible and that M4 deletions or disruptions of the lipid-binding site accelerate desensitization in ELIC, suggesting that lipid interactions shape the agonist response. Our data offer a structural context for understanding lipid modulation in pLGICs.


Assuntos
Ativação do Canal Iônico , Canais Iônicos/metabolismo , Lipídeos/química , Animais , Ligantes , Mutagênese , Xenopus
5.
Nat Commun ; 10(1): 4269, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537794

RESUMO

Embryonic development yields many different cell types in response to just a few families of inductive signals. The property of signal-receiving cells that determines how they respond to inductive signals is known as competence, and it differs in different cell types. Here, we explore the ways in which maternal factors modify chromatin to specify initial competence in the frog Xenopus tropicalis. We identify early-engaged regulatory DNA sequences, and infer from them critical activators of the zygotic genome. Of these, we show that the pioneering activity of the maternal pluripotency factors Pou5f3 and Sox3 determines competence for germ layer formation by extensively remodelling compacted chromatin before the onset of inductive signalling. This remodelling includes the opening and marking of thousands of regulatory elements, extensive chromatin looping, and the co-recruitment of signal-mediating transcription factors. Our work identifies significant developmental principles that inform our understanding of how pluripotent stem cells interpret inductive signals.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus/embriologia , Animais , Diferenciação Celular/genética , Cromatina/metabolismo , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/crescimento & desenvolvimento , Sequências Reguladoras de Ácido Nucleico/genética , Xenopus/genética
6.
Elife ; 82019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31364986

RESUMO

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.


Assuntos
Epigênese Genética , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , Transcrição Genética , Animais , Histonas/metabolismo , Xenopus
7.
Int J Mol Sci ; 20(17)2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31454910

RESUMO

The interaction between the pituitary hormone, adrenocorticotropin (ACTH), and melanocortin-2 receptor (MC2R) orthologs involves the H6 F7 R8 W9 and R/K15 K16 R17 R18 motifs in ACTH making contact with corresponding contact sites on MC2R. Earlier studies have localized the common HFRW binding site of all melanocortin receptors to residues in TM2, TM3, and TM6 that are located close to the extracellular space. The current study has identified residues in Xenopus tropicalis (xt) MC2R in TM4 (I158, F161), in EC2 (M166), and in TM5 (V172) that also are involved in activation of xtMC2R, and may be in the R/KKRR contact site of xtMC2R. These results are compared to earlier studies on the corresponding domains of human MC2R and rainbow trout MC2R in an effort to identify common features in the activation of teleost and tetrapod MC2R orthologs following stimulation with ACTH.


Assuntos
Receptor Tipo 2 de Melanocortina/metabolismo , Xenopus/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Sequência de Aminoácidos , Animais , Células CHO , Membrana Celular/metabolismo , Cricetulus , Humanos , Mutação , Receptor Tipo 2 de Melanocortina/agonistas , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/genética , Xenopus/genética
8.
Insect Biochem Mol Biol ; 114: 103204, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31422151

RESUMO

Ionotropic receptors (IRs), as a member of the conserved chemoreceptor families in the peripheral nervous system, play a critical role in the chemoreception of Drosophila. However, little is known about IRs in Hymenoptera insects. Here, we comprehensively characterized the gene structure, topological map and chemosensory roles of antennal IRs (MmedIRs) in the hymenopteran parasitoid wasp Microplitis mediator. We found that the IRs were conserved across various insect species. In the in situ hybridization assays, most IRs showed female antennae biased features, and there was no co-expression of the IRs and the olfactory receptor co-receptor (ORco). Moreover, three IR co-expressed complexes, IR75u-IR8a, IR64a1-IR8a and IR64a2-IR8a, were detected. Two genes with high similarity, IR64a1 and IR64a2, were located in distinct neurons but projected to the same sensillum. In two-electrode voltage-clamp recordings, IR64a1 was widely tuned to the chemicals from habitat cues released from host plants over long distances, whereas IR64a2 responded to a narrow range host cues and plant odors with low-volatility. Notably, IR64a2 was able to perceive Z9-14: Ald, a vital sex pheromone component that is released from Helicoverpa armigera, which is the preferred host of M. mediator. Furthermore, most ligands of IR64a1 and IR64a2 can trigger electrophysiological responses in female wasps. We propose that IR64a1 and IR64a2 collaboratively perceive habitat and host cues to assist parasitoids in efficiently seeking hosts.


Assuntos
Antenas de Artrópodes/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Vespas/metabolismo , Animais , Feminino , Masculino , Receptores Ionotrópicos de Glutamato/genética , Vespas/genética , Xenopus
9.
Plant Cell Physiol ; 60(11): 2423-2435, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292634

RESUMO

Control of K+ and Na+ transport plays a central role in plant adaptation to salinity. In the halophyte Hordeum maritimum, we have characterized a transporter gene, named HmHKT2;1, whose homolog HvHKT2;1 in cultivated barley, Hordeum vulgare, was known to give rise to increased salt tolerance when overexpressed. The encoded protein is strictly identical in two H. maritimum ecotypes, from two biotopes (Tunisian sebkhas) affected by different levels of salinity. These two ecotypes were found to display distinctive responses to salt stress in terms of biomass production, Na+ contents, K+ contents and K+ absorption efficiency. Electrophysiological analysis of HmHKT2;1 in Xenopus oocytes revealed distinctive properties when compared with HvHKT2;1 and other transporters from the same group, especially a much higher affinity for both Na+ and K+, and an Na+-K+ symporter behavior in a very broad range of Na+ and K+ concentrations, due to reduced K+ blockage of the transport pathway. Domain swapping experiments identified the region including the fifth transmembrane segment and the adjacent extracellular loop as playing a major role in the determination of the affinity for Na+ and the level of K+ blockage in these HKT2;1 transporters. The analysis (quantitative reverse transcription-PCR; qRT-PCR) of HmHKT2;1 expression in the two ecotypes submitted to saline conditions revealed that the levels of HmHKT2;1 transcripts were maintained constant in the most salt-tolerant ecotype whereas they decreased in the less tolerant one. Both the unique functional properties of HmHKT2;1 and the regulation of the expression of the encoding gene could contribute to H. maritimum adaptation to salinity.


Assuntos
Hordeum/metabolismo , Proteínas de Plantas/metabolismo , Animais , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Hordeum/genética , Proteínas de Plantas/genética , Potássio/metabolismo , Plantas Tolerantes a Sal/metabolismo , Sódio/metabolismo , Xenopus
10.
Int J Mol Sci ; 20(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261773

RESUMO

Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystallization or cryo-electronic microscopy will be attempted. This will be a way to gain information on the structure of the hERG active site and its modelization to obtain data on the pauses of various reference compounds from the pharmacopeia, as well as to gain information about the thermodynamics of the hERG/ligand relationship.


Assuntos
Canal de Potássio ERG1/genética , Engenharia de Proteínas/métodos , Animais , Fracionamento Químico/métodos , Cristalografia por Raios X/métodos , Canal de Potássio ERG1/química , Canal de Potássio ERG1/metabolismo , Células HEK293 , Humanos , Pichia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Xenopus
11.
Elife ; 82019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31329101

RESUMO

Upon membrane depolarization, the KCNQ1 potassium channel opens at the intermediate (IO) and activated (AO) states of the stepwise voltage-sensing domain (VSD) activation. In the heart, KCNQ1 associates with KCNE1 subunits to form IKs channels that regulate heart rhythm. KCNE1 suppresses the IO state so that the IKs channel opens only to the AO state. Here, we tested modulations of human KCNQ1 channels by an activator ML277 in Xenopus oocytes. It exclusively changes the pore opening properties of the AO state without altering the IO state, but does not affect VSD activation. These observations support a distinctive mechanism responsible for the VSD-pore coupling at the AO state that is sensitive to ML277 modulation. ML277 provides insights and a tool to investigate the gating mechanism of KCNQ1 channels, and our study reveals a new strategy for treating long QT syndrome by specifically enhancing the AO state of native IKs currents.


Assuntos
Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/tratamento farmacológico , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Animais , Membrana Celular/genética , Membrana Celular/fisiologia , Polaridade Celular/genética , Humanos , Síndrome do QT Longo/genética , Síndrome do QT Longo/patologia , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Piperidinas/farmacologia , Potássio/metabolismo , Tiazóis/farmacologia , Compostos de Tosil/farmacologia , Xenopus/genética
12.
Nature ; 572(7767): 131-135, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31316205

RESUMO

Pathogen-associated molecular patterns (PAMPs) activate innate immunity in both animals and plants. Although calcium has long been recognized as an essential signal for PAMP-triggered immunity in plants, the mechanism of PAMP-induced calcium signalling remains unknown1,2. Here we report that calcium nutrient status is critical for calcium-dependent PAMP-triggered immunity in plants. When calcium supply is sufficient, two genes that encode cyclic nucleotide-gated channel (CNGC) proteins, CNGC2 and CNGC4, are essential for PAMP-induced calcium signalling in Arabidopsis3-7. In a reconstitution system, we find that the CNGC2 and CNGC4 proteins together-but neither alone-assemble into a functional calcium channel that is blocked by calmodulin in the resting state. Upon pathogen attack, the channel is phosphorylated and activated by the effector kinase BOTRYTIS-INDUCED KINASE1 (BIK1) of the pattern-recognition receptor complex, and this triggers an increase in the concentration of cytosolic calcium8-10. The CNGC-mediated calcium entry thus provides a critical link between the pattern-recognition receptor complex and calcium-dependent immunity programs in the PAMP-triggered immunity signalling pathway in plants.


Assuntos
Arabidopsis/imunologia , Arabidopsis/metabolismo , Calmodulina/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Padrões Moleculares Associados a Patógenos/imunologia , Imunidade Vegetal/imunologia , Animais , Proteínas de Arabidopsis/agonistas , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio , Calmodulina/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/agonistas , Canais de Cátion Regulados por Nucleotídeos Cíclicos/antagonistas & inibidores , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Feminino , Imunidade Inata , Oócitos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Xenopus
13.
Sci Total Environ ; 689: 149-159, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31271984

RESUMO

Numerous studies suggest that amphibians are highly sensitive to endocrine disruptors (ED) but their precise role in population decline remains unknown. This study shows that frogs exposed to a mixture of ED throughout their life cycle, at environmentally relevant concentrations, developed an unexpected metabolic syndrome. Female Silurana (Xenopus) tropicalis exposed to a mixture of benzo[a]pyrene and triclosan (50 ng·L-1 each) from the tadpole stage developed liver steatosis and transcriptomic signature associated with glucose intolerance syndrome, and pancreatic insulin hyper secretion typical of pre-diabetes. These metabolic disorders were associated with delayed metamorphosis and developmental mortality in their progeny, both of which have been linked to reduced adult recruitment and reproductive success. Indeed, F1 females were smaller and lighter and presented reduced reproductive capacities, demonstrating a reduced fitness of ED-exposed Xenopus. Our results confirm that amphibians are highly sensitive to ED even at concentrations considered to be safe for other animals. This study demonstrates that ED might be considered as direct contributing factors to amphibian population decline, due to their disruption of energetic metabolism.


Assuntos
Benzo(a)pireno/toxicidade , Disruptores Endócrinos/toxicidade , Doenças Metabólicas/veterinária , Metamorfose Biológica/efeitos dos fármacos , Triclosan/toxicidade , Xenopus/metabolismo , Animais , Relação Dose-Resposta a Droga , Feminino , Fígado/efeitos dos fármacos , Fígado/fisiologia , Fígado/fisiopatologia , Doenças Metabólicas/induzido quimicamente , Doenças Metabólicas/metabolismo , Síndrome Metabólica/induzido quimicamente , Síndrome Metabólica/metabolismo , Síndrome Metabólica/veterinária , Reprodução/efeitos dos fármacos , Transcriptoma
14.
BMC Plant Biol ; 19(1): 268, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221084

RESUMO

BACKGROUND: Research on plant amino acid transporters was mainly performed in Arabidopsis, while our understanding of them is generally scant in rice. OsLHT1 (Lysine/Histidine transporter) has been previously reported as a histidine transporter in yeast, but its substrate profile and function in planta are unclear. The aims of this study are to analyze the substrate selectivity of OsLHT1 and influence of its disruption on rice growth and fecundity. RESULTS: Substrate selectivity of OsLHT1 was analyzed in Xenopus oocytes using the two-electrode voltage clamp technique. The results showed that OsLHT1 could transport a broad spectrum of amino acids, including basic, neutral and acidic amino acids, and exhibited a preference for neutral and acidic amino acids. Two oslht1 mutants were generated using CRISPR/Cas9 genome-editing technology, and the loss-of-function of OsLHT1 inhibited rice root and shoot growth, thereby markedly reducing grain yields. QRT-PCR analysis indicated that OsLHT1 was expressed in various rice organs, including root, stem, flag leaf, flag leaf sheath and young panicle. Transient expression in rice protoplast suggested OsLHT1 was localized to the plasma membrane, which is consistent with its function as an amino acid transporter. CONCLUSIONS: Our results indicated that OsLHT1 is an amino acid transporter with wide substrate specificity and with preference for neutral and acidic amino acids, and disruption of OsLHT1 function markedly inhibited rice growth and fecundity.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Aminoácidos/metabolismo , Animais , Sítios de Ligação , Técnicas de Inativação de Genes , Filogenia , Proteínas de Plantas/genética , Xenopus
15.
PLoS One ; 14(6): e0218806, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31233570

RESUMO

In the course of recent comparative genomic studies conducted on nervous systems across the phylogeny, current thinking is leaning in favor of more heterogeneity among nervous systems than what was initially expected. The isolation and characterization of molecular components that constitute the cnidarian neuron is not only of interest to the physiologist but also, on a larger scale, to those who study the evolution of nervous systems. Understanding the function of those ancient neurons involves the identification of neurotransmitters and their precursors, the description of nutrients used by neurons for metabolic purposes and the identification of integral membrane proteins that bind to those compounds. Using a molecular cloning strategy targeting membrane proteins that are known to be present in all forms of life, we isolated a member of the solute carrier family 6 from the scyphozoan jellyfish Cyanea capillata. The phylogenetic analysis suggested that the new transporter sequence belongs to an ancestral group of the nutrient amino acid transporter subfamily and is part of a cluster of cnidarian sequences which may translocate the same substrate. We found that the jellyfish transporter is expressed in neurons of the motor nerve net of the animal. To this end, we established an in situ hybridization protocol for the tissues of C. capillata and developed a specific antibody to the jellyfish transporter. Finally, we showed that the gene that codes for the jellyfish transporter also expresses a long non-coding RNA. We hope that this research will contribute to studies that seek to understand what constitutes a neuron in species that belong to an ancient phylum.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Cifozoários/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/genética , Animais , Clonagem Molecular , Evolução Molecular , Feminino , Células HEK293 , Humanos , Hibridização In Situ , Neurônios Motores/metabolismo , Rede Nervosa/metabolismo , Oócitos/metabolismo , Filogenia , RNA Longo não Codificante/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cifozoários/classificação , Cifozoários/genética , Homologia de Sequência de Aminoácidos , Xenopus
17.
Zygote ; 27(4): 195-202, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31250783

RESUMO

We isolated and characterized Xenopus tropicalis hb4 flanking DNA and showed that the -3076/+29 sequence was able to drive stage-specific transcription in the developmental process. Transgenic reporter analysis indicated that green fluorescent protein was expressed in the ovaries of female frogs at 3 months of age and in both the ovaries and testis of frogs at 6 months of age. A series of experiments with deletion of the flanking sequence and a subsequent luciferase reporter assay revealed that there were two positive regulatory regions and that the most proximal sequence of the promoter region had a certain level of transcriptional activity in oocytes. Subsequently, we showed that a conserved sequence containing Nobox-binding element (NBE) was essential for transcriptional activation and that Nobox expressed in the ovary had a crucial role in hb4 transcription through the NBE sequence.


Assuntos
Proteínas Nucleares/genética , Oócitos/metabolismo , Regiões Promotoras Genéticas/genética , Elementos Reguladores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Ovário/citologia , Ovário/metabolismo , Ativação Transcricional , Xenopus laevis/genética
18.
Insect Biochem Mol Biol ; 111: 103176, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31150760

RESUMO

The Oriental armyworm, Mythimna separata, has been described to emit geographic population-specific sex pheromones, with either Z11-16:Ald or Z11-16:Ac as the major component. Using a comprehensive set of electrophysiological, behavioral, and genetic analyses, we study the sex pheromone communication of M. separata in North China from pheromone receptors and antennal lobe to behavior. GC-EAD results show that Z11-16:Ald is the only compound eliciting electrophysiological responses in pheromone gland extracts. Further in vivo optical imaging studies reveal that Z11-16:Ald activates the cumulus of the MGC and show dose-dependent responses. The wind tunnel tests demonstrate that Z11-16:Ald alone is sufficient to induce the entire sequence of male sexual behaviors. Transcriptome and q-PCR results show that MsepOR3 is specifically and abundantly expressed in male antennae. By using the Xenopus oocytes and two-electrode voltage-clamp recording, we finally validate that the oocytes expressing MsepOR3/ORco gave dose dependent responses to Z11-16:Ald. We suggest single Z11-16:Ald could be used for monitoring the population of M. separata in North China.


Assuntos
Mariposas/efeitos dos fármacos , Atrativos Sexuais/farmacologia , Comportamento Sexual Animal/efeitos dos fármacos , Animais , Antenas de Artrópodes/fisiologia , China , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Feminino , Masculino , Mariposas/genética , Oócitos , Técnicas de Patch-Clamp , Xenopus
19.
PLoS Negl Trop Dis ; 13(6): e0007432, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31158225

RESUMO

BACKGROUND: Multiple mutations in the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides in a major human disease vector Aedes aegypti. One mutation, V1016G, confers sodium channel resistance to pyrethroids, but a different substitution in the same position V1016I alone had no effect. In pyrethroid-resistant Ae. aegypti populations, V1016I is often linked to another mutation, F1534C, which confers sodium channel resistance only to Type I pyrethroids including permethrin (PMT), but not to Type II pyrethroids including deltamethrin (DMT). Mosquitoes carrying both V1016G and F1534C exhibited a greater level of pyrethroid resistance than those carrying F1534C alone. More recently, a new mutation T1520I co-existing with F1534C was detected in India. However, whether V1016I or T1520I enhances pyrethroid resistance of sodium channels carrying F1534C remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: V1016I, V1016G, T1520I and F1534C substitutions were introduced alone and in various combinations into AaNav1-1, a sodium channel from Aedes aegypti. The mutant channels were then expressed in Xenopus oocytes and examined for channel properties and sensitivity to pyrethroids using the two-electrode voltage clamping technique. The results showed that V1016I or T1520I alone did not alter the AaNav1-1 sensitivity to PMT or DMT. However, the double mutant T1520I+F1534C was more resistant to PMT than F1534C, but remained sensitive to DMT. In contrast, the double mutant V1016I+F1534C was resistant to DMT and more resistant to PMT than F1534C. Furthermore, V1016I/G and F1534C channels, but not T1520I, were resistant to dichlorodiphenyltrichloroethane (DDT). Cryo-EM structures of sodium channels suggest that T1520I allosterically deforms geometry of the pyrethroid receptor site PyR1 in AaNav1-1. The small deformation does not affect binding of DDT, PMT or DMT, but in combination with F1534C it increases the channel resistance to PMT and DDT. CONCLUSIONS/SIGNIFICANCE: Our data corroborated the previously proposed sequential selection of kdr mutations in Ae. aegypti. We proposed that mutation F1534C first emerged in response to DDT/pyrethroids providing a platform for subsequent selection of mutations V1016I and T1520I that confer greater and broader spectrum of pyrethroid resistance.


Assuntos
Aedes/genética , DDT/farmacologia , Evolução Molecular , Resistência a Inseticidas , Inseticidas/farmacologia , Piretrinas/farmacologia , Canais de Sódio/genética , Aedes/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Células Cultivadas , Expressão Gênica , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Canais de Sódio/metabolismo , Xenopus
20.
Dev Cell ; 49(4): 643-650.e3, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31112700

RESUMO

It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present an analysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does not support the authors' general conclusion, which was based on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.


Assuntos
Imunidade Inata/imunologia , Morfolinos/imunologia , Morfolinos/metabolismo , Animais , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Imunidade Inata/fisiologia , Oligonucleotídeos Antissenso/genética , Processamento de RNA , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Transcriptoma/genética , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética
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