RESUMO
Protein evolution rate is negatively correlated with several effectors, such as expression level, expression distribution, protein-protein interactions (PPIs), and essentiality for survival. These effectors can characterize the signaling pathways mediated by ligand-receptor binding. However, it is unclear whether these effectors are constraining factors on the pathway-specific evolution of ligands and receptors. To clarify the relation between the effectors and protein evolution (dN /dS ratio) in ligands and their receptors considering each signaling pathway, we investigated 377 proteins in 20 peptide/protein ligand groups and their receptor groups using 15 primate sequences. The dN /dS ratios between peptide/protein ligand groups and their receptor groups were positively correlated, suggesting the protein evolution under the influence of signaling pathway to which they belong. Comparing each signaling pathway, ligands and receptors mainly related to development and growth (FGF/Hedgehog/Notch/WNT groups) showed lower dN /dS ratios, higher PPI numbers, and higher essentiality, whereas those mainly related to immune process (CSF/IFN/IL/TNF groups) showed higher dN /dS ratios, lower PPI numbers, and lower essentiality. Most ligands and receptors were poorly expressed, and expression level was not a constraining factor on the protein evolution. These findings indicate that PPI and essentiality are constraining factors that characterize the pathway-specific evolution of ligands and receptors.
Assuntos
Evolução Molecular , Primatas , Animais , Ligantes , Proteínas/genética , Transdução de SinaisRESUMO
To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.
Assuntos
Evolução Molecular , Genoma/genética , Filogenia , Tetraploidia , Xenopus laevis/genética , Animais , Cromossomos/genética , Sequência Conservada/genética , Elementos de DNA Transponíveis/genética , Diploide , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Cariótipo , Anotação de Sequência Molecular , Mutagênese/genética , Pseudogenes , Xenopus/genéticaRESUMO
Foam nests of frogs are natural biosurfactants that contain potential compounds for biocompatible materials, Drug Delivery System (DDS), emulsifiers, and bioremediation. To elucidate the protein components in the foam nests of Rhacophorus arboreus, which is an endemic Japanese frog species commonly seen during the rainy season, we performed amino acid analysis, SDS-PAGE electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry using intact foam nests. Many proteins were detected in these foam nests, ranging from a few to several hundred kDa, with both essential and non-essential amino acids. Next, we performed transcriptome analysis using a next-generation sequencer on total RNAs extracted from oviducts before egg-laying. The soluble foam nests were purified by LC-MS and analyzed using Edman degradation, and the identified N-terminal sequences were matched to the transcriptome data. Four proteins that shared significant sequence homologies with extracellular superoxide dismutase of Nanorana parkeri, vitelline membrane outer layer protein 1 homolog of Xenopus tropicalis, ranasmurfin of Polypedates leucomystax, and alpha-1-antichymotrypsin of Sorex araneus were identified. Prior to purification of the foam nests, they were treated with both a reducing reagent and an alkylating agent, and LC-MS/ MS analyses were performed. We identified 22 proteins in the foam nests that were homologous with proteinase inhibitors, ribonuclease, glycoproteins, antimicrobial protein and barrier, immunoglobulin-binding proteins, glycoprotein binding protein, colored protein, and keratin-associated protein. The presence of these proteins in foam nests, along with small molecules, such as carbohydrates and sugars, would protect them against microbial and parasitic attack, oxidative stress, and a shortage of moisture.
Assuntos
Anuros/metabolismo , Comportamento de Nidação/fisiologia , Oviductos/metabolismo , Proteoma , Animais , Anuros/genética , Feminino , Perfilação da Expressão GênicaRESUMO
Quality control for human induced pluripotent stem cells (hiPSCs) is important for efficient and stable production of hiPSC-derived cell therapy products to be used for transplantation. During cell culture, hiPSCs spontaneously undergo morphological changes and lose pluripotent properties. Such cells are termed deviated cells, which are altered from the undifferentiated state of hiPSCs, and express the early differentiation marker stage-specific embryonic antigen 1 (SSEA-1). In this study, we searched for soluble SSEA-1+ glycoproteins secreted from deviated cells generated by culturing hiPSCs in cell culture medium containing heat-inactivated supplements. Glycoproteins obtained from cell culture supernatants of SSEA-1+ deviated cells were enriched by an O-glycan binding lectin and blotted with anti-SSEA-1 antibody. A single protein band at >250 kDa specifically detected by anti-SSEA-1 antibody was identified as fibronectin (FN) by LC-MS/MS analysis and immunoprecipitation combined with western blotting, indicating that FN is a carrier protein of SSEA-1. We then constructed a sandwich enzyme-linked immunosorbent assay to detect SSEA-1+ FN secreted from deviated cells. This FN-SSEA-1 test proved to be both sensitive and specific, allowing for non-destructive detection of SSEA-1+ deviated cells within mixed cell population, with a lower limit of detection of 100 cells/mL. The developed assay may provide a standard technology for quality control of hiPSCs used for regenerative medicine.
Assuntos
Fibronectinas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Antígenos CD15/metabolismo , Western Blotting , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Cromatografia Líquida , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Medicina Regenerativa/métodos , Espectrometria de Massas em TandemRESUMO
In recent advanced information society, it is important to individually identify products or living organisms automatically and quickly. However, with the current identifying technology such as RFID tag or biometrics, it is difficult to apply to amphibians such as frogs or newts because of its size, stability, weakness under a wet environment and so on. Thus, this research aims to establish a system that can trace small amphibians easily even in a wet environment and keep stable sensing for a long time. The magnetism was employed for identification because it was less influenced by water for a long time. Here, a novel magnetization-free micro-magnetic tag is proposed and fabricated with low cost for installation to a living target sensed by Magneto-Optical sensor for high throughput sensing. The sensing ability of the proposed method, which was evaluated by image analysis, indicated that it was less than half of the target value (1 mm) both in the water and air. The FEM analysis showed that it is approximately twice the actual identification ability under ideal conditions, which suggests that the actual sensing ability can be extended by further improvement of the sensing system. The developed magnetization-free micro-magnetic tag can contribute to keep up the increasing demand to identify a number of samples under a wet environment especially with the development of gene technology.
Assuntos
Organismos Aquáticos/isolamento & purificação , Técnicas Biossensoriais , Dispositivos Ópticos , Organismos Aquáticos/química , Imãs , Dispositivo de Identificação por Radiofrequência , Água/químicaRESUMO
Two siamois-related homeobox genes siamois (sia1) and twin (sia2), have been reported in Xenopus laevis. These genes are expressed in the blastula chordin- and noggin-expressing (BCNE) center and the Nieuwkoop center, and have complete secondary axis-inducing activity when over-expressed on the ventral side of the embryo. Using whole genome sequences of X. tropicalis and X. laevis, we identified two additional siamois-related genes, which are tandemly duplicated near sia1 and sia2 to form the siamois gene cluster. Four siamois genes in X. tropicalis are transcribed at blastula to gastrula stages. In X. laevis, the siamois gene cluster is present on both homeologous chromosomes, XLA3L and XLA3S. Transcripts from seven siamois genes (three on XLA3L and four on XLA3S) in X. laevis were detected at blastula to gastrula stages. A transcribed gene, sia1p. S, encodes an inactive protein without a homeodomain. When over-expressed ventrally, all siamois-related genes tested in this study except for sia1p. S induced a complete secondary axis, indicating that X. tropicalis and X. laevis have four and six active siamois-related genes, respectively. Of note, each gene required different amounts of mRNA for full activity. These results suggest the possibility that siamois cluster genes have functional redundancy to endow robustness and quickness to organizer formation in Xenopus species.
Assuntos
Proteínas de Homeodomínio/genética , Família Multigênica , Proteínas de Xenopus/genética , Xenopus/genética , Sequência de Aminoácidos , Animais , Blástula/metabolismo , Padronização Corporal/genética , Mapeamento Cromossômico , Sequência Conservada , Diploide , Embrião não Mamífero/metabolismo , Gástrula/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Organizadores Embrionários , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Tetraploidia , Via de Sinalização Wnt , Xenopus/embriologia , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
The zinc finger and BTB domain-containing 11 gene (zbtb11) is expressed in the Xenopus anterior neuroectoderm, but the molecular nature of the Zbtb11 protein during embryonic development remains to be elucidated. Here, we show the role of Zbtb11 in anterior patterning of the neuroectoderm and the cooperative action with the transcription factor Otx2. Both overexpression and knockdown of zbtb11 caused similar phenotypes: expanded expression of the posterior gene gbx2 in the neural plate, and later microcephaly with reduced eyes, suggesting that a proper level of zbtb11 expression is necessary for normal patterning of the neuroectoderm, including eye formation. Co-immunoprecipitation assays showed that Zbtb11 formed a complex with itself and with a phosphomimetic and repressive form of Otx2, suggesting that Zbtb11 forms a dimer or oligomer and interacts with Otx2 in a phosphorylation-dependent manner. Reporter analysis further showed that Zbtb11 enhanced the activity of the phosphomimetic Otx2 to repress a silencer element of the posterior gene meis3. These data suggest that Zbtb11 coordinates with phosphorylated Otx2 to specify the anterior neuroectoderm by repressing posterior genes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Placa Neural , Fatores de Transcrição Otx , Proteínas de Xenopus , Animais , Fatores de Transcrição Otx/metabolismo , Fatores de Transcrição Otx/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Placa Neural/metabolismo , Placa Neural/embriologia , Xenopus laevis , Ligação Proteica , Fosforilação , Padronização Corporal/genéticaRESUMO
Overproduction of isopentenyl diphosphate by the amplification of the genes for the methylerythritol 4-phosphate pathway, dxs and dxr, is known to be deleterious for the growth of Escherichia coli. We hypothesized that overproduction of one of the endogenous isoprenoids, in addition to isopentenyl diphosphate itself, might be the cause of the reported reduced growth rate and attempted to identify the causative agent. In order to analyze polyprenyl phosphates, they were methylated by the reaction with diazomethane. The resulting dimethyl esters of polyprenyl phosphates with carbon numbers from 40 to 60 were quantitated by high-performance liquid chromatography-mass spectrometric analysis detecting ion peaks of the sodium ion adducts. The E. coli was transformed by a multi-copy plasmid carrying both the dxs and dxr genes. Amplification of dxs and dxr significantly increased the levels of polyprenyl phosphates and 2-octaprenylphenol. The levels of Z,E-mixed polyprenyl phosphates with carbon numbers of 50-60 in the strain in which ispB was co-amplified with dxs and dxr were lower than those in the control strain where only dxs and dxr were amplified. The levels of (all-E)-octaprenyl phosphate and 2-octaprenylphenol in the strains in which ispU/rth or crtE was co-amplified with dxs and dxr were lower than those in the control strain. Although the increase in the level of each isoprenoid intermediate was blocked, the growth rates of these strains were not restored. Neither polyprenyl phosphates nor 2-octaprenylphenol can be determined to be the cause of the growth rate reduction seen with dxs and dxr amplification.
Assuntos
Escherichia coli , Fosfatos Açúcares , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfatos/metabolismo , Terpenos , Fosfatos Açúcares/metabolismo , Eritritol , Cromatografia Líquida , Transferases/genéticaRESUMO
Steroid hormones play essential roles in a wide variety of biological processes in multicellular organisms. The principal steroid hormones in nematodes and arthropods are dafachronic acids and ecdysteroids, respectively, both of which are synthesized from cholesterol as an indispensable precursor. The first critical catalytic step in the biosynthesis of these ecdysozoan steroids is the conversion of cholesterol to 7-dehydrocholesterol. However, the enzymes responsible for cholesterol 7,8-dehydrogenation remain unclear at the molecular level. Here we report that the Rieske oxygenase DAF-36/Neverland (Nvd) is a cholesterol 7,8-dehydrogenase. The daf-36/nvd genes are evolutionarily conserved, not only in nematodes and insects but also in deuterostome species that do not produce dafachronic acids or ecdysteroids, including the sea urchin Hemicentrotus pulcherrimus, the sea squirt Ciona intestinalis, the fish Danio rerio, and the frog Xenopus laevis. An in vitro enzymatic assay system reveals that all DAF-36/Nvd proteins cloned so far have the ability to convert cholesterol to 7-dehydrocholesterol. Moreover, the lethality of loss of nvd function in the fruit fly Drosophila melanogaster is rescued by the expression of daf-36/nvd genes from the nematode Caenorhabditis elegans, the insect Bombyx mori, or the vertebrates D. rerio and X. laevis. These data suggest that daf-36/nvd genes are functionally orthologous across the bilaterian phylogeny. We propose that the daf-36/nvd family of proteins is a novel conserved player in cholesterol metabolism across the animal phyla.
Assuntos
Colesterol/metabolismo , Sequência Conservada , Oxigenases/química , Oxigenases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Linhagem Celular , Desidrocolesteróis/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Evolução Molecular , Microssomos/metabolismo , Dados de Sequência Molecular , Oxigenases/genética , Transporte Proteico , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
X chromosome inactivation (XCI), determined during development, remains stable after embryonic cell divisions. However, primordial germ cells (PGCs) are exceptions in that XCI is reprogrammed and inactivated X chromosomes are reactivated. Although interactions between PGCs and somatic cells are thought to be important for PGC development, little is known about them. Here, we performed imaging of X chromosome reactivation (XCR) using the 'Momiji' mouse system, which can monitor the X chromosome's inactive and active states using two color fluorescence reporter genes, and investigated whether interactions would affect XCR in PGCs. Based on their expression levels, we found that XCR of the Pgk1 locus began at embryonic day (E)10.5 and was almost complete by E13.5. During this period, PGCs became distributed uniformly in the genital ridge, proliferated, and formed clusters; XCR progressed accordingly. In addition, XCR of the Pgk1 locus preceded that of the Hprt locus, indicating that the timing of epigenetic memory erasure varied according to the locus of each of these X-linked genes. Our results indicate that XCR proceeds along with the proliferation of PGCs clustered within the genital ridge. This article has an associated First Person interview with the first author of the paper.
Assuntos
Genes Ligados ao Cromossomo X , Células Germinativas/metabolismo , Ativação Transcricional , Inativação do Cromossomo X/genética , Animais , Desenvolvimento Embrionário/genética , Feminino , Loci Gênicos , Camundongos , Fosfoglicerato Quinase/genéticaRESUMO
We determined the complete mitochondrial genome sequence of the Japanese forest green tree frog (Rhacophorus arboreus). The mitochondrial genome is 22,236 bp in length, which encodes 13 protein-coding genes, 2 rRNA, and 22 tRNA genes, and two control regions (D-loops). The whole gene arrangement of R. arboreus was the same as that of Rhacophorus omeimontis and Rhacophorus schlegelii.
RESUMO
The recombinant N-terminal domain of BC2L-C lectin (rBC2LCN) is useful for detecting not only human pluripotent stem cells but also some cancers. However, the cancer types and stages that can be detected by rBC2LCN remain unclear. In this study, we identified the human breast carcinoma subtypes and stages that can be detected by rBC2LCN. Compared with rBC2LCN-negative breast carcinoma cell lines, the rBC2LCN-positive cells expressed higher levels of human epidermal growth factor receptor 2 (HER2) and epithelial marker genes. Importantly, rBC2LCN histochemical staining of human breast carcinoma tissues demonstrated the utility of rBC2LCN in detecting breast carcinoma types that express HER2 and have not spread much in the early phase of growth. We conclude that rBC2LCN may have potential as a detection probe and a drug delivery vehicle to identify and treat early-stage HER2-positive breast carcinoma.
Assuntos
Proteínas de Bactérias/química , Neoplasias da Mama/diagnóstico , Lectinas/química , Sondas Moleculares/química , Antineoplásicos/administração & dosagem , Proteínas de Bactérias/genética , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Burkholderia cenocepacia , Portadores de Fármacos/química , Estudos de Viabilidade , Feminino , Humanos , Lectinas/genética , Células MCF-7 , Sondas Moleculares/genética , Estadiamento de Neoplasias , Receptor ErbB-2/análise , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análise Serial de Tecidos/métodosRESUMO
INTRODUCTION: Tumourigenesis attributed to residual undifferentiated cells in a graft is considered to be a significant issue in cell therapy using human pluripotent stem cells. To ensure the safety of regenerative medicine derived from pluripotent stem cells, residual undifferentiated cells must be eliminated in the manufacturing process. We previously described the lectin probe rBC2LCN, which binds harmlessly and specifically to the cell surface of human pluripotent stem cells. We report here a technique using rBC2LCN to remove pluripotent cells from a heterogenous population to reduce the chance of teratoma formation. METHODS: We demonstrate a method for separating residual tumourigenic cells using rBC2LCN-bound magnetic beads. This technology is a novel use of their previous discovery that rBC2LCN is a lectin that selectively binds to pluripotent cells. We optimize and validate a method to remove hPSCs from a mixture with human fibroblasts using rBC2LCN-conjugated magnetic beads. RESULTS: Cells with the potential to form teratoma could be effectively eliminated from a heterogeneous cell population with biotin-labelled rBC2LCN and streptavidin-bound magnetic beads. The efficiency was measured by FACS, ddPCR, and animal transplantation, suggesting that magnetic cell separation using rBC2LCN is quite efficient for eliminating hPSCs from mixed cell populations. CONCLUSIONS: The removal of residual tumourigenic cells based on rBC2LCN could be a practical option for laboratory use and industrialisation of regenerative medicine using human pluripotent stem cells.
RESUMO
The Bestrophin-1/VMD2 gene has been implicated in Best disease, a juvenile-onset vitelliform macular dystrophy. The Bestrophin proteins have anion channel activity, and the four mammalian members share sequence homologies in multiple transmembrane domains and an RFP-tripeptide motif. The expression patterns and functions of the Bestrophin genes in retinal pigment epithelium have been studied intensively, whereas little is known about their roles in vertebrate embryogenesis. This study examined the roles of four Xenopus tropicalis homologs of BEST genes. The xtBest genes showed spatially and temporally distinct expression. xtBest-2 was the only maternally expressed Best gene, and both xtBest-2 and the Xenopus laevis Best-2 gene were expressed at the edge of the blastopore lip including the organizer. Ectopic expression of xBest-2 caused defects in dorsal axis formation and in mesodermal gene expression during gastrulation. These results suggest a new role of the Bestrophin family genes in early vertebrate embryogenesis.
Assuntos
Canais de Cloreto/genética , Regulação da Expressão Gênica no Desenvolvimento , Xenopus/embriologia , Sequência de Aminoácidos , Animais , Expressão Gênica , Dados de Sequência Molecular , Xenopus/genéticaRESUMO
hox genes are found as clusters in the genome in most bilaterians. The order of genes in the cluster is supposed to be correlated with the site of expression along the anterior-posterior body axis and the timing of expression during development, and these correlations are called spatial and temporal collinearity, respectively. Here we studied the expression dynamics of all hox genes of the diploid species Xenopus tropicalis in four Hox clusters (A-D) by analyzing high-temporal-resolution RNA-seq databases and the results showed that temporal collinearity is not supported, which is consistent with our previous data from allotetraploid X enopus laevis Because the temporal collinearity hypothesis implicitly assumes the collinear order of gene activation, not mRNA accumulation, we determined for the first time the timing of when new transcripts of hox genes are produced, by detecting pre-spliced RNA in whole embryos with reverse transcription and quantitative PCR (RT-qPCR) for all hoxa genes as well as several selected hoxb, hox c and hoxd genes. Our analyses showed that, coinciding with the RNA-seq results, hoxa genes started to be transcribed in a non-sequential order, and found that multiple genes start expression almost simultaneously or more posterior genes could be expressed earlier than anterior ones. This tendency was also found in hoxb and hoxc genes. These results suggest that temporal collinearity of hox genes is not held during early development of Xenopus.
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RecQ-like helicase 4 (RECQL4) is mutated in patients suffering from the Rothmund-Thomson syndrome, a genetic disease characterized by premature aging, skeletal malformations, and high cancer susceptibility. Known roles of RECQL4 in DNA replication and repair provide a possible explanation of chromosome instability observed in patient cells. Here, we demonstrate that RECQL4 is a microtubule-associated protein (MAP) localizing to the mitotic spindle. RECQL4 depletion in M-phase-arrested frog egg extracts does not affect spindle assembly per se, but interferes with maintaining chromosome alignment at the metaphase plate. Low doses of nocodazole depolymerize RECQL4-depleted spindles more easily, suggesting abnormal microtubule-kinetochore interaction. Surprisingly, inter-kinetochore distance of sister chromatids is larger in depleted extracts and patient fibroblasts. Consistent with a role to maintain stable chromosome alignment, RECQL4 down-regulation in HeLa cells causes chromosome misalignment and delays mitotic progression. Importantly, these chromosome alignment defects are independent from RECQL4's reported roles in DNA replication and damage repair. Our data elucidate a novel function of RECQL4 in mitosis, and defects in mitotic chromosome alignment might be a contributing factor for the Rothmund-Thomson syndrome.
Assuntos
Metáfase/genética , Proteínas Associadas aos Microtúbulos/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Síndrome de Rothmund-Thomson/enzimologia , Animais , Cromatina/metabolismo , Instabilidade Cromossômica/genética , Segregação de Cromossomos/genética , Códon sem Sentido/genética , Reparo do DNA , Replicação do DNA , Mutação da Fase de Leitura/genética , Células HEK293 , Células HeLa , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Óvulo/enzimologia , Fuso Acromático/enzimologia , Xenopus/genéticaRESUMO
Urodele newts have unique biological properties, notably including prominent regeneration ability. The Iberian ribbed newt, Pleurodeles waltl, is a promising model amphibian distinguished by ease of breeding and efficient transgenic and genome editing methods. However, limited genetic information is available for P. waltl. We conducted an intensive transcriptome analysis of P. waltl using RNA-sequencing to build and annotate gene models. We generated 1.2 billion Illumina reads from a wide variety of samples across 12 different tissues/organs, unfertilized egg, and embryos at eight different developmental stages. These reads were assembled into 1,395,387 contigs, from which 202,788 non-redundant ORF models were constructed. The set is expected to cover a large fraction of P. waltl protein-coding genes, as confirmed by BUSCO analysis, where 98% of universal single-copy orthologs were identified. Ortholog analyses revealed the gene repertoire evolution of urodele amphibians. Using the gene set as a reference, gene network analysis identified regeneration-, developmental-stage-, and tissue-specific co-expressed gene modules. Our transcriptome resource is expected to enhance future research employing this emerging model animal for regeneration research as well as for investigations in other areas including developmental biology, stem cell biology, and cancer research. These data are available via our portal website, iNewt (http://www.nibb.ac.jp/imori/main/).
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Pleurodeles/genética , Regeneração/genética , Transcriptoma , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Especificidade de Órgãos , Filogenia , Análise de Sequência de RNARESUMO
Smad-interacting protein-1 (SIP1), also known as deltaEF2, ZEB2 and zfhx1b, is essential for the formation of the neural tube and the somites. Overexpression of Xenopus SIP1 causes ectopic neural induction via inhibition of bone morphogenetic protein (BMP) signaling and inhibition of Xbra expression. Here, we report the functional analyses of 4 domain-deletion mutants of XSIP1. Deletion of the N-terminus zinc finger domain suppressed neural induction and BMP inhibition, but these were not affected by deletion of the other domains (the Smad binding domain, the DNA-binding homeodomain together with the CtBP binding site and the C-terminus zinc finger). Therefore SIP1 does not inhibit BMP signaling by binding to Smad proteins. In contrast, all of the deletion constructs inhibited Xbra expression. These results suggest that the N-terminus zinc finger domain of XSIP1 has an important role in neural induction and that Xbra suppression occurs via a mechanism separate from the neural inducing activity.
Assuntos
Indução Embrionária , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Sistema Nervoso/embriologia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Animais , Embrião não Mamífero , Proteínas de Homeodomínio/genética , Mutação , Sistema Nervoso/metabolismo , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas com Domínio T/genética , Xenopus/embriologia , Proteínas de Xenopus/genética , Dedos de ZincoRESUMO
The diploid Xenopus tropicalis, with its small nuclear genomic size and short generation time compared to the traditional experimental amphibian X. laevis, is considered a next-generation model animal. Several experimental X. tropicalis lines have been used in research studies. Previous studies showed that the mtDNA sequence of the Asashima line is divergent from other lines and that this line may represent a distinct species. Here, we report the complete nucleotide sequence of this unique X. tropicalis experimental line. The genome is 17,700 bp in length and contains 37 genes commonly found in animal mtDNAs. The 16S rRNA gene sequence in Asashima line differed by over 6% from the standard Nigerian lines (a 3% difference is considered the species threshold in anurans), suggesting that this experimental line is a distinct species from the true X. tropicalis.
Assuntos
Genoma Mitocondrial , Pipidae/classificação , Pipidae/genética , Animais , Composição de Bases , Códon , Ordem dos Genes , Rearranjo Gênico , Genes Mitocondriais , Tamanho do Genoma , Fases de Leitura Aberta , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Sequenciamento Completo do GenomaRESUMO
Pxt peptides (Pxt-1 through Pxt-12) have been isolated from amphibian, Xenopus tropicalis Pxt-related peptides (Pxt-2, Pxt-5, Pxt-12, reverse Pxt-2, reverse Pxt-5 and reverse Pxt-12) with significant foaming properties were further characterized. In the physicochemical experiments, all Pxt-related peptides formed significant amphiphilic α-helices in 50% 2,2,2-trifluoroethanol by circular dichroism measurements. Among Pxt-related peptides, both Pxt-5 and reverse Pxt-5 were the most effective in reducing their surface tensions. Moreover, Pxt-2, Pxt-5 and reverse Pxt-5 produced constant surface tensions above their critical association concentrations, suggesting the micelle-like assemblies. In the biological experiments, Pxt-5 possessed the most potent hemolytic activity, while reverse Pxt-5 exhibited the most remarkable gene expression of interleukin 8 and heme oxygenase 1 and the most potent cytotoxicity in HaCaT cells. In contrast, Pxt-12 and reverse Pxt-12 were much weaker in antimicrobial assays for Gram-negative bacteria, Gram-positive bacteria and yeasts, as well as in hemolytic, cell viability and cytotoxicity assays in HaCaT cells. All Pxt-related peptides exhibited about 20-50% of the total cellular histamine release at 10(-5) M, as well as mastoparan and melittin in mast cells. Real-time polymerase chain reaction analysis confirmed the gene expressions of Pxt-5 in testis and Pxt-12 in muscle, in addition to skin, while Pxt-2 was only in skin.