RESUMO
A novel jumbo bacteriophage (myovirus) is described. The lytic phage of Tenacibaculum maritimum, which is the etiological agent of tenacibaculosis in a variety of farmed marine fish worldwide, was plaque-isolated from seawater around a fish aquaculture field in Japan. The phage had an isometric head 110-120 nm in diameter, from which several 50- to 100-nm-long flexible fiber-like appendages emanate, and a 150-nm-long rigid contractile tail. The full genomes of the two representative phages (PTm1 and PTm5) were 224,680 and 226,876 bp long, respectively, both with 29.7% GC content, and the number of predicted open reading frames (ORFs) was 308 and 306, respectively. The average nucleotide sequence identity between PTm1 and PTm5 was 99.95%, indicating they are quite similar to each other. A genetic relationship was found in 15.0-16.6% of the predicted ORFs among the T. maritimum phages PTm1 and PTm5, the Tenacibaculum spp. phage pT24, and the Sphingomonas paucimobilis phage PAU. Phylogenetic analysis based on the terminase large subunit genes revealed that these four phages (PTm1, PTm5, pT24 and PAU) are more closely related than the other 10 jumbo myoviruses that have similar genome sizes. Transmission electron microscopy observations suggest that the head fibers of the T. maritimum phage function as tentacles to search and recognize the host cell surface to facilitate infection.
Assuntos
Bacteriófagos/genética , Genoma Viral/genética , Tenacibaculum/genética , Animais , Aquicultura , Composição de Bases , Sequência de Bases/genética , Peixes/virologia , Japão , Fases de Leitura Aberta/genética , Filogenia , Análise de Sequência de DNA/métodos , Proteínas Virais/genéticaRESUMO
Synaptic plasticity in the cerebellar cortex contributes to motor learning. In particular, long-term depression at excitatory parallel fiber - Purkinje neuron synapses has been intensively studied as a primary cellular mechanism for motor learning. Recent studies showed that synaptic plasticity other than long-term depression such as long-term potentiation at inhibitory interneuron - Purkinje neuron synapses called rebound potentiation is also involved in motor learning. It was suggested that long-term depression and rebound potentiation might synergistically support motor learning. Here, we have examined induction conditions of long-term depression and rebound potentiation in cultured rat Purkinje neurons, and found that both of them were induced simultaneously by certain patterns of depolarization of a Purkinje neuron. Further, we found that long-term depression was induced by shorter depolarizing pulses causing a smaller intracellular Ca(2+) increase than rebound potentiation. These results support an idea that long-term depression and rebound potentiation synergistically contribute to motor learning, and suggest that long-term depression may play a primary role in wider variety of motor learning paradigms than rebound potentiation.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Inibição Neural/fisiologia , Células de Purkinje/fisiologia , Animais , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores/fisiologia , RatosRESUMO
Long-term depression (LTD) at excitatory synapses between parallel fibers and a Purkinje cell has been regarded as a critical cellular mechanism for motor learning. However, it was demonstrated that normal motor learning occurs under LTD suppression, suggesting that cerebellar plasticity mechanisms other than LTD also contribute to motor learning. One candidate for such plasticity is rebound potentiation (RP), which is long-term potentiation at inhibitory synapses between a stellate cell and a Purkinje cell. Both LTD and RP are induced by the increase in postsynaptic Ca(2+) concentration, and work to suppress the activity of a Purkinje cell. Thus, LTD and RP might work synergistically, and one might compensate defects of the other. RP induction is dependent on the interaction between GABAA receptor and GABAA receptor binding protein (GABARAP). Transgenic mice expressing a peptide which inhibits binding of GABARAP and GABAA receptor only in Purkinje cells show defects in both RP and adaptation of vestibulo-ocular reflex (VOR), a motor learning paradigm. However, another example of motor learning, adaptation of optokinetic response (OKR), is normal in the transgenic mice. Both VOR and OKR are reflex eye movements suppressing the slip of visual image on the retina during head movement. Previously, we reported that delphilin knockout mice show facilitated LTD induction and enhanced OKR adaptation, but we recently found that VOR adaptation was not enhanced in the knockout mice. These results together suggest that animals might use LTD and RP differently depending on motor learning tasks.
Assuntos
Cerebelo/fisiologia , Aprendizagem/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Atividade Motora/fisiologia , Animais , Cerebelo/citologia , Humanos , Camundongos , Camundongos Transgênicos , Nistagmo Optocinético/fisiologia , Receptores de GABA/genética , Reflexo Vestíbulo-Ocular/fisiologiaRESUMO
A novel Gram-stain-negative, rod-shaped (0.3 × 4-6 µm), non-flagellated, aerobic strain with gliding motility, designated JBKA-6T, was isolated in 1991 from a yellowtail fish, Seriola quinqueradiata, showing symptoms of bacterial haemolytic jaundice. 16S rRNA gene sequence analysis showed that strain JBKA-6T was related most closely to members of the family Flavobacteriaceae in the phylum 'Bacteroidetes'. Furthermore, based on gyrB gene sequence analysis, JBKA-6T was classified into a single clade within the order Flavobacteriales, which was distinct from the known clades of the families Flavobacteriaceae, Blattabacteriaceae and Cryomorphaceae. The predominant isoprenoid quinone was identified as MK-6 (97.9 %), and the major cellular fatty acids (>10 %) were C14 : 0 and iso-C15 : 0. The main polar lipids were phosphatidylethanolamine, three unidentified phospholipids, two unidentified aminophospholipids and two unidentified polar lipids. The DNA G+C content of JBKA-6T, as derived from its whole genome, was 33.4âmol%. The distinct phylogenetic position and phenotypic traits of strain JBKA-6T distinguish it from all other described species of the phylum 'Bacteroidetes', and therefore it was concluded that strain JBKA-6T represents a new member of the phylum 'Bacteroidetes', and the name Ichthyobacterium seriolicida gen. nov., sp. nov. is proposed. The type strain of Ichthyobacterium seriolicida is JBKA-6T ( = ATCC BAA-2465T = JCM 18228T). We also propose that Icthyobacterium gen. nov. is the type genus of a novel family, Ichthyobacteriaceae fam. nov.
Assuntos
Bacteroidetes/classificação , Perciformes/microbiologia , Filogenia , Animais , Técnicas de Tipagem Bacteriana , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Doenças dos Peixes/microbiologia , Icterícia/microbiologia , Fosfatidiletanolaminas/química , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
Bluefin tuna are high-performance swimmers and top predators in the open ocean. Their swimming is grounded by unique features including an exceptional glycolytic potential in white muscle, which is supported by high enzymatic activities. Here we performed high-throughput RNA sequencing (RNA-Seq) in muscles of the Pacific bluefin tuna (Thunnus orientalis) and Pacific cod (Gadus macrocephalus) and conducted a comparative transcriptomic analysis of genes related to energy production. We found that the total expression of glycolytic genes was much higher in the white muscle of tuna than in the other muscles, and that the expression of only six genes for glycolytic enzymes accounted for 83.4% of the total. These expression patterns were in good agreement with the patterns of enzyme activity previously reported. The findings suggest that the mRNA expression of glycolytic genes may contribute directly to the enzymatic activities in the muscles of tuna.
Assuntos
Proteínas de Peixes/genética , Genoma , Músculos/metabolismo , RNA Mensageiro/genética , Transcriptoma , Atum/genética , Animais , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Peixes/metabolismo , Ontologia Genética , Glicólise/genética , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Natação/fisiologia , Atum/metabolismoRESUMO
Tunas are migratory fishes in offshore habitats and top predators with unique features. Despite their ecological importance and high market values, the open-ocean lifestyle of tuna, in which effective sensing systems such as color vision are required for capture of prey, has been poorly understood. To elucidate the genetic and evolutionary basis of optic adaptation of tuna, we determined the genome sequence of the Pacific bluefin tuna (Thunnus orientalis), using next-generation sequencing technology. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified five common fish visual pigment genes: red-sensitive (middle/long-wavelength sensitive; M/LWS), UV-sensitive (short-wavelength sensitive 1; SWS1), blue-sensitive (SWS2), rhodopsin (RH1), and green-sensitive (RH2) opsin genes. Sequence comparison revealed that tuna's RH1 gene has an amino acid substitution that causes a short-wave shift in the absorption spectrum (i.e., blue shift). Pacific bluefin tuna has at least five RH2 paralogs, the most among studied fishes; four of the proteins encoded may be tuned to blue light at the amino acid level. Moreover, phylogenetic analysis suggested that gene conversions have occurred in each of the SWS2 and RH2 loci in a short period. Thus, Pacific bluefin tuna has undergone evolutionary changes in three genes (RH1, RH2, and SWS2), which may have contributed to detecting blue-green contrast and measuring the distance to prey in the blue-pelagic ocean. These findings provide basic information on behavioral traits of predatory fish and, thereby, could help to improve the technology to culture such fish in captivity for resource management.
Assuntos
Evolução Molecular , Proteínas de Peixes/genética , Pigmentos da Retina/genética , Atum/genética , Animais , Sequência de Bases , Visão de Cores/genética , Visão de Cores/fisiologia , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Dados de Sequência Molecular , Opsinas/genética , Filogenia , Comportamento Predatório/fisiologia , Atum/fisiologiaRESUMO
Pseudomonas plecoglossicida is a lethal pathogen of ayu (Plecoglossus altivelis) in Japan and is responsible for substantial economic costs to ayu culture. Previously, we demonstrated the efficacy of phage therapy against P. plecoglossicida infection using two lytic phages (PPpW-3 and PPpW-4) (S. C. Park, I. Shimamura, M. Fukunaga, K. Mori, and T. Nakai, Appl Environ Microbiol 66:1416-1422, 2000, http://dx.doi.org/10.1128/AEM.66.4.1416-1422.2000; S. C. Park and T. Nakai, Dis Aquat Org 53:33-39, 2003, http://dx.doi.org/10.3354/dao053033). In the present study, the complete genome sequences of these therapeutic P. plecoglossicida phages were determined and analyzed for deleterious factors as therapeutic agents. The genome of PPpW-3 (myovirus) consisted of 43,564 bp with a GC content of 61.1% and 66 predicted open reading frames (ORFs). Approximately half of the genes were similar to the genes of the Escherichia coli phage vB_EcoM_ECO1230-10 (myovirus). The genome of PPpW-4 (podovirus) consisted of 41,386 bp with a GC content of 56.8% and 50 predicted ORFs. More than 70% of the genes were similar to the genes of Pseudomonas fluorescens phage ÏIBB-PF7A and Pseudomonas putida phage Ï15 (podoviruses). The whole-genome analysis revealed that no known virulence genes were present in PPpW-3 and PPpW-4. An integrase gene was found in PPpW-3, but other factors used for lysogeny were not confirmed. The PCR detection of phage genes in phage-resistant variants provided no evidence of lysogenic activity in PPpW-3 and PPpW-4. We conclude that these two lytic phages qualify as therapeutic agents.
Assuntos
DNA Viral/química , DNA Viral/genética , Genoma Viral , Fagos de Pseudomonas/genética , Animais , Composição de Bases , Japão , Dados de Sequência Molecular , Fases de Leitura Aberta , Osmeriformes/microbiologia , Pseudomonas/virologia , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
Edwardsiellosis, which is caused by Edwardsiella tarda, a Gram-negative bacterium, is one of the most serious infectious diseases in both marine and freshwater fish farms worldwide. Previously, we reported the complete genome sequences of three E. tarda-lytic bacteriophages (two podoviruses and a myovirus), which were isolated from fish tissues and fish-rearing seawater. Further genomic information regarding E. tarda phages is important for understanding phage-host interactions as well as for applications of the phages for the control of disease. Here, we report the complete genome sequence of a novel E. tarda phage (GF-2) of myovirus morphology (family Myoviridae), isolated from tissue homogenates of a cultured Japanese flounder (Paralichthys olivaceus) that succumbed to edwardsiellosis in Japan. The size of the entire genome was 43,129 bp, with a GC content of 51.3 % and containing 82 open reading frames (ORFs). The GF-2 genome possesses lysogeny-related genes that have not been found in the reported Edwardsiella phage genomes. Comparative genomics of Edwardsiella myophages suggest that the C-terminal domains of the tail fiber proteins have relevance to their host specificity. Thus, GF-2 genome information provides a novel resource for our understanding of the molecular mechanisms involved in their host specificity and for detection of E. tarda in aquaculture environments.
Assuntos
Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Edwardsiella tarda/virologia , Edwardsiella/virologia , Genoma Viral , Myoviridae/genética , Myoviridae/isolamento & purificação , Sequência de Aminoácidos , Bacteriófagos/classificação , Bacteriófagos/ultraestrutura , Sequência de Bases , Dados de Sequência Molecular , Myoviridae/classificação , Myoviridae/ultraestrutura , Fases de Leitura Aberta , Filogenia , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
BACKGROUND: Recent advancements in next-generation sequencing technology have enabled cost-effective sequencing of whole or partial genomes, permitting the discovery and characterization of molecular polymorphisms. Double-digest restriction-site associated DNA sequencing (ddRAD-seq) is a powerful and inexpensive approach to developing numerous single nucleotide polymorphism (SNP) markers and constructing a high-density genetic map. To enrich genomic resources for Japanese eel (Anguilla japonica), we constructed a ddRAD-based genetic map using an Ion Torrent Personal Genome Machine and anchored scaffolds of the current genome assembly to 19 linkage groups of the Japanese eel. Furthermore, we compared the Japanese eel genome with genomes of model fishes to infer the history of genome evolution after the teleost-specific genome duplication. RESULTS: We generated the ddRAD-based linkage map of the Japanese eel, where the maps for female and male spanned 1748.8 cM and 1294.5 cM, respectively, and were arranged into 19 linkage groups. A total of 2,672 SNP markers and 115 Simple Sequence Repeat markers provide anchor points to 1,252 scaffolds covering 151 Mb (13%) of the current genome assembly of the Japanese eel. Comparisons among the Japanese eel, medaka, zebrafish and spotted gar genomes showed highly conserved synteny among teleosts and revealed part of the eight major chromosomal rearrangement events that occurred soon after the teleost-specific genome duplication. CONCLUSIONS: The ddRAD-seq approach combined with the Ion Torrent Personal Genome Machine sequencing allowed us to conduct efficient and flexible SNP genotyping. The integration of the genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits and for investigating comparative genomics of the Japanese eel.
Assuntos
Anguilla/genética , Evolução Biológica , Genoma , Animais , Mapeamento Cromossômico , Feminino , Duplicação Gênica , Biblioteca Gênica , Ligação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Japão , Masculino , Repetições de Microssatélites , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
In rearing systems for the Japanese eel Anguilla japonica, although it is assumed that microorganisms influence larval survival and mortality, particularly during the early stages of growth, the effects of bacterial communities on larval survival have yet to be sufficiently determined. In this study, we compared the bacterial communities associated with larval survival at three stages of eel growth. To artificially alter bacterial communities and assess larval survival, eel larvae were treated with 11 types of antibiotic, and larval survival and bacterial characteristics were compared between the antibiotic-treated and antibiotic-free control groups. Throughout the three growth stages, eels treated with four antibiotics (polymyxin B, tetracycline, novobiocin, and erythromycin) had survival rates higher than those in the control groups. The bacterial communities of surviving larvae in the control and antibiotic groups and dead larvae in the control groups were subsequently analyzed using 16S rRNA gene amplicon sequencing. PERMANOVA analysis indicated that these three larval groups were characterized by significantly different bacterial communities. We identified 14 biomarker amplicon sequence variants (ASVs) of bacterial genera such as Oceanobacter, Alcanivorax, Marinobacter, Roseibium, and Sneathiella that were enriched in surviving larvae in the antibiotic treatment groups. In contrast, all four biomarker ASVs enriched in dead larvae of the control groups were from bacteria in the genus Vibrio. Moreover, 52 bacterial strains corresponding to nine biomarkers were isolated using a culture method. To the best of our knowledge, this is the first study to evaluate the bacterial communities associated with the survival and mortality of larvae in during the early stages of Japanese eel growth and to isolate biomarker bacterial strains. These findings will provide valuable insights for enhancing larval survival in the eel larval rearing systems from a microbiological perspective.
Assuntos
Anguilla , Antibacterianos , Biomarcadores , Larva , Animais , Larva/microbiologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Anguilla/microbiologia , Anguilla/crescimento & desenvolvimento , Antibacterianos/farmacologia , RNA Ribossômico 16S/genética , Bactérias/genética , Bactérias/efeitos dos fármacos , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/crescimento & desenvolvimentoRESUMO
Pacific saury (Cololabis saira) is an important fish in several countries. Notably, the catch of this fish has markedly decreased recently, which might be due to environmental changes, including feeding habitat changes. However, no clear correlation has been observed. Therefore, the physiological basis of Pacific saury in relation to possible environmental factors must be understood. We sequenced the genome of Pacific saury and extracted RNA from nine tissues (brain, eye, gill, anterior/posterior guts, kidney, liver, muscle, and ovary). In 1.09 Gb assembled genome sequences, a total of 26,775 protein-coding genes were predicted, of which 26,241 genes were similar to known genes in a public database. Transcriptome analysis revealed that 24,254 genes were expressed in at least one of the nine tissues, and 7,495 were highly expressed in specific tissues. Based on the similarity of the expression profiles to those of model organisms, the transcriptome obtained was validated to reflect the characteristics of each tissue. Thus, the present genomic and transcriptomic data serve as useful resources for molecular studies on Pacific saury. In particular, we emphasize that the gene expression data, which serve as the tissue expression panel of this species, can be employed in comparative transcriptomics on marine environmental responses.
Assuntos
Genoma , Transcriptoma , Animais , Perfilação da Expressão Gênica , Peixes/genética , Peixes/metabolismo , Especificidade de ÓrgãosRESUMO
This is a case report of a man in his 60s who was diagnosed with a small bowel obstruction due to an internal hernia caused by a ureterocutaneous fistula. Internal hernia caused by the ureter following urinary diversion is rare, posing challenges in preoperative diagnosis and carrying the risk of intraoperative injury due to the resemblance of a ureterocutaneous fistula to an adhesive band. The presentation and surgical management are discussed in this case report.
RESUMO
BACKGROUND: Edwardsiella tarda is an enterobacterium which causes edwardsiellosis, a fatal disease of cultured fishes such as red sea bream, eel, and flounder. Preventing the occurrence of E. tarda infection has thus been an important issue in aquaculture. E. tarda has been isolated from other animals and from many environments; however, the relationship between the genotype and evolutionary process of this pathogen is not fully understood. To clarify this relationship, we sequenced and compared the genomes of pathogenic and non-pathogenic E. tarda strains isolated from fish, human, and eel pond using next-generation sequencing technology. RESULTS: Eight strains of E. tarda were sequenced with high accuracy (>99.9%) with coverages from 50- to 400-fold. The obtained reads were mapped to a public reference genome. By comparing single nucleotide and insertion/deletion polymorphisms, we found that an attenuated strain of E. tarda had a loss-of-function mutation in a gene related to the type III secretion system (T3SS), suggesting that this gene is involved in the virulence of E. tarda. A comprehensive gene comparison indicated that fish pathogenic strains possessed a type VI secretion system (T6SS) and pilus assembly genes in addition to the T3SS. Moreover, we found that an E. tarda strain isolated from red sea bream harbored two pathogenicity islands of T3SS and T6SS, which were absent in other strains. In particular, this T3SS was homologous to the locus of enterocyte effacement (LEE) in enteropathogenic and enterohemorrhagic Escherichia coli. Evolutionary analysis suggested that this locus, here named Et-LEE (E. tarda LEE), was introgressed into the E. tarda genome through horizontal transfer. CONCLUSIONS: We found significant differences in the presence/absence of virulence-related genes among E. tarda strains, reflecting their evolutionary relationship. In particular, a single genotype previously proposed for fish-pathogenic strains may be further divided into two subgroups. Furthermore, the current study demonstrated, for the first time, that a fish pathogenic bacterium carried a LEE-like pathogenicity island which was previously reported only in zoonotic pathogenic enterobacteria. These findings will contribute to the exploration of strain-specific drug targets against E. tarda in aquafarms, while also shedding light on the evolution of pathogenesis in enterobacteria.
Assuntos
Proteínas de Bactérias/genética , Edwardsiella tarda/genética , Peixes/microbiologia , Transferência Genética Horizontal , Genoma Bacteriano , Ilhas Genômicas/genética , Genômica , Animais , Análise por Conglomerados , Biologia Computacional , Edwardsiella tarda/classificação , Edwardsiella tarda/patogenicidade , Ordem dos Genes , Anotação de Sequência Molecular , Mutação , Filogenia , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Virulência/genéticaRESUMO
Molluscan reproduction has been a target of biological research because of the various reproductive strategies that have evolved in this phylum. It has also been studied for the development of fisheries technologies, particularly aquaculture. Although fundamental processes of reproduction in other phyla, such as vertebrates and arthropods, have been well studied, information on the molecular mechanisms of molluscan reproduction remains limited. The recently released draft genome of the pearl oyster Pinctada fucata provides a novel and powerful platform for obtaining structural information on the genes and proteins involved in bivalve reproduction. In the present study, we analyzed the pearl oyster draft genome to screen reproduction-related genes. Analysis was mainly conducted for genes reported from other molluscs for encoding orthologs of reproduction-related proteins in other phyla. The gene search in the P. fucata gene models (version 1.1) and genome assembly (version 1.0) were performed using Genome Browser and BLAST software. The obtained gene models were then BLASTP searched against a public database to confirm the best-hit sequences. As a result, more than 40 gene models were identified with high accuracy to encode reproduction-related genes reported for P. fucata and other molluscs. These include vasa, nanos, doublesex- and mab-3-related transcription factor, 5-hydroxytryptamine (5-HT) receptors, vitellogenin, estrogen receptor, and others. The set of reproduction-related genes of P. fucata identified in the present study constitute a new tool for research on bivalve reproduction at the molecular level.
Assuntos
Genoma , Pinctada/genética , Pinctada/metabolismo , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica/fisiologia , Gônadas/crescimento & desenvolvimento , Gônadas/metabolismo , Dados de Sequência Molecular , Filogenia , Reprodução/genética , Reprodução/fisiologiaRESUMO
Horizontal gene transfer is one of the main mechanisms contributing to microbial genome diversification. To clarify the overall picture of interspecific gene flow among prokaryotes, we developed a new method for detecting horizontally transferred genes and their possible donors by Bayesian inference with training models for nucleotide composition. Our method gives the average posterior probability (horizontal transfer index) for each gene sequence, with a low horizontal transfer index indicating recent horizontal transfer. We found that 14% of open reading frames in 116 prokaryotic complete genomes were subjected to recent horizontal transfer. Based on this data set, we quantitatively determined that the biological functions of horizontally transferred genes, except mobile element genes, are biased to three categories: cell surface, DNA binding and pathogenicity-related functions. Thus, the transferability of genes seems to depend heavily on their functions.
Assuntos
Transferência Genética Horizontal , Algoritmos , Família MultigênicaRESUMO
Tunas (genus Thunnus) are one of the most ecologically and commercially important fish worldwide. To establish a biological basis for reproduction in this globally essential species, we have recently studied crucial reproductive aspects of the Pacific bluefin tuna (T. orientalis; PBT), as a model of tuna species, based on our closed-cycle aquaculture technology. In this study, we clarified the global expression profile of the genes regulating gonadal sex differentiation in PBT, as this developmental process is vital to sexual reproduction. Based on the results of our comparative (RNA-sequencing) and temporal (qRT-PCR) transcriptome analyses using the updated genome dataset, we propose the molecular mechanisms of gonadal sex differentiation in PBT. In female gonads, foxl2 and cyp19a1a (coding aromatase) are expressed at the onset of sex differentiation. Active aromatase-mediated estrogen biosynthesis, which includes positive regulation of cyp19a1a expression by Foxl2, induces ovarian differentiation. By contrast, dmrt1 and gsdf are upregulated in differentiating male gonads lacking active estrogen synthesis. Dmrt1 and Gsdf would mainly promote testicular differentiation. Furthermore, androgen biosynthesis is upregulated in differentiating male gonad. Endogenous androgens may also be vital to testicular differentiation. This study provides the first comprehensive data clarifying the molecular basis for gonadal sex differentiation in tunas.
Assuntos
Aromatase , Atum , Feminino , Masculino , Animais , Atum/genética , Aromatase/genética , Transcriptoma , Diferenciação Sexual/genética , Gônadas , EstrogêniosRESUMO
The order Parmales (class Bolidophyceae) is a minor group of pico-sized eukaryotic marine phytoplankton that contains species with cells surrounded by silica plates. Previous studies revealed that Parmales is a member of ochrophytes and sister to diatoms (phylum Bacillariophyta), the most successful phytoplankton group in the modern ocean. Therefore, parmalean genomes can serve as a reference to elucidate both the evolutionary events that differentiated these two lineages and the genomic basis for the ecological success of diatoms vs. the more cryptic lifestyle of parmaleans. Here, we compare the genomes of eight parmaleans and five diatoms to explore their physiological and evolutionary differences. Parmaleans are predicted to be phago-mixotrophs. By contrast, diatoms have lost genes related to phagocytosis, indicating the ecological specialization from phago-mixotrophy to photoautotrophy in their early evolution. Furthermore, diatoms show significant enrichment in gene sets involved in nutrient uptake and metabolism, including iron and silica, in comparison with parmaleans. Overall, our results suggest a strong evolutionary link between the loss of phago-mixotrophy and specialization to a silicified photoautotrophic life stage early in diatom evolution after diverging from the Parmales lineage.
Assuntos
Diatomáceas , Estramenópilas , Diatomáceas/genética , Estramenópilas/genética , Fitoplâncton/metabolismo , Genoma , Dióxido de SilícioRESUMO
Many cancer-testis antigen genes have been identified; however, few human leukocyte antigen (HLA)-A24-restricted cytotoxic T cell (CTL) epitope peptides are available for clinical immunotherapy. To solve this problem, novel tools increasing the efficacy and accuracy of CTL epitope detection are needed. In the present study, we utilized a highly active dendritic cell (DC)-culture method and an in silico HLA-A24 peptide-docking simulation assay to identify novel CTL epitopes from MAGE-A6 and MAGE-A12 antigens. The highly active DCs, called α-type-1 DCs, were prepared using a combination of maturation reagents to produce a large amount of interleukin-12. Meanwhile, our HLA-A24 peptide-docking simulation assay was previously demonstrated to have an obvious advantage of accuracy over the conventional prediction tool, bioinformatics and molecular analysis section. For CTL induction assays, peripheral blood mononuclear cells derived from six cases of HLA-A24(+) melanoma were used. Through CTL induction against melanoma cell lines and peptide-docking simulation assays, two peptides (IFGDPKKLL from MAGE-A6 and IFSKASEYL from MAGE-A12) were identified as novel CTL epitope candidates. Finally, we verified that the combination of the highly active DC-culture method and HLA-A24 peptide-docking simulation assay might be tools for predicting CTL epitopes against cancer antigens.
Assuntos
Antígenos de Neoplasias/imunologia , Epitopos de Linfócito T/imunologia , Antígeno HLA-A24/imunologia , Proteínas de Neoplasias/imunologia , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Epitopos de Linfócito T/metabolismo , Feminino , Antígeno HLA-A24/metabolismo , Humanos , Interleucina-12/imunologia , Interleucina-12/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Melanócitos/imunologia , Melanócitos/metabolismo , Melanoma/imunologia , Melanoma/metabolismo , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Linfócitos T Citotóxicos/metabolismoRESUMO
Carcinoembryonic antigen (CEA) is a very common tumor marker because many types of solid cancer usually produce a variety of CEA and a highly sensitive measuring kit has been developed. However, immunological responses associated with CEA have not been fully characterized, and specifically a weak immunogenicity of CEA protein as a tumor antigen is reported in human leukocyte antigen (HLA)-A24-restricted CEA peptide-based cancer immunotherapy. These observations demonstrated that immunogenic and potent HLA-A24-restricted CTL epitope peptides derived from CEA protein are seemingly difficult to predict using a conventional bioinformatics approach based on primary amino acid sequence. In the present study, we developed an in silico docking simulation assay system of binding affinity between HLA-A24 protein and A24-restricted peptides using two software packages, AutoDock and MODELLER, and a crystal structure of HLA-A24 protein obtained from the Protein Data Bank. We compared the current assay system with HLA-peptide binding predictions of the bioinformatics and molecular analysis section (BIMAS) in terms of the prediction capability using MHC stabilization and peptide-stimulated CTL induction assays for CEA and other HLA-A24 peptides. The MHC stabilization score was inversely correlated with the affinity calculated in the docking simulation alone (r = -0.589, P = 0.015), not with BIMAS score or the IFN-γ production index. On the other hand, BIMAS was not significantly correlated with any other parameters. These results suggested that our in silico assay system has potential advantages in efficiency of epitope prediction over BIMAS and ease of use for bioinformaticians.
Assuntos
Antígeno Carcinoembrionário/metabolismo , Antígenos HLA-A/metabolismo , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Antígeno Carcinoembrionário/química , Simulação por Computador , Bases de Dados Factuais , Antígenos HLA-A/química , Antígeno HLA-A24 , Humanos , Interferon gama/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Fragmentos de Peptídeos , Conformação Proteica , Linfócitos T Citotóxicos/imunologiaRESUMO
Fish species have a variety of sex determination systems. Tunas (genus Thunnus) have an XY genetic sex determination system. However, the Y chromosome or responsible locus has not yet been identified in males. In a previous study, a female genome of Pacific bluefin tuna (T. orientalis) was sequenced, and candidates for sex-associated DNA polymorphisms were identified by a genome-wide association study using resequencing data. In the present study, we sequenced a male genome of Pacific bluefin tuna by long-read and linked-read sequencing technologies and explored male-specific loci through a comparison with the female genome. As a result, we found a unique region carrying the male-specific haplotype, where a homolog of estrogen sulfotransferase gene was predicted to be encoded. The genome-wide mapping of previously resequenced data indicated that, among the functionally annotated genes, only this gene, named sult1st6y, was paternally inherited in the males of Pacific bluefin tuna. We reviewed the RNA-seq data of southern bluefin tuna (T. maccoyii) in the public database and found that sult1st6y of southern bluefin tuna was expressed in all male testes, but absent or suppressed in the female ovary. Since estrogen sulfotransferase is responsible for the inactivation of estrogens, it is reasonable to assume that the expression of sult1st6y in gonad cells may inhibit female development, thereby inducing the individuals to become males. Thus, our results raise a promising hypothesis that sult1st6y is the sex determination gene in Thunnus fishes or at least functions at a crucial point in the sex-differentiation cascade.