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Find video protocols related to scientific articles indexed in Pubmed.
Sequencing and characterization of the transcriptome of half-smooth tongue sole (Cynoglossus semilaevis).
BMC Genomics
PUBLISHED: 06-04-2014
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Half-smooth tongue sole (Cynoglossus semilaevis) is a valuable fish for aquaculture in China. This fish exhibits sexual dimorphism, particularly different growth rates and body sizes between two genders. Thus, C. semilaevis is a good model that can be used to investigate mechanisms responsible for such dimorphism, this model can also be utilized to answer fundamental questions in evolution and applied fields of aquaculture. Hence, advances in second-generation sequencing technology, such as 454 pyrosequencing, could provide a robust tool to study the genome characteristics of non-model species.
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Molecular cloning, expression profiles and promoter analysis of insulin-like growth factor binding protein-4 (IGFBP-4) in Japanese flounder (Paralichthys olivaceus).
Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
PUBLISHED: 05-24-2014
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We cloned and characterized cDNA sequence of insulin-like growth factor binding protein-4 (IGFBP-4) from Japanese flounder (Paralichthys olivaceus). The 1493 bp full-length cDNA sequence contained an open reading frame (ORF) of 780 bp, which encoded a protein of 259 amino acids. The deduced amino acid sequences included a putative signal peptide of 28 amino acid residues resulting in a mature protein of 231 amino acids. Twenty cysteine residues and two conserved IGFBPs motif (GCGCCXXC and CWCV) were found in the N- and C-terminal domain. In the over 13 kbp genomic sequence, four exons, three introns, and 5'-/3'-flanking sequences were identified. Sequence alignment and phylogenetic analysis showed that Japanese flounder IGFBP-4 was indeed the ortholog of the human IGFBP-4 gene and shared high identities with other teleost IGFBP-4 genes. The promoter region was also analyzed and several potential transcription factor (TF) binding sites were determined which may modulate the IGFBP-4 expression. Quantitative real-time PCR analysis revealed that IGFBP-4 mRNA was observed in various tissues, with intestine showing the highest expression. The maternal transcripts of IGFBP-4 gene existed in the early embryonic stages and then increased in the following stages until hatching, suggesting that IGFBP-4 may be involved in the fish early development. The expression level of IGFBP-4 mRNA was relatively higher at 3 days post hatching (dph) and 15 dph, and gradually decreased during the metamorphosis period. All these results indicated that IGFBP-4 plays a significant role in IGF regulating vertebrate growth and development.
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Identification and characterization of a Sox2 homolog in the Japanese flounder Paralichthys olivaceus.
Gene
PUBLISHED: 04-15-2014
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Sox2 plays an essential role in maintaining the pluripotency of embryonic and neural stem cells as well as in the neurogenesis. While it has been well studied in mammals, information from lower invertebrate especially marine fish is still limited. In this study, we cloned and sequenced the full-length cDNA and partial 5'-flanking region of the Japanese flounder Sox2. Phylogenetic, gene structure, and protein comparison analyses revealed that Paralichthys olivaceus Sox2 (Po-Sox2) was homologous to mammalian Sox2. Quantitative real-time RT-PCR results showed that Po-Sox2 was not maternal inherited, and the transcripts were present from high blastula-stage onwards, with the highest level at the mid-gastrula stage. Tissue distribution analysis revealed that Po-Sox2 was present not only in neural tissues, but also in gonadal and gill tissues. In addition, we analyzed the Po-Sox2 promoter region for several species-conserved motifs as well as various transcription factor binding sites. The overall hypomethylation status of the identified CpG sites in the 5'-regulatory region revealed that it was not involved in the transcriptional modulation of Po-Sox2. All these results suggest that Po-Sox2 may have a conserved function in neurogenesis and early embryonic development.
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Characterisation of kisspeptin system genes in an ovoviviparous teleost: Sebastes schlegeli.
Gen. Comp. Endocrinol.
PUBLISHED: 02-20-2014
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Kisspeptins are neuropeptides that play important roles in the reproduction and the onset of puberty in vertebrate by activating their receptor, Kissr. In the present study, we first isolated kiss1 and kissr4 genes from an ovoviviparous fish, the black rockfish (Sebastes schlegeli) by homologue cloning. Phylogenetic analysis indicated that the kiss and kissr of S.schlegeli belonged to kiss1 and kissr4 respectively. Quantitative real-time PCR analysis showed that the kissr4 was expressed mainly in the brain and testis, while the kiss1 was expressed predominantly in the heart of both sexes. As for the different gonadal maturation stages the kiss1 showed different expression patterns in different tissues. During the early development stage, expression levels of the ligand and receptor genes showed similar increasing trends. The promoter region of kissr4 contained several putative transcription factor (TF) binding sites which may have the function of regulating kisspeptin system gene expression, providing potential targets for future in-depth investigation. These results together confirmed that the kisspeptin system in S.schlegeli may be involved in reproduction and other activities. Furthermore, our study laid the groundwork for further learning about the evolution and function of kisspeptin system in fish even vertebrate.
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The impact of exogenous DNA on the structure of sperm of olive flounder (Paralichthys olivaceus).
Anim. Reprod. Sci.
PUBLISHED: 02-13-2014
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Sperm-mediated gene transfer (SMGT) is a promising transgenic technology that relies on the capability of sperm to internalize exogenous DNA. In marine fish, however, the interaction between sperm and exogenous DNA appears to be deficient. Here, we demonstrated significant DNase activity in the seminal plasma of the olive flounder. When incubated with naked-DNA, the spermatozoa lost their structural integrity, including the head, mitochondria and flagellum, in an incubation time-dependent manner. However, internalization of a liposome-DNA complex resulted in the structural integrity of the spermatozoa being maintained, even when using incubation times of up to 50min. We concluded that in the olive flounder, SMGT is possible by integrating liposome-DNA complexes, rather than naked-DNA alone, into the sperm. In brief, removal of the seminal plasma and packaging the exogenous DNA were necessary for successful SMGT in the olive flounder.
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Characterization of the Dmrt1 gene in the black rockfish Sebastes schlegeli revealed a remarkable sex-dimorphic expression.
Fish Physiol. Biochem.
PUBLISHED: 02-10-2014
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The Dmrt genes encode a large family of transcription factors with a conserved zinc finger-like DNA-binding DM domain. The function of Dmrt1, one of the family members, in sexual development has been well studied in invertebrates and vertebrates. In the present study, the full-length cDNA of Dmrt1 was isolated from the testis of Sebastes schlegeli. The full-length cDNA of S. schlegeli Dmrt1 (SsDmrt1) was 1,587 bp and contained a 189-bp 5' UTR, a 489-bp 3' UTR and a 909-bp open reading frame, which encoded 302 amino acids with a conserved DM domain and an male-specific motif domain. Phylogenetic analysis showed the evolutionary relationships of SsDmrt1 with other known Dmrt genes in fish and tetrapods. Several transcriptional factor-binding sites in the 5' promoter were identified that might regulate SsDmrt1 expression. Quantitative real-time PCR analysis indicated that SsDmrt1 was expressed in all of the inspected larval developmental stages from 1 to 35 days after birth and that the level of expression gradually decreased. The expression of SsDmrt1 in adult gonads was sexually dimorphic with extremely high expression in the testis, but very low expression in the ovary. No expression was detected in other tissues. Using in situ hybridization, we demonstrated that SsDmrt1 was specifically expressed in the germ cells of both the testis and the ovary. Thus, our results suggest that SsDmrt1 may have an important role in the differentiation of both the testis and the ovary of S. schlegeli.
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Molecular characterization of heat shock protein 70 (HSP 70) promoter in Japanese flounder (Paralichthys olivaceus), and the association of Pohsp70 SNPs with heat-resistant trait.
Fish Shellfish Immunol.
PUBLISHED: 02-04-2014
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Ambient temperature is one of the major abiotic environmental factors determining the main parameters of fish vital activity. HSP70 plays an essential role in heat response. In this investigation, the promoter and structure of Paralichthys olivaceus hsp70 (Pohsp70) gene was cloned and predicted. 2558 bp upstream regulatory region of Pohsp70 was annotated with four potential promoter elements and four putative binding sites of transcription factors heat shock elements (HSE, nGAAn) in the upstream of the transcription start site. In addition, one intron with 454 bp in the 5'-noncoding region was found. Quantitative Real Time PCR analysis indicated that the transcript level of Pohsp70 was raised markedly after 1 h by heat shocked. Furthermore, 25 SNPs were identified in Pohsp70 by resequencing, seven of which was associated with heat resistance. In addition, two of the seven SNPs, namely SNP14 and SNP16, were observed in strong linkage disequilibrium. The haplotype with association analysis showed TAGGAG haplotype was more represented in heat susceptible group while (DEL/T) GAATA haplotype was more frequent in heat resistant group. The heat resistant SNPs and haplotype could be candidate markers potentially serving for selective breeding programs of Japanese flounder aimed at improving anti-stress and production.
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Sexually dimorphic expression of vasa isoforms in the tongue sole (Cynoglossus semilaevis).
PLoS ONE
PUBLISHED: 01-01-2014
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The vasa gene encodes an ATP-dependent RNA helicase of the DEAD box protein family that functions in a broad range of molecular events involving duplex RNA. In most species, the germline specific expression of vasa becomes a molecular marker widely used in the visualization and labeling of primordial germ cells (PGCs) and a tool in surrogate broodstock production through PGC transplantation. The vasa gene from tongue sole (Cynoglossus semilaevis) was characterized to promote the development of genetic breeding techniques in this species. Three C. semilaevis vasa transcripts were isolated, namely vas-l, vas-m, and vas-s. Quantitative real-time PCR results showed that C. semilaevis vasa transcripts were prevalently expressed in gonads, with very weak expression of vas-s in other tissues. Embryonic development expression profiles revealed the onset of zygotic transcription of vasa mRNAs and the maternal deposit of the three transcripts. The genetic ZW female juvenile fish was discriminated from genetic ZZ males by a pair of female specific primers. Only the expression of vas-s can be observed in both sexes during early gonadal differentiation. Before PGCs started mitosis, there was sexually dimorphic expression of vas-s with the ovary showing higher levels and downward trend. The results demonstrated the benefits of vasa as a germline specific marker for PGCs during embryonic development and gonadal differentiation. This study lays the groundwork for further application of C. semilaevis PGCs in fish breeding.
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Reference gene selection for quantitative real-time RT-PCR normalization in the half-smooth tongue sole (Cynoglossus semilaevis) at different developmental stages, in various tissue types and on exposure to chemicals.
PLoS ONE
PUBLISHED: 01-01-2014
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Quantitative real time RT-PCR has been described as the most sensitive method for the detection of low abundance mRNA. To date, no reference genes have been screened in the half-smooth tongue sole (Cynoglossus semilaevis). The aim of this study was to select the most stable genes for quantitative real-time RT-PCR. Eight housekeeping genes (18S, TUBA, B2M, ACTB, EF1A, GAPDH, RPL17 and UBCE) were tested at different developmental stages, in different tissues, and following exposure to the drug SB-431542. Using geNorm, BestKeeper and NormFinder software, GAPDH/B2M, GAPDH/18S and UBCE/GAPDH were identified as the most suitable genes from samples taken of different developmental stages while 18S/RPL17 were consistently ranked as the best reference genes for different tissue types. Furthermore, TUBA/B2M, TUBA/UBCE and B2M/TUBA were found to be the most suitable genes in samples treated with the drug, SB-431542 by geNorm, BestKeeper and NormFinder respectively. Across both different developmental stages and tissue types, the combination of 18S and GAPDH was the most stable reference gene analyzed by Ref-Finder. To test and verify the screened reference genes, the expression profiles of LEFTY-normalized to the combination of GAPDH/18S and ACTB were presented. These results will be useful for future gene-expression studies in the half-smooth tongue sole.
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Identification and characterization of a nanog homolog in Japanese flounder (Paralichthys olivaceus).
Gene
PUBLISHED: 06-21-2013
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The homeodomain-containing transcription factor nanog plays a key role in maintaining the pluripotency and self-renewal of embryonic stem cells in mammals. Stem cells offered as a significant and effective tool for generation of transgenic animals and preservation of genetic resources. The molecular genetic organization and expression of nanog gene in marine fish have not been reported yet. In this study, we isolated and characterized the flounder nanog gene as a first step towards understanding the mechanism of the plurpotency of fish stem cells and develop a potential molecular marker to identify the stem cells in vivo and in vitro. Phylogenetic, gene structure and chromosome synteny analysis provided the evidence that Po-nanog is homologous to the mammalian nanog gene. Protein sequence comparison showed that flounder Nanog shared low similarity with other vertebrate orthologs except for a conserved homeodomain. Quantitative RT-PCR analysis showed that flounder nanog was maternally expressed, and the transcripts were present from the one-cell stage to the neurula stage with the peaking at blastula stage. Whole mount in situ hybridization analyses demonstrated that the transcripts were present in all blastomeres of the early embryo. Tissue distribution analysis indicated that nanog was detectable only in gonads. Further, the expression was significantly high in ovary than in testis. In situ hybridization revealed that the transcripts were located in the cytoplasm of the oogonia and oocytes in ovary, only in the spermatogonia but no spermatocytes or spermatids in testis. The promoter region was also analyzed to have several basal core promoter elements and transcription factor binding sites. All these results suggest that Po-Nanog may have a conservative function between teleosts and mammals.
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Selection of reference genes for reverse transcription quantitative real-time PCR normalization in black rockfish (Sebastes schlegeli).
Mar Genomics
PUBLISHED: 06-08-2013
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Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a technique widely used for quantification of mRNA transcription. Data normalization is an indispensable process for RT-qPCR and reference genes are most commonly used to normalize RT-qPCR and to reduce possible errors generated in the quantification of genes among several proposed methods. To date, RT-qPCR has been used in terms of gene expression studies in black rockfish (Sebastes schlegeli) but the majority of published RT-qPCR studies still lack proper validation of the reference genes. In the present study, mRNA transcription profiles of eight putative reference genes (18S rRNA, ACTB, GAPDH, TUBA, RPL17, EF1A, HPRT, and B2M) were examined using RT-qPCR in different tissues and larvae developmental stages of black rockfish. Three common statistical algorithms (geNorm, NormFinder, and BestKeeper) were used to assess expression stability and select the most stable genes for gene normalization. Two reference genes, RPL17 and EF1A showed high stability in black rockfish tissue analysis, while GAPDH was the least stable gene. During larvae developmental stages, EF1A, RPL17 and ACTB were identified as the optimal reference genes for data normalization, whereas B2M appeared unsuitable as the reference gene. In summary, our results could provide a useful guideline for reference gene selection and enable more accurate normalization of gene expression data in gene expression studies of black rockfish.
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Cloning, expression promoter analysis of vasa gene in Japanese flounder (Paralichthys olivaceus).
Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
PUBLISHED: 03-24-2013
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Vasa is a DEAD box helicase and has shown essential functions during gametogenesis and embryogenesis. In most species, research revealed a specific expression of vasa gene in the germ cells. Thus, vasa has become the candidate gene in identifying germ cells. In this study, the vasa gene was isolated from gonads of Japanese flounder (Paralichthys olivaceus). In the 11.4kb genomic sequence, 23 exons were identified besides 5 and 3 flanking regions. The promoter region contained several putative TF binding sites which may have the function of regulating vasa expression. Quantitative real-time PCR analysis showed that vasa gene expression was restricted to adult gonads, with a higher level in the ovary. Development expression profiling revealed a maternal deposit and constant embryonic expression at early stages, but the relative mRNA amount decreased after gastrula. Nine other PoVasa transcripts were detected and their expression in gonads and during early development was not all the same, implying potential different functions during gametogenesis or early embryonic development. These results together confirmed the feasibility of using vasa as a marker of germ cells and that vasa gene had an important role in spermatogenesis and oogenesis. Furthermore, our study laid the groundwork for identifying fish primordial germ cells (PGCs) and investigating germ cell biology.
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Polymorphism in a serine protease inhibitor gene and its association with disease resistance in the eastern oyster (Crassostrea virginica Gmelin).
Fish Shellfish Immunol.
PUBLISHED: 01-06-2011
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Serine protease inhibitors (SPIs) are a superfamily of structurally related but functionally diverse proteins found in almost all organisms ranging from viruses to humans. Some of them play important roles in host defense. A recently identified SPI from the eastern oyster (Crassostrea virginica), cvSI-1, has been shown to inhibit the proliferation of the Dermo pathogen Perkinsus marinus in vitro, although direct evidence linking it to disease resistance is lacking. In this study, we identified polymorphism in the cvSI-1 gene and studied its association with improved survival after disease-caused mortalities and in disease-resistant eastern oyster strains. Full-cDNA sequence of cvSI-1 was sequenced in a diverse panel of oysters, revealing 12 single-nucleotide polymorphisms (SNPs) in the 273 bp coding region: five were synonymous and seven non-synonymous. The Dn/Ds ratio, 1.4, suggests that cvSI-1 is under positive selection. Selected SNPs were genotyped in families before and after disease-caused mortalities as well as in disease-resistant and susceptible strains. At SNP198, the C allele consistently increased in frequency after mortalities that are caused primarily by Dermo and possibly also by MSX. Its frequency in the disease-resistant strain is significantly higher than that in the susceptible strains and the base population from which the selected strains were derived. These results indicate that polymorphism at cvSI-1 is associated with Dermo (possibly also MSX) resistance in the eastern oyster. SNP198 is a synonymous mutation, and its association with disease resistance may be due to its close linkage to a functional polymorphism nearby.
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HbMT2, an ethephon-induced metallothionein gene from Hevea brasiliensis responds to H(2)O(2) stress.
Plant Physiol. Biochem.
PUBLISHED: 04-15-2010
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Metallothioneins (MTs) are the cysteine-rich proteins with low molecular weight, which play important roles in maintaining intracellular ion homeostasis, detoxification of heavy metal ions and protecting against intracellular oxidative damages. In this study a novel ethephon-induced metallothionein gene, designated as HbMT2, was isolated and characterized from Hevea brasiliensis. The HbMT2 cDNA contained a 237 bp open reading frame encoding 78 amino acids and the deduced protein showed high similarity to the type 2 MTs from other plant species. Expression analysis revealed more significant accumulation of HbMT2 transcripts in leaves and latex than in roots and barks. The transcription of HbMT2 in latex was strongly induced by ethephon and hydrogen peroxide (H(2)O(2)) stress. Overproduction of recombinant HbMT2 protein gave the Escherichia coli cells more tolerance on Cu(2+) and Zn(2+), and the recombinant HbMT2 could scavenge the reactive oxidant species (ROS) in vitro. All these results indicated that HbMT2 could respond to ethephon stimulation and H(2)O(2) stress as a ROS scavenger in H. brasiliensis. It is also suggested that HbMT2 function in improving the tolerance of rubber trees to heavy metal ions, and repressing the ethephon-induced senilism and tapping panel dryness (TPD) development by ROS scavenge system in H. brasiliensis.
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Identification of differential genes in the ovary relative to the testis and their expression patterns in half-smooth tongue sole (Cynoglossus semilaevis).
J Genet Genomics
PUBLISHED: 03-16-2010
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Half-smooth tongue sole (Cynoglossus semilaevis) is a rare marine flatfish whose mature ovary and testis greatly differ in volume and weight. The length and weight of mature females are over twice greater than those of mature males. To obtain sufficient information on gonad differentiation and the relationship between gonad development and growth in the fish, we compared gene expression between the ovary and testis using suppression subtractive hybridization (SSH). Testis cDNAs are subtracted from ovary cDNAs and are used to establish an ovary testis-subtracted cDNA library. A total of 41 nonredundant clones are identified, including 20 known cDNAs, 9 uncharacterized cDNAs (EST clones), and 12 novel sequences. For selected genes such as ZPC, RacGAP, survivin, aquaporin, CPEB, O5, O15, and O18, gene expression patterns are analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). The results confirm that these genes are only expressed in the ovary and not in the testis, or at higher levels in the ovary than in the testis. At the same time, expressions of certain genes such as ZPC, survivin, aquaporin, CPEB, and O15 are demonstrated to possess sexual dimorphism in the kidney or muscle, and spleen. The results suggest that these genes could play key roles not only in the ovary but in other female tissues as well.
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Cloning and characterization of type II interleukin-1 receptor cDNA from Japanese flounder (Paralichthys olivaceus).
Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
PUBLISHED: 02-18-2010
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The type II interleukin-1 receptor (IL-1RII) cDNA was cloned from Japanese flounder (Paralichthys olivaceus) by mRNA differential display (DD-PCR) and rapid amplification of cDNA ends (RACE). The full-length cDNA is 1793 bp in length, including a 100 bp 5-terminal untranslated region (UTR), a 430 bp 3-terminal UTR, and a 1263 bp open reading frame (ORF). The ORF encodes 420 amino acid residues with an estimated molecular mass of 46.65 kDa. The protein possesses signature features of the IL-R family, consisting of one N-terminal signal peptide, one transmembrane (TM) domain, two Ig-like domains in its extracellular region, one short cytoplasmic tail of 17 amino acids and four conserved proline residue sites. The predicted amino acid sequence has 65.3%, 52.5% and 51.6% identity with gilthead seabream, rainbow trout and Atlantic salmon IL-1RII, respectively. Phylogenetic analysis supported a close relation to mammalian IL-1RII. Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis indicated that the IL-1RII gene expression of Japanese flounder was weakly up-regulated and reached the peak expression in the kidney, spleen, and gill at 6 h after infection with Vibrio anguillarum, at 1.9, 2.0 and 1.5 times relative to the uninfected fish, respectively. These results suggest that IL-1RII has a signal transduction function and possibly plays a minor role in immune regulation against bacterial(s) infection in Japanese flounder.
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Molecular characterization and expression pattern of two zona pellucida genes in half-smooth tongue sole (Cynoglossus semilaevis).
Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
PUBLISHED: 02-06-2010
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Zona pellucida (ZP) proteins are glycoproteins in fish chorion that are encoded by multiple gene families, mainly including zp1, zp2, and zp3. In the present study, we cloned two zp genes of half-smooth tongue sole, Cynoglossus semilaevis, using reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The two genes were demonstrated to belong to zp3 isoforms by utilizing BLAST, alignment of amino acid sequence, and phylogenetic analysis. The two genes were named C. semilaevis zp3a and C. semilaevis zp3b. C. semilaevis zp3a encoded 519 amino acid protein, including a signal peptide and a ZP domain with 257 amino acids, which was 62% identical to the medaka ZPC5. Incontrast, C. semilaevis zp3b encoded a 313 amino acid protein, including a signal peptide and a ZP domain with 233 amino acids, which was 63% identical to the medaka (Oryzias latipes) ZPC1. Alignment analysis showed that the ZP domain of the two ZP proteins contained eight conserved cysteines. RT-PCR indicated that the C. semilaevis zp3a was highly expressed in the ovary and kidney of females, and weakly expressed in female muscle and spleen. The C. semilaevis zp3b mRNA was expressed in several tissues of females,including a high expression level in the ovary and kidney, and a relatively low expression level in the heart,brain, muscle, spleen, gill, and intestine. Interestingly, the zp3b mRNA was slightly detected in the testis and kidney of males. Therefore, molecular cloning and characterization of zp genes could lay a foundation for our understanding of the regulation of zp gene evolution and the regulatory mechanism of fertilization.
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Fosmid library construction and initial analysis of end sequences in female half-smooth tongue sole (Cynoglossus semilaevis).
Mar. Biotechnol.
PUBLISHED: 05-20-2009
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Half-smooth tongue sole (Cynoglossus semilaevis: Pleuronectiformes) is a commercially important cultured marine flatfish in China and forms an important fishery resource, but the research of its genome is underdeveloped. In this study, we constructed a female C. semilaevis fosmid library and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of 49,920 clones with an average insert size of about 39 kb, amounting to 3.23 genome equivalents. Fosmid stability assays indicate that female C. semilaevis DNA was stable during propagation in the fosmid system. Library screening with eight microsatellite markers yielded between two and five positive clones, and none of those tested was absent from the library. End-sequencing of both 5 and 3 ends of 1,152 individual clones generated 2,247 sequences after trimming, with an average sequence length of 855 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 259 (11.53%) and 287 (12.77%) significant hits (E < e (-5)), respectively. Repetitive sequences analysis resulted in 5.23% of base pairs masked using both the Fugu and Danio databases, repetitive elements were composed of retroelements, DNA transposons, satellites, simple repeats, and low-complexity sequences. The fosmid library, in conjunction with the fosmid end sequences, will serve as a useful resource for large-scale genome sequencing, physical mapping, and positional cloning, and provide a better understanding of female C. semilaevis genome.
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Isolation and characterization of Toll-like receptor 9 in half-smooth tongue sole Cynoglossus semilaevis.
Fish Shellfish Immunol.
PUBLISHED: 04-03-2009
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Toll-like receptors (TLRs) are considered as key sensors to trigger the hosts innate immune system and adaptive immune responses by recognizing various PAMPs and initiating signal transduction. TLR9, as a member of TLR family, mediates the recognition of unmethylated CpG dinucleotide motifs commonly found in both bacterial and viral genomes. In the current study, the TLR9 gene was isolated from one of flatfish species, half-smooth tongue sole (Cynoglossus semilaevis). In the 4588 bp genomic sequence, three exons, two introns, and 5 UTR of 23 bp and 3 UTR of 342 bp were identified. Putative amino acid sequence was 1062 residues long, including a typical conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, 14 leucine-rich repeat (LRR) motifs, with greater than 60% identity to gilthead sea bream Sparus aurata and Japanese flounder Paralichthys olivaceus orthologs. Quantitative RT-PCR analysis indicated a broad expression of csTLR9, especially in spleen and gonads. No statistically significant changes were observed for csTLR9 mRNA levels in spleen and head kidney after inactive Vibrio anguillarum immunisation. In C. semilaevis ontogeny, the expression of csTLR9 appeared to be developmentally regulated. The presence of maternal TLR9 mRNA and the dramatic decrease of TLR9 expression at metamorphic stage indicated TLR9 might be involved in C. semilaevis development. Comparing sequence and expression profile of csTLR9 with mammalian and other piscine TLR9s suggested that the main function of TLR9 might be conserved across vertebrates, although species-specific features were present.
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Intragenomic variability and pseudogenes of ribosomal DNA in stone flounder Kareius bicoloratus.
Mol. Phylogenet. Evol.
PUBLISHED: 03-21-2009
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Molecular study of Stone flounder Kareius bicoloratus revealed a high level of intra-individual polymorphism of ribosomal DNA (18S rDNA, ITS1 and 5.8S rDNA). This polymorphism includes not only multiple functional genes but also putative pseudogenes and recombinants. Three major types (Type I, II and III) of 18S rDNA differing in lengths coexisted in the genome of each studied individual. Type II and Type III have a 15-bp-deletion and Type III has an additional 7-bp-deletion relative to Type I. Expression analyses gave clear evidence that Type I 18S rDNA was abundantly expressed. In contrast, transcription of Type II and III 18S rDNA were not detected. Further sequence analysis revealed that Type I was the expected and functional 18S rDNA in K. bicoloratus, while Type II and Type III were pseudogenes. Phylogeny of the ITS1 sequences formed three groups (Group I, II and III), of which Group I linking with Type I 18S rDNA were functional, whereas the remaining two Groups (II and III) linking with Type II and Type III 18S rDNA were pseudogenes. Groups II and III ITS1 sequences were shorter (685-717bp in length) than Group I ITS1 (762-776bp), but had much higher sequence divergence (9.8%) than the later ones did (4.9%). Group II sequences were recombinants between Group I and Group III sequences, providing the possibility that the rDNA of K. bicoloratus is in a transition stage of concerted evolution. In addition, by comparing standard PCR and high-stringency PCR, we concluded that DMSO might prevent functional ITS1 sequences from being amplified instead of enhancing its amplification efficiency. The analysis suggests a non-concerted evolution of rDNA in K. bicoloratus. Features of rDNA functional genes and pseudogenes are described. Possible causes for the lack of homogenization of rDNA paralogues within individuals are discussed.
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A microsatellite genetic linkage map of half smooth tongue sole (Cynoglossus semilaevis).
Mar Genomics
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Half smooth tongue sole, Cynoglossus semilaevis (Pleuronectiformes, Cynoglossidae), is an important aquaculture species throughout coastal areas of China which has a high nutritional and economic value. Genetic linkage map is an important tool for accelerating aquatic breeding process through marker assisted selection (MAS) and quantitative trait locus (QTL). Here, 325 polymorphic microsatellite markers were explored and sex-specific genetic linkage map were constructed using these markers. The female map contained 193 markers located at 21 linkage groups, with a total length of 1041cM and an average resolution of 6.8cM; the male map contained 195 markers located at 21 linkage groups, with a total length of 1154cM and an average resolution of 7.2cM, they covered approximately 76.72% and 78.12% genomes, respectively. The recombination ratio of female/male was about 1:1.02 estimated in the sex-specific frame map. All developed microsatellite markers and this linkage map could serve as the foundation for further study in high density linkage map construction.
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Mutation in promoter region of a serine protease inhibitor confers Perkinsus marinus resistance in the eastern oyster (Crassostrea virginica).
Fish Shellfish Immunol.
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Protease inhibitors from the host may inhibit proteases from invading pathogens and confer resistance. We have previously shown that a single-nucleotide polymorphism (SNP198C) in a serine protease inhibitor gene (cvSI-1) is associated with Perkinsus marinus resistance in the eastern oyster. As SNP198 is synonymous, we studied whether its linkage to polymorphism at the promoter region could explain the resistance. A 631 bp fragment of the promoter region was cloned by genome-walking and resequenced, revealing 22 SNPs and 3 insertion/deletions (indels). A 25 bp indel at position -404 was genotyped along with SNP198 for association analysis using before- and after-mortality samples. After mortalities that were primarily caused by P. marinus, the frequency of deletion allele at -404indel increased by 15.6% (p = 0.0437), while that of SNP198C increased by only 3.4% (p = 0.5756). The resistance alleles at the two loci were coupled in 79.6% of the oysters. Oysters with the deletion allele at -404indel showed significant (p = 0.0189) up-regulation of cvSI-1 expression under P. marinus challenge. Our results suggest that mutation at the promoter region causes increased transcription of cvSI-1, which in turn confers P. marinus resistance in the eastern oyster likely through inhibiting pathogenic proteases from the parasite.
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Molecular cloning, tissue distribution, and ontogeny of gonadotropin-releasing hormone III gene (GnRH-III) in half-smooth tongue sole (Cynoglossus semilaevis).
Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
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Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a vital role in hypothalamus-pituitary-gonad (HPG) axis. In the present study, the GnRH-III gene was isolated from half-smooth tongue sole (Cynoglossus semilaevis). In the 1160 bp genomic sequence, four exons, three introns, and 5-/3-flanking sequences were identified. The putative peptide was 92 residues long, including a putative signal peptide containing 23 amino acids, the GnRH decapeptide, a proteolytic cleavage site of three amino acids and a GnRH associated peptide of 56 amino acids. The overall amino acid sequence of C. semilaevis GnRH-III (csGnRH-III) was highly conserved with other teleost GnRH-III genes. Phylogenetic analysis showed the evolutionary relationships of csGnRH-III with other known GnRH genes. A 320 bp promoter sequence of the csGnRH-III was also analyzed, and several potential regulatory motifs were identified which were conserved in the GnRH promoters of other teleosts. Quantitative real-time PCR analysis indicated csGnRH-III was expressed only in brain and gonads. In C. semilaevis, the csGnRH-III transcript was maternally deposited and appeared to be developmentally regulated during embryogenesis and early larval development. Comparing sequence and expression patterns of csGnRH-III with other teleosts GnRH-IIIs suggested that the main function of GnRH-III might be conserved in teleosts.
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Identification and characterization of a hepcidin from half-smooth tongue sole Cynoglossus semilaevis.
Fish Shellfish Immunol.
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Hepcidin, an antimicrobial peptide, has a dual function including innate immunity and iron regulation. Here, based on the sequence of an EST database, we have isolated and characterized a hepcidin gene (referred to as CsHepcidin) from half-smooth tongue sole (Cynoglossus semilaevis). Analysis of the coding regions indicated CsHepcidin gene comprised 3 exons and 2 introns. The putative CsHepcidin showed a great similarity to other hepcidin orthologues, particularly with respect to its 24 aa signal peptide, typical RX(K/R)R motif and eight conserved cysteine residues in the mature cationic peptide. Phylogenic analysis indicated that CsHepcidin was a hepcidin 1-type peptide of acanthopterygians, with highly homologous with Solea senegalensis hepcidin. In C. semilaevis ontogeny, CsHepcidin mRNA was detected at a low level in unfertilized eggs, increased on 6 d after hatching, and decreased remarkably at metamorphic stage. CsHepcidin transcripts showed a constitutive basal expression in most of the tissues, especially in liver. Challenge with formalin-inactivated Vibrio anguillarum led to significantly up-regulations of CsHepcidin gene in liver, head kidney and spleen in time-dependent manners. Biological activity analysis showed that recombinant CsHEP exhibited direct antimicrobial activity against bacterial pathogens in vitro, particularly showed strong activity against the principal fish pathogens, V. anguillarum and Edwardsiella tarda. All these results suggest that CsHepcidin may be involved in the initial response to invasion of microbial pathogens. Further exploration to elucidate the role of CsHepcidin in iron regulation and embryogenesis in C. semilaevis are needed.
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Molecular cloning and characterization of interleukin-1? in half-smooth tongue sole Cynoglossus semilaevis.
Vet. Immunol. Immunopathol.
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As a member of the interleukin (IL)-1 family, IL-1? is a prototypical proinflammatory cytokine, which plays a crucial role in immune responses. Herein, we reported the full length cDNA of IL-1? in half-smooth tongue sole (Cynoglossus semilaevis). The csIL-1? cDNA contained a 130 bp 5 UTR, a 417 bp 3 UTR and a 741 bp coding sequence (CDS) that translated into a polypeptide of 246 amino acids. The protein sequence included a typical IL-1 family signature and lacked an interleukin-converting enzyme (ICE) cut site. RT-PCR analysis indicated a broad expression of csIL-1?, especially in immune-related organs. After injection with inactive Vibrio anguillarum, the expression of csIL-1? was induced in the head kidney and spleen and reached the highest level at 8 h post injection. Higher expression of csIL-1? was observed at gastrula stage, eye-bud stage and hatching stage and lower level of csIL-1? mRNA was detected at metamorphic stage. The expression of csIL-1? during development suggested IL-1? might be involved not only in immunity but also development. Taken together, the present study indicated that csIL-1? played an important role in immune responses and development like its mammalian counterparts, although species-specific features were present.
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Identification and characterization of IL-1 receptor-associated kinase-4 (IRAK-4) in half-smooth tongue sole Cynoglossus semilaevis.
Fish Shellfish Immunol.
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As a crucial component in TLR/IL-1R signaling pathways, IRAK-4 plays a central role in innate and adaptive immunity. In the present study, the cDNA of IRAK-4 was cloned for the first time from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of csIRAK-4 was 2149 bp and contained a 168 bp 5 UTR, a 580 bp 3 UTR and a 1401 bp CDS. The predicted protein sequence of csIRAK-4 had two typical domains, a death domain (DD) at the N terminus and a serine/threonine/tyrosine protein kinase domain (STYKc) at the C terminus. RT-PCR showed that csIRAK-4 mRNA was detected in all tested tissues, especially in immune-related organs, gonads and brain. After injected with inactivated Vibrio anguillarum, the expressions of csIRAK-4 were up-regulated significantly (P<0.05) in spleen and head kidney. During development, csIRAK-4 was expressed at all selected stages and low-level expression was detected at metamorphosis. Taken together, the present study indicated that csIRAK-4 played a crucial role in immune responses and might be involved in the process of development.
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