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Amur catfish is extensively distributed and cultured in Asian countries. Despite of economic importance, the genomic information of this species remains limited. A reference transcriptome of Amur catfish was assembled and the sex-biased gene expression in the gonads was characterized using RNA-sequencing. The assembled transcriptome of Amur catfish consisted of 74,840 transcripts. The N50, mean length and max length of transcripts are 1970, 1235 and 16,748 bp. Putative sex-specific transcripts were identified and sex-specific expression of the representative genes was verified by RT-PCR. Differential expression analysis identified 5401 ovary-biased and 5618 testis-biased genes. The ovary-biased genes were mainly enriched in pathways such as RNA transport and ribosome biogenesis in eukaryotes. The testis-biased genes were enriched in calcium signaling and cytokine-cytokine receptor interaction, etc. Our data provide a valuable genomic resource for further investigating the genetic basis of sex determination, sex differentiation and sexual dimorphism of catfish.

Galectins constitute a group of lectins with binding specificity for beta-galactoside sugars. Galectin-1 is a prototype galectin and the multifunctionality of mammalian galectin-Is is well-known, but only a few of fish galectin-Is have been identified. In this study, we obtained the full-length cDNA and genomic sequence of the galectin-1 gene (designated as Pdlgals1) from large scale loach (Paramisgurnus dabryanus), performed phylogenetic analysis, and characterized the expression pattern and the transcriptional activity of its 5' flanking region. The Pdlgals1 gene contains 4 exons that encode a peptide of 132 amino acids with all the galectin signature motifs. Phylogenetic analysis and sequence alignment indicated that Pdlgals1 is a homologue of human LGALS1. RT-PCR and whole-mount in situ hybridization revealed that Pdlgals1 is mainly expressed in the skin, muscle, intestine and cavum oropharyngeum. Transcriptional activity assays demonstrated that the basal promoter of Pdlgals1 is located in a region from -500 bp to its transcriptional start site. Potential binding sites for transcription factors including C/EBP, AP-1, GATA, Oct-1, delta EF1 NF-kappa B, c-Myb, SP-1, AP-2, AML-1 alpha, and AP-4 were identified in the basal promoter, suggesting that these factors are associated with the regulation of Pdlgals1. These results provided clues for further investigation of galectin-1 functions in loaches (c) 2015 Elsevier B.V. All rights reserved.

Microcystin-LR (MC-LR) is one of the most common microcystins (MCs), which are hepatotoxic and released into a water body during a period of cyanobacterial blooms. These toxicants can be accumulated in aquatic animals and transferred along the food chain and thus pose adverse effects on aquatic environment and public health. Zebrafish Abcb4 is reported to mediate the cellular efflux of ecotoxicologically relevant compounds including galaxolide, tonalide and phenanthrene; however, it remains unclear whether Abcb4 functions in the detoxification of MC-LR. Here, we demonstrated the role of zebrafish Abcb4 in cellular efflux of MC-LR. Transcripts of zebrafish abcb4 were detected in all of adult tissues examined. MC-LR was able to induce the expression of abcb4 gene and overexpression of Abcb4 significantly decreased the cytotoxicity and accumulation of MC-LR in LLC-PK1 cells and developing embryos. In contrast, overexpression of an Abcb4-G1177D mutant abolished its transporter function but not substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to this cyanobacterial toxin. Moreover, ATPase activity in developing embryos can be induced by MC-LR. Thus, zebrafish Abcb4 plays crucial roles in cellular efflux of MC-LR and is a potential molecular marker for the monitoring of cyanobacteria contamination in the aquatic environment. (C) 2014 Elsevier Inc. All rights reserved.

Conditional expression of a target gene during zebrafish development is a powerful approach to elucidate gene functions. The tetracycline-controlled systems have been successfully used in the modulation of gene expression in mammalian cells, but few lines of zebrafish carrying these systems are currently available. In this study, we had generated a stable transgenic zebrafish line that ubiquitously expressed the second-generation of reverse Tet transactivator (rtTA-M2). Southern blotting analysis and high-throughput genome sequencing verified that a single copy of rtTA-M2 gene had stably integrated into the zebrafish genome. After induction with doxycycline (Dox), a strong green fluorescent protein (GFP) was seen in rtTA-transgenic eggs injected with pTRE-EGFP plasmids. The fluorescent signal gradually decreased after the withdrawal of Dox and disappeared. However, leaky expression of GFP was undetectable before Dox-induction. Additionally, transgenic embryos expressing rtTA-M2 exhibited no obvious defects in morphological phenotypes, hatching behavior and expression patterns of developmental marker genes, suggesting that rtTA-M2 had little effect on the development of transgenic zebrafish. Moreover, expressed Dickkopf-1 (DKK1) in pTRE-DKK1-injected embryos led to alterations in the expression of marker genes associated with Wnt signaling. Thus, this rtTA-transgenic zebrafish can be utilized to dissect functions of genes in a temporal manner.

Expression-independent gene or polyadenylation [poly(A)] trapping is a powerful tool for genome-wide mutagenesis regardless of whether a targeted gene is expressed. Although a number of poly(A)-trap vectors have been developed for the capture and mutation of genes across a vertebrate genome, further efforts are needed to avoid the 3'-terminal insertion bias and the splice donor (SD) read-through, and to improve the mutagenicity. Here, we present a Sleeping Beauty (SB) transposon-based vector that can overcome these limitations through the inclusion of three functional cassettes required for gene-finding, gene-breaking and large-scale mutagenesis, respectively. The functional cassette contained a reporter/selective marker gene driven by a constitutive promoter in front of a strong SD signal and an AU-rich RNA-destabilizing element (ARE), which greatly reduced the SD read-through events, except that the internal ribosomal entry site (IRES) element was introduced in front of the SD signal to overcome the phenomenon of 3'-bias gene trapping. The breaking cassette consisting of an enhanced splicing acceptor (SA), a poly(A) signal coupled with a transcriptional terminator (TT) effectively disrupted the transcription of trapped genes. Moreover, the Hsp70 promoter from tilapia genome was employed to drive the inducible expression of SB11, which allows the conditional remobilization of a trap insert from a non-coding region. The combination of three cassettes led to effective capture and disruption of endogenous genes in HeLa cells. In addition, the Cre/LoxP system was introduced to delete the Hsp70-SB11 cassette for stabilization of trapped gene interruption and biosafety. Thus, this poly(A)-trap vector is an alternative and effective tool for identification and mutation of endogenous genes in cells and animals.

p53R2 is a newly identified small subunit of ribonucleotide reductase and plays a pivotal role in the supply of dNTPs for genomic DNA repair and mitochondrial DNA synthesis, but little is known about its functions in zebrafish. Herein, we obtained the cDNA of zebrafish p53R2 that shares 72.8% and 72.5% amino acid identities with human p53R2 and zebrafish R2, respectively. Residues crucial for enzymatic activity are highly conserved among p53R2 proteins from different species. p53R2 in zebrafish was maternally expressed, its transcripts were detected in developing embryos and all adult tissues examined. A 250-bp minimal promoter upstream of the translational initiation site was identified to drive basal expression of p53R2 in a p53-independent manner. Expression of p53R2 was induced by DNA-damaging reagents CPT or MMS, but suppressed by p53-knockdown in zebrafish embryos. Moreover, p53R2 was mainly distributed in the cytoplasm of cells under normal condition and upon DNA damage. Furthermore, overexpression of p53R2 attenuated apoptosis of embryonic cells caused by CPT or MMS treatment and protected developing embryos from death. Therefore, functions of p53R2 in zebrafish are closely associated with its activity in DNA repair and synthesis. (C) 2010 Elsevier B.V. All rights reserved.

Caveolin-1 (Cav-1) is the principal component of plasma membrane caveolae that negatively regulates a number of cellular signaling events including canonical Wnt signaling Activation of the Wnt/beta-catenin pathway is essential for dorsal organizer formation and specification in early vertebrate embryos, but it remains not well understood what controls dorsal activity of maternal beta-catenin and how Cav-1 functions in zebrafish embryogenesis Here, we report that Cav-1 is required for proper dorsoventral patterning in zebrafish Both Win and BMP signals act coordinately to negatively control transcriptional expression of cav-1 during embryonic development. Ectopic expression of Cav-1 alpha or -1 beta resulted in formation of typical ventralized embryos, whereas Cav-1 knockdown led to abnormal embryos with expanded expression of dorsal genes Cav-1 overexpression disrupts the nuclear translocation of beta-catenin through the interaction of its scaffolding domain with Cav-1 binding motif of beta-catenin This reciprocal interaction is necessary for the ventralizing activity of Cav-1 We have further demonstrated that human Cav-1 proteins have conserved ventralizing activity in zebrafish embryogenesis. Thus, maternally expressed zebrafish Cav-1 regulates dorsoventral patterning by limiting nuclear translocation of active beta-catenin (C) 2010 Published by Elsevier Inc

Transforming growth factor-beta (TGF-beta) is a prototypical tumour-suppressor cytokine with cytostatic and pro-apoptotic effects on most target cells; however, mechanisms of its pro-survival/anti-apoptotic signalling in certain cell types and contexts remain unclear. In human lung fibroblasts, TGF-beta1 is known to induce myofibroblast differentiation in association with the delayed activation of focal adhesion kinase (FAK) and protein kinase B (PKB/AKT). Here, we demonstrate that FAK and AKT are independently regulated by early activation of SMAD3 and p38 MAPK, respectively. Pharmacologic or genetic approaches that disrupt SMAD3 signalling block TGF-beta1-induced activation of FAK, but not AKT; in contrast, disruption of early p38 MAPK signalling abrogates AKT activation, but does not alter FAK activation. TGF-beta1 is able to activate AKT in cells expressing mutant FAK or in cells treated with an RGD-containing peptide that interferes with integrin signalling, inhibits FAK activation and induces anoikis (apoptosis induced by loss of adhesion signalling). TGF-beta1 protects myofibroblasts from anoikis, in part, by activation of the PI3K-AKT pathway. Thus, TGF-beta1 co-ordinately and independently activates the FAK and AKT protein kinase pathways to confer an anoikis-resistant phenotype to myofibroblasts. Activation of these pro-survival/anti-anoikis pathways in myofibroblasts likely contributes to essential roles of TGF-beta1 in tissue fibrosis and tumour-promotion.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine involved in differentiation, growth, and survival of mesenchymal cells while inhibiting growth/survival of most other cell types. The mechanism(s) of pro-survival signaling by TGF-beta1 in mesenchymal cells is unclear. In this report, we demonstrate that TGF-beta1 protects against serum deprivation-induced apoptosis of mesenchymal cells isolated from patients with acute lung injury and of normal human fetal lung fibroblasts (IMR-90). TGF-beta receptor(s)-activated signaling in these cells involves rapid activation of the Smad and p38 MAPK pathways within minutes of TGF-beta1 treatment followed by a more delayed activation of the pro-survival phosphatidylinositol 3-kinase-protein kinase B (PKB)/Akt pathway. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a p38 kinase-deficient mutant protein inhibits TGF-beta1-induced PKB/Akt phosphorylation. Conditioned medium from TGF-beta1-treated cells rapidly induces PKB/Akt activation in an SB203580- and suramin-sensitive manner, suggesting p38 MAPK-dependent production of a secreted growth factor that activates this pro-survival pathway by an autocrine/paracrine mechanism. Inhibition of the phosphatidylinositol 3-kinase-PKB/Akt pathway blocks TGF-beta1-induced resistance to apoptosis. These results demonstrate the activation of a novel TGF-beta1-activated pro-survival/anti-apoptotic signaling pathway in mesenchymal cells/fibroblasts that may explain cell-specific actions of TGF-beta1 and provide mechanistic insights into its pro-fibrotic and tumor-promoting effects.

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.

Myofibroblast differentiation and activation by transforming growth factor-beta1 (TGF-beta1) is a critical event in the pathogenesis of human fibrotic diseases, but regulatory mechanisms for this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-beta1 and adhesion-dependent signals. TGF-beta1-induced myofibroblast differentiation of lung fibroblasts is blocked in non-adherent cells despite the preservation of TGF-beta receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-beta1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) including that of its autophosphorylation site, Tyr-397, an effect that is dependent on cell adhesion and is delayed relative to early Smad signaling. Pharmacologic inhibition of FAK or expression of kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit TGF-beta1-induced alpha-smooth muscle actin expression, stress fiber formation, and cellular hypertrophy. Basal expression of alpha-smooth muscle actin is elevated in cells grown on fibronectin-coated dishes but is decreased on laminin and poly-d-lysine, a non-integrin binding polypeptide. TGF-beta1 up-regulates expression of integrins and fibronectin, an effect that is associated with autophosphorylation/activation of FAK. Thus, a safer and more effective therapeutic strategy for fibrotic diseases characterized by persistent myofibroblast activation may be to target this integrin/FAK pathway while not interfering with tumor-suppressive functions of TGF-beta1/Smad signaling.

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates. (C) 2002 Elsevier Science Ltd. All rights reserved.