Cloning and analysis of expression of a gilthead sea bream (Sparus aurata) Mx cDNA

In the current work, we have cloned and sequenced the full cDNA for a Mx protein in the gilthead sea bream (Sparus aurata) by RACE PCR. The Mx cDNA of 2182 bp contained an open reading frame of 1857 bp that codes for a protein of 618 aa. Within the coding sequence, characteristic features of Mx proteins were found, such as a tripartite guanosine-5'-triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD), the signature of the dynamin family, LPRG(S/K)GIVTR, and a sequence that codes for a leucine zipper at the C-terminal region of the protein. An RT-PCR was optimised to estimate the level of expression of Mx protein in sea bream. Through this method we determined that Mx is constitutively expressed in head kidney, liver, spleen, heart, gills, muscle and brain of healthy sea bream. Intramuscular challenge of sea bream with polyinosinic:polycytidylic acid (Poly I:C) up-regulated Mx expression in liver, head kidney, spleen and muscle. Constitutive expression was also found in isolated head kidney macrophages and blood leukocytes. This expression was significantly up-regulated by addition of Poly I:C. Mx was not constitutively expressed in the sea bream established cell line, SAF-1, but Poly I:C and nodavirus were also capable of inducing Mx expression in this cell line.     

Cystatin B homolog from rock bream Oplegnathus fasciatus: genomic characterization, transcriptional profiling and protease-inhibitory activity of recombinant protein

Cysteine proteases are present in all living organisms and, in animals, function in a vast array of physiological and pathological processes. Cysteine protease inhibitors act upon the cysteine proteases to regulate their activity. The cystatin superfamily of cysteine protease inhibitors has members represented in all living organisms studied to date. Here, we report the identification of a new member of the family 1 cystatin in Oplegnathus fasciatus rock bream (denoted as RbCyt B) and the characterization at the molecular level. The complete genomic sequence of RbCyt B consists of three exons and a promoter region. The open reading frame (ORF) encodes for a 100 amino acids length polypeptide with a single cystatin-like domain and a cysteine protease inhibitor signature motif. The conserved N-terminal glycine, glutamine-valine-glycine motif, QxVxG, and a variant of the proline-tryptophan, PW, motif were identified. RbCyt B showed closest phylogenetic distance to Dicentrarchus labrax cystatin B, and shared up to 73% amino acid identity and 90% amino acid similarity with known cystatin B genes. RbCyt B mRNA expression was detected in nine different tissues and was highly expressed in liver, spleen, gill, brain, intestine, kidney, head kidney, and blood, as compared with muscle. In vivo immune stimulation with Edwardsiella tarda bacteria caused significant up-regulation of RbCyt B mRNA in head kidney and spleen at 24h post-infection (P<0.05). Recombinant RbCyt B was expressed in Escherichia coli, and the purified protein demonstrated 82% papain inhibitory activity at 500 × 10(-3) μg μL(-1) in a concentration-dependent manner. These results suggest that RbCyt B is a member of family 1 cystatin with high homology to cystatin B, and is a biologically active protein possessing papain inhibitory activity and potentially involved in immune responses against invading Gram-negative bacteria in rock bream.     

Molecular cloning and characterization of gilthead sea bream (Sparus aurata) growth hormone receptor (GHR). Assessment of alternative splicing

The full-length growth hormone receptor (GHR) of gilthead sea bream (Sparus aurata) was cloned and sequenced by RT-PCR and rapid amplification of 5'and 3'ends. The open reading frame codes for a mature 609 amino acid protein with a hydrophobic transmembrane region and all the characteristic motifs of GHRs. Sequence analysis revealed a 96 and 76% of amino acid identity with black sea bream (Acanthopagrus schlegeli) and turbot (Scophthalmus maximus) GHRs, respectively, but this amino acid identity decreases up to 52% for goldfish (Carassius auratus) GHR. By means of real-time PCR assays, concurrent changes in the hepatic expression of GHRs and insulin-like growth factor-I (IGF-I) was evidenced. Moreover, their regulation occurred in conjunction with the summer spurt of growth rates and circulating levels of GH and IGF-I. Search of alternative splicing was carried out exhaustively for gilthead sea bream GHR, but Northern blot and 3' RACE failed to demonstrate the occurrence of short alternative messengers. Besides, RT-PCR screening did not reveal deletions or insertions that could lead to alternative reading frames. In agreement with this, cross-linking assays only evidenced two protein bands that match well with the size of glycosylated and non-glycosylated forms of the full-length GHR. If so, it appears that alternative splicing at the 3'end does not occur in gilthead sea bream, although different messengers for truncated or longer GHR variants already exist in turbot and black sea bream, respectively. The physiological relevance of this finding remains unclear, but perhaps it points out large inter-species differences in the heterogeneity of the GHR population.     

Molecular cloning and genomic structure of the betaTRCP2 gene on chromosome 5q35.1

Beta-catenin, IkappaBalpha, and HIV Vpu are recruited to the ubiquitin-proteasome degradation pathway by betaTRCP, one of the components of the ubiquitin ligase complex. betaTRCP2, a related gene of betaTRCP, was cloned and characterized. Three isoforms, betaTRCP2A, betaTRCP2B, and betaTRCP2C, were identified. All of these betaTRCP2 isoforms consist of an F-box and seven WD repeats. Human betaTRCP2A shows 86% total amino acid identity with human betaTRCP. betaTRCP2 mRNA of 4.5 kb in size was detected almost ubiquitously. Sequence analyses on betaTRCP2 genomic clones revealed that the betaTRCP2 gene consists of at least 14 exons. Exons 1 and 4-14 are shared among all betaTRCP2 isoforms. betaTRCP2A of 508 amino acids lacks exons 2 and 3, betaTRCP2B of 529 amino acids contains exon 3, and betaTRCP2C of 542 amino acids contains exon 2. These results indicate that three betaTRCP2 isoforms are transcribed due to alternative splicing. The betaTRCP2 gene has been mapped to human chromosome 5q35.1 by fluorescence in situ hybridization.     

赤眼鳟Mx1基因的cDNA克隆及表达特性

为探究赤眼鳟(Squaliobarbus curriculus)是否存在Mx1 (Myxovirus resistance)基因及参与抗病毒免疫反应, 研究利用RACE技术获得了赤眼鳟Mx1基因(ScMx1)的cDNA全长序列, 并对其进行了生物信息学分析; 采用荧光定量PCR技术, 检测了ScMx1在赤眼鳟健康组织中的表达情况以及感染GCRV后ScMx1和ScIFN-Ⅰ的表达特征。结果表明, ScMx1的cDNA全长为3000 bp, 包含5′非编码区124 bp, 开放阅读框1893 bp, 3′非编码区983 bp, 共编码630个氨基酸。预测的ScMx1蛋白包含GTP酶结合区域、中央核心结构域和GTP酶效应结构域。ScMx1与青鱼Mx1的相似性最高(97%), 与ScMx的相似性仅为50%。ScMx1在所检测的10种组织中均有表达, 其中在脾脏中表达量最高。经GCRV感染开始至168h, ScMx1和ScIFN-Ⅰ在肝脏和体肾中的表达量持续上调; 在脾脏和头肾中于感染后72h达到峰值。相关性分析显示脾脏中ScMx1和ScIFN-Ⅰ的表达水平呈显著相关(r=0.94, P=0.018)。研究发现赤眼鳟存在Mx1基因, 且可能参与了抗GCRV免疫应答反应。     

Identification of four functional NR3B isoforms in developing white matter reveals unexpected diversity among glutamate receptors

Functional neurotransmitter receptors are expressed in central white matter, where they mediate ischemic damage to glia and may be involved in cell-cell signalling. In this study, we analysed NMDA receptor NR1, NR2B-C and NR3A-B subunit expression in the brain and optic nerve by molecular cloning. In addition to the canonical forms of NR1 and NR2, four previously unknown NR3B variants, generated by alternative splicing, were identified. The variants encoded for isoforms with deletions of 8/15 amino acids in the N-terminal domain or 200/375 amino acids removing one or three transmembrane domains and part of the C-terminal domain, as compared with the previously characterized NR3B isoform. Co-expression of NR3B isoforms with NR1/NR2A-C modulated the amplitude and Mg(2+)-sensitivity of glutamate responses in a NR2 subunit-dependent fashion, with significant variations in the effects produced by different isoforms. These effects were not the result of reduced surface expression of the receptor complex since all NR3B isoforms reduced surface expression by a similar degree. These data reveal previously uncharacterized regulation of NMDA receptor function by alternative splicing of the NR3B subunit.     

Molecular cloning of rock bream's (Oplegnathus fasciatus) tumor necrosis factor receptor-associated factor 2 and its role in NF-κB activiation

Tumor necrosis factor receptor-associated factor 2 (TRAF2) acts as a transducer of tumor necrosis factor-α (TNF-α)-triggered cell signals which results in inflammation, cell proliferation and antiapoptotic response. In this study, we have cloned cDNA of rock bream (Oplegnathus fasciatus) TRAF2, and analyzed its function in activation of NF-κB. The full length cDNA of rock bream TRAF2 consisted of 95 bp 5' UTR, 335 bp 3' UTR, and 1563 bp ORF encoding 520 amino acids that contained N-terminal RING-type and TRAF-type zinc finger domains and a C-terminal TRAF domain. The deduced amino acid sequence of rock bream TRAF2 showed more than 75% identity with other fish TRAF2s, and even as high as 56% identity with mouse and human TRAF2 proteins. To know whether the rock bream TRAF2 involves in NF-κB activation, Epithelioma papulosum cyprini (EPC) cells harboring an NF-κB reporting vector were transfected with a vector expressing rock bream TRAF2 or a control empty vector. NF-κB activity of EPC cells was significantly increased by exposure to the rock bream recombinant TNF-α. EPC cells transfected with the vector expressing rock bream TRAF2 showed significantly higher NF-κB activity by stimulation with the recombinant TNF-α than cells transfected with a control empty vector, suggesting the present rock bream TRAF2 acts as a transducer of TNF-α-mediated cell signals that enhance NF-κB activation.