Molecular cloning,characterization and expression analysis of cytoplasmic Cu/Zn-superoxid dismutase (SOD) from pearl oyster Pinctada fucata

Because of its capacity to rapidly convert superoxide to hydrogen peroxide, superoxide dismutase (SOD) is crucial in both intracellular signalling and regulation of oxidative stress. In this paper we report the cloning of a Cu/Zn SOD (designated as pfSOD) from the pearl oyster (Pinctada fucata) using rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of this Cu/Zn SOD contains an open reading frame (ORF) of 471 bp coding for 156 amino acids. No signal peptide was identified at the N-terminal amino acid sequence of Cu/Zn SOD indicating that this pfSOD encodes a cytoplasmic Cu/Zn SOD. This is supported by the presence of conserved amino acids required for binding copper and zinc. Semi-quantitative analysis in adult tissues showed that the pfSOD mRNA was abundantly expressed in haemocytes and gill and scarcely expressed in other tissues tested. After challenge with lipopolysaccharide (LPS), expression of pfSOD mRNA in haemocytes was increased, reaching the highest level at 8 h, then dropping to basal levels at 36 h. These results suggest that Cu/Zn SOD might be used as a bioindicator of the aquatic environmental pollution and cellular stress in pearl oyster.     

食蚊鱼CYP19b基因克隆及环境激素暴露对其表达的影响

为探讨17-雌二醇(E2)、17-甲基睾酮(MT)和雄烯二酮(AED)暴露对食蚊鱼脑组织CYP19b表达的影响,首次克隆和分析了食蚊鱼(Gambusia affinis) CYP19b cDNA的全系列。采用静水暴露方法, 分别设置2、10、50和100 ng/L E2、MT和AED各4个实验组, 并设置对照组和平行组, 定量测定了1、3、5和7d脑组织中的CYP19b mRNA表达水平的变化。成功克隆了食蚊鱼CYP19b基因全长, 获得基因片段总长1670 bp, ORF(OpenReading Frame)是从52 bp到1554 bp, 共编码501个氨基酸。通过与其他相关鱼类CYP19b基因编码的氨基酸序列相似性作比较, 食蚊鱼CYP19b基因氨基酸序列的同源性很高。结果显示, 短时间的暴露, E2较低浓度组(2和10 ng/L)对食蚊鱼作用不明显, 但E2相对较高浓度组(50和100 ng/L)能显著地上调食蚊鱼CYP19b基因的表达(P0.01)。暴露早期MT对CYP19b表达无明显影响, 随着暴露时间的持续延长, 除了MT较高浓度组(50 ng/L)外, 其他各浓度组MT对CYP19b的表达有显著的抑制作用(P0.01), 并呈一定的剂量相关关系。AED作为雄激素类似物与MT的作用效果相似, 相对较高浓度(50和100 ng/L)的AED抑制CYP19b的表达(P0.01)。研究结果初步表明, 3种典型环境激素对食蚊鱼CYP19b基因表达的影响十分显著, 但不同的暴露浓度及不同的取样时间点可能表现为促进或抑制CYP19b基因的表达。     

Molecular cloning and characterization of a cDNA for an iron-superoxide dismutase in rice (Oryza sativa L.)

We have isolated a cDNA encoding Fe-SOD from rice (Oryza sativa L.). The deduced amino acid sequence consists of a polypeptide with 255 amino acids, including a putative transit peptide (40 a.a.) in amino-terminal residues. This sequence is similar to the known plant Fe-SODs but not classified in the group of known Fe-SODs. The metal analysis and SOD assays of the partial purified recombinant protein expressed in E. coli showed that this cDNA encodes an iron-containing SOD. However this SOD activity was not inhibited by the treatment with hydrogen peroxide, which was expected to inhibit known Fe-SOD activity. mRNA of rice Fe-SOD was detected in all vegetative tissues examined, being especially abundant in calli, and strongly increased by light induction. These results suggested that this cDNA encodes rice Fe-SOD, which is apparently distinct from known plant Fe-SODs.     

Molecular cloning of the alcohol/hydroxysteroid form (hSTa) of sulfotransferase from human liver.

A cDNA encoding the human alcohol/hydroxysteroid sulfotransferase (h-ST-a), which catalyzes the sulfo-conjugation of many drugs and hormones, was isolated from a human liver cDNA library using a rat STa (rSTa) cDNA probe. The cDNA, designated as hSTa, consists of 1069 base pairs (bp) and contains an 855-nucleotide open reading frame beginning at nucleotide 65, which encodes a 285 amino acid polypeptide of 33.76 kDa. A second cDNA clone (1563 bp) was truncated 5' at nucleotide 231 (lacking the first 15 amino acids) with identical coding region, however, it had a much longer 3' untranslated region (UTR). Both clones contained a short segment of poly(A)+ tail. Northern blot analysis of an adult human liver showed that there are at least 2 mature mRNA with sizes ranging from approximately 1.1 kb to 1.7 kb, verifying the authenticity of the obtained cDNA clones. From the sequence alignment, the hSTa shares 62%/74%, 39%/59%, 35%/48%, 36%/54% identity with rSTa, rSTp (phenol), rSTe (estrogen), and bovine STe (bSTe) at the deduced amino acid and DNA levels, respectively, indicating that there are at least three subfamilies (alcohol, phenol and estrogen) of genes that encode for sulfotransferases in mammals.     

Molecular characterization and mRNA expression of glutathione peroxidase and glutathione S-transferase during osmotic stress in olive flounder (Paralichthys olivaceus)

Glutathione peroxidase (GPX) and glutathione S-transferase (GST) are key enzymes of cellular detoxification systems that defend cells against reactive oxygen species (ROS). In this study, we isolated the GPX and GST full-length cDNA and investigated the expression of these mRNAs from livers of olive flounder during salinity changes (35, 17.5, 8.75, 4 and 0 psu) by quantitative PCR (QPCR). GPX cDNA consists of 429 base pairs (bp) and encodes a protein of 142 amino acids. GST cDNA consists of 663 bp and encodes a protein of 220 amino acids. Both of GPX and GST mRNA expressions were the highest in 4 psu and then decreased in 0 psu. Also, the levels of Na(+) and Cl(-) decreased, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased during the experimental period. These findings provide molecular characterization of GPX and GST in olive flounder and suggest that GPX and GST play important roles in detoxification of ROS, thereby these maybe indicators of oxidative stress responses by salinity changes in olive flounder.     

Expression and localization of transcripts of MT5-MMP and its related MMP in the ovary of the medaka fish Oryzias latipes

cDNA clones of MT5-matrix metalloproteinase (MT5-MMP) and a related protein (designated MT5-MMP-del) were isolated by screening the cDNA library and by 3'-rapid amplification of cDNA ends using an ovary RNA of the medaka fish Oryzias latipes. The MT5-MMP clone encodes a protein of 546 amino acids while the MT5-MMP-del clone encodes a protein of 431 amino acids. Compared with mammalian counterparts, the fish MT5-MMP and MT5-MMP-del both lack the signal peptide and a part of the prodomain. The fish MT5-MMP and MT5-MMP-del were different in that the latter did not have the stem/transmembrane/cytoplasmic domain. The two fish MMPs were expressed in the ovary, testis, brain, and intestine. In the ovary, MT5-MMP mRNA was expressed in the oocytes of small growing follicles. In contrast, MT5-MMP-del mRNA was found in the stromal interstitial cells. These results strongly suggest that a MT5-MMP/gelatinase A cascade may possibly operate in the process of spawning and/or other events associated with ovulated oocytes or fertilized eggs of the medaka fish.     

Cloning of profilin (FcPFN) from the shrimpFenneropenaeus chinensis, a highly expressed protein in white spot syndrome virus (WSSV)-infected shrimp

We isolated and characterized the profilin (FcPFN) cDNA from hemocytes ofFenneropenaeus chinensis, a unique shrimp species from the Yellow Sea. The FcPFN cDNA consists of 830 bp and encodes a polypeptide of 125 amino acids, having a predicted isoelectric point of 5.06. The deduced amino acid sequence of FcPFN shows 36% and 90% amino acid sequence identity to the profilin genes of Pacific white shrimpLitopenaeus vannamei and black tiger shrimpPenaeus monodon, respectively. The FcPFN mRNA was highly expressed in hemocytes and hepatopancreas and moderately in muscle of normal shrimp. The higher expression of FcPFN mRNA is observed in shrimp infected with the white spot syndrome virus (WSSV), which is a major concern in all shrimp-growing regions of the world. These results suggest a potential role for FcPFN in viral host defense mechanisms.     

三疣梭子蟹Profilin基因全长cDNA的克隆与表达分析

三疣梭子蟹（Portunus trituberculatus）是我国沿海重要养殖品种之一,近年来养殖病害呈逐年上升趋势,制约了三疣梭子蟹养殖产业的健康可持续发展。克隆三疣梭子蟹免疫相关基因,研究免疫基因的功能和作用机制,可为三疣梭子蟹养殖病害的防治奠定基础。本研究从三疣梭子蟹血细胞全长cDNA文库中克隆了742 bp 的profilin基因全长cDNA。Profilin全长cDNA中开放阅读框长375 bp,编码125 aa。推导的三疣梭子蟹profilin理论等电点pI 5.87,氨基酸序列与冈比亚按蚊（Anopheles gambiae）profilin同源性最高,序列一致性为42.9%。荧光定量RT-PCR分析结果显示在正常的三疣梭子蟹机体中,血细胞profilin表达水平最高,其次为肝胰脏;在致病菌副溶血弧菌（Vibrio parahaemolyticus）诱导后,血细胞中profilin表达量显著上升（P<0.01）,表明profilin可能参与了三疣梭子蟹的免疫防御反应,是一个免疫相关因子。     

Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA from Zhikong scallop Chlamys farreri

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter for the TLR/IL-1R family. In this report, the first mollusk Myd88 ortholog (named as CfMyd88) was cloned from Zhikong scallop (Chlamys farreri). The full-length cDNA of CfMyd88 was of 1554 bp, including a 5'-terminal untranslated region (UTR) of 427 bp, a polyA tail, and an open reading frame (ORF) of 1104 bp encoding a polypeptide of 367 amino acids containing the typical TLR and IL-1R-related (TIR) domain and death domain (DD). Homology analysis revealed that the predicted amino acid sequence of CfMyd88 was homologous to a variety of previously identified Myd88s with more than 30% identity. The temporal expressions of CfMyd88 mRNA in the mixed primary cultured haemocytes stimulated by lipopolysaccharide (LPS) and peptidoglycans (PGN) were measured by real-time RT-PCR system. The mRNA expression of CfMyd88 decreased after stimulation with both LPS and PGN, and the lowest level was about 1/3 times (at 6 h) and 1/10 times (at 9 h) to that in the control group, respectively. The expression then recovered and was upregulated to two-fold at 9 h after LPS stimulation or to the original level at 12 h after PGN stimulation. The results suggest that the MyD88-dependent signaling pathway exists in scallop and was involved in the defense system.     

Cadmium affects the expression of metallothionein (MT) and glutathione peroxidase (GPX) mRNA in goldfish, Carassius auratus

Cadmium (Cd) is a widespread non-essential heavy metal that enters the aquatic environment as a result of natural and human-caused activities, including industrial effluent, mining, and agricultural runoff. In the present study, we investigated time and dose-related effect of CdCl(2) on metallothionein (MT) and glutathione peroxidase (GPX) mRNA levels in a number of goldfish tissues, in vivo. Basal MT and GPX mRNA levels remained unchanged in the tissues tested throughout the experiment. Injection with CdCl(2) significantly increased MT mRNA levels in the brain, liver, kidney and intestine in a dose-dependant manner at all time tested (6, 12, 24 and 36 h). We isolated the full length GPX cDNA from goldfish kidneys, and found it to contain 785 nucleotides, including an open reading frame, predicted to encode a protein of 142 amino acids. In contrast, injection with CdCl(2) significantly decreased GPX mRNA levels in the liver and kidney in a time-, and dose-, dependant, and became undetectable after 12, 24 and 36 h. The findings provide molecular characterization of MT and GPX in goldfish and suggest that exposure to Cd results in significant physiological changes in goldfish.     

Characterization and transcriptional regulation of thioredoxin reductase 1 on exposure to oxidative stress inducing environmental pollutants in Chironomus riparius

We characterized thioredoxin reductase 1 (TrxR1) from Chironomus riparius (CrTrxR1) and studied its expression under oxidative stress. The full-length cDNA is 1820 bp long and contains an open reading frame (ORF) of 1488 bp. The deduced CrTrxR1 protein has 495 amino acids and a calculated molecular mass of 54.41 kDa and an isoelectric point of 6.15. There was a 71 bp 5’ and a 261 bp 3' untranslated region with a polyadenylation signal site (AATAAA). Homologous alignments showed the presence of conserved catalytic domain Cys-Val-Asn-Val-Gly-Cys (CVNVGC), the C-terminal amino acids ‘CCS’ and conserved amino acids required in catalysis. The expression of CrTrxR1 is measured using quantitative real-time PCR after exposure to 50 and 100 mg/L of paraquat (PQ) and 2, 10 and 20 mg/L of cadmium chloride (Cd). CrTrxR1 mRNA was upregulated after PQ exposure at all conditions tested. The highest level of CrTrxR1 expression was observed after exposure to 10 mg/L of Cd for 24 h followed by 20 mg/L for 48 h. Significant downregulation of CrTrxR1 was observed after exposure to 10 and 20 mg/L of Cd for 72 h. This study shows that the CrTrxR1 could be potentially used as a biomarker of oxidative stress inducing environmental contaminants.     

Molecular cloning and characterization of carp (Cyprinus carpio L.) CD8beta and CD4-like genes

Partial cDNA sequences of both CD8beta and CD4-like (CD4L) genes of common carp (Cyprinus carpio L.) were isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp CD8beta and CD4L were obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp CD8beta is 1164 bp and encodes 207 amino acids including a signal peptide region of 24 amino acids, a transmembrane region of 23 amino acids from aa 167 to aa189 and an immunoglobulin V-set from aa 19 to aa 141. Similar to other species CD8betas, carp CD8beta also lacks p56(lck) domain in the cytoplasmic region. The full length cDNA of carp CD4L is 2001 bp and encodes 458 amino acids including four immunoglobulin (Ig)-like domains in the extracellular region, a transmembrane region of 23 amino acids at the C-terminal region from aa 402 to aa 424 and a cytoplasmic tail. Similar to mammalian, avian CD4s and fugu CD4L, carp CD4L also has the conserved p56(lck) tyrosine kinase motif (C-X-C) in the cytoplasmic region. RT-PCR analysis demonstrated that carp CD8beta and CD4L genes were both expressed predominantly in thymus. The results from this study can be used to understand the evolution of both the CD8beta and CD4 molecules which can be used as markers for cytotoxic and helper T cells in carp.