Immunoglobulin joining (J) chain and its expression in the Chinese soft-shelled turtle (Pelodiscus sinensis)

The immunoglobulin (Ig) joining (J) chain plays an important role in the formation of polymeric Igs and their transport into secretions. In the present study, the cDNA sequence of J chain has been cloned from the Chinese soft-shelled turtle (Pelodiscus sinensis) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The cDNA sequence is 2347 bp in length and contains an open reading frame of 480 bp encoding 160 aa including the signal sequence. The deduced amino acid sequence has a high degree of homology with that of an already reported turtle J chain (80.7%), and of chicken (71.3%). By using real-time quantitative RT-PCR analysis, a significant up-regulation of J-chain transcripts was observed in spleen, kidney and blood of turtles injected with inactivated Aeromonas hydrophila, indicating the immune role of J chain in response to bacterial infection. (C) 2009 Elsevier B.V. All rights reserved.

Molecular characterization and expression pattern of AFPIV during embryogenesis in gibel carp(Carassiu auratus gibelio)

As a new type of AFPs, AFPIV has been firstly identified in longhorn sculpin (Myoxocephalus octodecimspinosus), and in recent years, its cDNA and amino acid sequence have been reported, and its pancreatic synthesis has been firstly reported in polar fish. However, its expression patterns during fish embryogenesis have not been elucidated yet. By differential screening, we cloned the CagAFPIV in gibel carp, Carassius auratus gibelio, demonstrated its predominant expression during embryogenesis. RT-PCR detection revealed that CagAFPIV was first transcribed from blastula stage and kept a high level during embryogenesis and declined remarkably in hatched larva. In situ hybridization revealed that CagAFPIV transcripts were firstly distributed over the margin and marginal blastomere in blastula stage embryos, at the early-gastrula stage the positive signals distributed in the marginal cells and the internalization cells, and later restricted to the cells the yolk syncytial layer (YSL) from later gastrula stage to larva stage. Consistently, the CagAFPIV protein also kept a high level during embryogenesis, and the high protein level retained some days after the larva hatched. Our work, for the first time, revealed the dynamic expression and distribution of CagAFPIV during embryogenesis.

Molecular characterization and expression profiles in response to bacterial infection of Chinese soft-shelled turtle interleukin-8 (IL-8), the first reptilian chemokine gene

In this study, an IL-8 homologue has been cloned and identified from a reptile, Chinese soft-shelled turtle for the first time. The full-length cDNA of turtle IL-8 was 1188 bp and contained a 312 bp open reading frame (ORF) coding for a protein of 104 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in turtle IL-8. The 4924 bp genomic DNA of turtle IL-8 contained four exons and three introns. Phylogenetic analysis showed that the amino acid sequence of turtle IL-8 clustered together with birds. RT-PCR analysis showed that turtle IL-8 mRNA was constitutively expressed liver, spleen, kidney, heart, blood and intestine tissues of control turtles. Real-time quantitative PCR analysis further indicated that the turtle IL-8 mRNA expression was apparent in various tissues at 8 h and up-regulated significantly during 8 h-7 d after Aeromonas hydrophila infection. The present studies will help us to understand the evolution of IL-8 molecule and the inflammatory response mechanism in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

Grass carp reovirus activates RNAi pathway in rare minnow, Gobiocypris rarus

Dicer catalyzes the initiation step of RNA interference (RNAi) which is known to play a significant role in innate immune response to viral infection in many organisms. To study the RNAi-related pathway after virus infection in fish, we identified a partial cDNA sequence of dicer from rare minnow, Gobiocypris rants. Real-time quantitative RT-PCR (qRT-PCR) demonstrated the Dicer transcript level was the highest at zygote stage, decreased at prim-5 stage, and was stable from the protruding mouth to adult stage. Regular RT-PCR analysis showed that the Dicer gene expressed widely in the tested tissues, including brain, gill, heart, intestine, kidney, liver, muscle, ovary, spleen and testis. The expression of Dicer mRNA was significantly increased in the early period of Grass carp reovirus (GCRV) infection, and declined from 24 It post-injection (h p.i.) (P<0.05). The mRNA expression returned to control levels at 48 h p.i. (P>0.05). Under transmission electron microscope, virions were difficulty to find out in 12 h p.i., and virus inclusion bodies and few scattered viral particles were easily visualized from 24 h p.i. to moribund. These results implied GCRV triggered the RNAi pathway in the early stages of infection and perhaps virus inclusion bodies suppressed the antiviral functions of RNAi mechanism. (C) 2009 Published by Elsevier B.V.

Identification and characterization of hypoxia-induced genes in Carassius auratus blastulae embryonic cells using suppression subtractive hybridization

Organisms living in water are inevitably exposed to periods of hypoxia. Environmental hypoxia has been an important stressor having manifold effects on aquatic life. Many fish species have evolved behavioral, physiological, biochemical and molecular adaptations that enable them to cope with hypoxia. However, the molecular mechanisms of hypoxia tolerance in fish, remain unknown. in this study, we used suppression subtractive hybridization to examine the differential gene expression in CAB cells (Carassius auratus blastulae embryonic cells) exposed to hypoxia for 24 h. We isolated 2100 clones and identified 211 differentially expressed genes (e-value <= 5e-3; Identity > 45%). Among the genes whose expression is modified in cells, a vast majority involved in metabolism, signal transduction, cell defense, angiogenesis, cell growth and proliferation. Twelve genes encoding for ERO1-L, p53, CPO, HO-1, MKP2, PFK-2, cystatin B, GLUT1, BTG1, TGF beta 1, PGAM1, hypothetical protein F1508, were selected and identified to be hypoxia-induced using semi-quantitive RT-PCR and real-time PCR. Among the identified genes, two open reading frames (ORFs) encoding for CaBTG1 and Cacystatin B were obtained. The deduced amino acid sequence of CaBTG1 had 94.1%, 72.8%, 72.8%, 72.8%, 68.6% identity with that of DrBTG1, HsBTG1, BtBTG1, MmBTG1 and XIBTG1. Comparison of Cacystatin B with known cystatin B, the molecules exhibited 49.5 to 76.0% identity overall. These results may provide significant information for further understanding of the adaptive mechanism by which C. auratus responds to hypoxia. (c) 2008 Elsevier Inc. All rights reserved.

Metallothionein-2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization

The metallothionein-2 (MT-2) gene was isolated from the mandarin fish, one of the most important industrial aquatic animals in China, by using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of MT-2 comprised 60 amino acids and showed approximately 62.3% identity to human metallothionein. Its promoter region was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The MT-2 gene consists of 3 exons and 2 introns, extending approximately 900 bp of genomic sequence. Phylogenetic analysis clearly demonstrated that MT-2 formed a clade with fish metallothionein. The promoter region contained 5 putative metal-regulatory elements (MREs) and 1 TATA box. Real-time quantitative RT-PCR analysis revealed that MT-2 transcripts were significantly increased in the brain and gills and were stable in the muscles, liver, and trunk kidney in Cd2+-stimulated fish. Western blotting analysis demonstrated that the protein of the MT-2 gene was expressed mainly in the gills, liver, heart, trunk kidney, muscle, and intestine; it was weakly detected in the brain and head kidney. Moreover, the MT-2 protein was immunohistochemically detected in the cytoplasm in the liver and trunk kidney. All the above results revealed that the mandarin fish MT-2 would be a useful biomarker for metal pollution. (C) 2008 Published by Elsevier Inc.

Identification of differentially expressed immune-relevant genes in Chinese soft-shelled turtle (Trionyx sinensis) infected with Aeromonas hydrophila

Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and profiling gene expression. Aeromonas hydrophila, a ubiquitous waterborne bacterium, is one of the most frequent pathogens isolated from diseased aquatic organisms. In order to understand the molecular mechanism of anti-bacteria immune response in reptile, we have investigated the differentially expressed genes in Chinese soft-shelled turtle (Trionyx sinensis) experimentally infected with A. hydrophila by suppression subtractive hybridization (SSH). Forty-two genes were identified from more than 200 clones, of which 25 genes are found for the first time in reptiles, and classified into 6 categories: 18 in defense/immunity. 4 in catalysis, 2 in retrotransposon; 2 in cell signal transduction, 5 in cell metabolism, 10 in protein expression, and 1 in cell structure. Of the 42 differentially expressed genes, 6 genes, IL-8, serum amyloid A (SAA), CD9, CD59, activating transcription factor 4 (ATF4) and cathepsin L genes, were further observed to be up-regulated in the infected turtles by virtual Northern hybridization and RT-PCR assays. (C) 2008 Elsevier B.V. All rights reserved.

Molecular characterization and expression pattern of fetuin-B in gibel carp (Carassius auratus gibelio)

Fetuin-B has recently been cloned and identified from rats, mice, and humans; their expression patterns, however, have not been elucidated yet. In this study, Cagfetuin-B has been cloned in gibel carp. RT-PCR and Western blot detection revealed that Cagfetuin-B is first transcribed from the blastula stage and at a relatively stable level afterward during embryogenesis and the larval stage. Cagfetuin-B transcripts are predominantly distributed over the yolk syncytial layer in the early embryos and later restricted to the cells of liver and brain in newly hatched larvae. Moreover, a dynamic distribution of Cagfetuin-B protein was observed in brain, kidney, liver, and skin during morphogenesis. In adult fish, Cagfetuin-B transcripts are restricted in liver and ovary. Our work, for the first time, revealed the extra-hepatic transcription and a dynamic distribution of fetuin-B during embryogenesis and in adults, which indicates the potential roles of fetuin-B in fish organogenesis.

Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress

Heme oxygenase-1 is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide and free divalent iron. In this study, we cloned heme oxygenase isoform 1 (CaHO-1) from a hypoxia-tolerant teleost fish Carassius auratus. The full-length cDNA of CaHO-1 is 1247 bp and encodes a protein of 272 amino acids. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and induction of gene transcription was observed predominantly in posterior kidney under hypoxic stress. Moreover, the hypoxia-induced transcription was confirmed in goldfish larvae and in in vitro cultured CAB cells. Fluorescence of the HO-1-GFP fusion protein revealed a cytoplasmic and plasma membrane localization, which was consistent with the putative transmembrane structure. Subsequently, we established a stably transfected CAB/pcDNA3.1-HO-1 cell line and a control CAB/pcDNA3.1 cell line, and found that the number of dead cells was obviously reduced in the pcDNA3.1-HO-1-transfected group following 4 days of hypoxic (1% O-2) treatment in comparison with numerous detached dead cells in the control pcDNA3.1-transfected cells. Furthermore, a significant cell viability difference between the two kinds of transfected cells during hypoxia-reoxygenation was revealed. Therefore, the data suggest that fish HO-1 might play a significant protective role in cells in response to hypoxic stress.

Toll-like receptor 3 regulates Mx expression in rare minnow Gobiocypris rarus after viral infection

Toll-like receptor 3 (TLR3) plays a key role in activating immune responses during viral infection. To study the genes involved in the regulatory function of TLR3 in the rare minnow Gobiocypris rarus after viral infection, a full-length cDNA of TLR3 (GrTLR3) with a splice variant (GrTLR3s) was identified by homologous cloning and RACE techniques. The antiviral effector molecule Mx gene was cloned and partially sequenced. The mRNA expression levels of GrTLR3, GrTLR3s, and Mx were studied in different tissues before and after virus infection by real-time quantitative RT-PCR. The transcripts of all three genes in liver were significantly increased following GCRV infection (P<0.05). The mRNA levels in liver were upregulated at 24 h post-injection for GrTLR3 and GrTLR3s, and at 12 h for Mx. The upregulated expression levels were several folds for GrTLR3s, tens of folds for GrTLR3, and hundreds of folds for Mx. By semi-quantitative RT-PCR, GrTLR3 and Mx expressed at all the developmental stages, whereas GrTLR3s could only be detected at later developmental stages. Using RNAi and transgenic techniques, GrTLR3 mediated Mx expression but GrTLR3s did not. The time-dependent upregulation of receptor and effector, and the Mx over-expression dependent on TLR3, indicated that GrTLR3 regulated Mx expression in viral infection through a configuration change in rare minnow, and its splice variant did not contribute to the process.

Characterization and expression analysis of TNFR-associated factor 1 (TRAF1) in grass carp Ctenopharyngodon idella

TNF receptor associated factor 1 (TRAF1) plays an important role in regulating the TNF signaling and protecting cells from apoptosis. In the present study, a TRAF1 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA is 2235 bp, including a 250 bp 5' UTR (untranslated region), a 1659 bp open reading frame, and a 326 bp 3'UTR. The polyadenylation signal (AATAAA, AATAA) and one mRNA instability motif (AUUUA) were found followed by a poly (A) tail in the 3'UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF1 (gcTRAF1). The putative amino acids of gcTRAF1 share 72% identity with the homologue in zebrafish. It is characterized by a zinc finger at the N-terminus and a TRAF domain (contains one TRAF-C and one TRAF-N) at the C-terminus. The identity of the TRAF domain among all the TRAF1 homologues in vertebrates varies from 52% to 58%, while the identities of TRAF-C were almost the same as 70%. The recombinant gcTRAF1 has been constructed successfully and expressed in Escherichia coli by using pET-32a expression vector. The polyclonal antibody for rabbit has been successfully obtained. The expression of gcTRAF1 in different organs was examined by real-time quantitative PCR and Western blotting, respectively. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of TRAF1 homologue molecule found in fish. (c) 2007 Elsevier B.V. All rights reserved.

Antisense for gonadotropin-releasing hormone reduces gonadotropin synthesis and gonadal development in transgenic common carp (Cyprinus carpio)

Generating transgenic fish with desirable traits (e.g., rapid growth, larger size, etc.) for commercial use has been hampered by concerns for biosafety and competition if these fish are released into the environment. These obstacles may be overcome by producing transgenic fish that are sterile, possibly by inhibiting hormones related to reproduction. In vertebrates, synthesis and release of gonadotropin (GtH) and other reproductive hormones is mediated by gonadotropin-releasing hormone (GnRH). Recently two cDNA sequences encoding salmon-type GnRH (sGnRH) decapeptides were cloned from common carp (Cyprinus carpio). This study analyzed the expression of these two genes using real-time polymerase chain reaction (RT-PCR) in different tissues carp at varying developmental stages. Transcripts of both genes were detected in ovary and testis in mature and regressed, but not in juvenile carp. To evaluate the effects of sGnRH inhibition, the recombinant gene CAsGnRHpc-antisense, expressing antisense sGnRH RNA driven by a carp beta-actin promoter, was constructed. Blocking sGnRH expression using antisense sGnRH significantly decreased GtH in the blood of male transgenic carp. Furthermore, some antisense transgenic fish had no gonadal development and were completely sterile. These data demonstrate that sGnRH is important for GtH synthesis and development of reproductive organs in carp. Also, the antisense sGnRH strategy may prove effective in generating sterile transgenic fish, eliminating environmental concerns these fish may raise. (c) 2007 Published by Elsevier B.V.

Molecular cloning, promoter analysis and induced expression of the complement component C9 gene in the grass carp Ctenopharyngodon idella

Complement-mediated killing of pathogens through lytic pathway is an important effector mechanism of innate immune response. C9 is the ninth member of complement components, creating the membrane attack complex (MAC). In the present study, a putative cDNA sequence encoding the 650 amino acids of C9 and its genomic organization were identified in grass carp Ctenopharyngodon idella. The deduced amino acid sequence of grass carp C9 (gcC9) showed 48% and 38.5% identity to Japanese flounder and human C9, respectively. Domain search revealed that gcC9 contains a LDL receptor domain, an EGF precursor domain, a MACPF domain and two TSP domain located in the N-terminal and C-terminal, respectively. Phylogenetic analysis demonstrated that gcC9 is clustered in a same clade with Japanese flounder, pufferfish and rainbow trout C9. The gcC9 gene consists of 11 exons with 10 introns, spacing over approximately 7 kb of genomic sequence. Analysis of gcC9 promoter region revealed the presence of a TATA box and some putative transcription factor such as C/EBP, HSF, NF-AT, CHOP-C, HNF-3B, GATA-2, IK-2, EVI- 1, AP-1, CP2 and OCT-1 binding sites. The first intron region contains C/EBPb, HFH-1 and Oct-1 binding sites. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcC9 gene have similar expression patterns, being constitutively expressed in all organs examined of healthy fish, with the highest level in hepatopancreas. By real-time quantitative RT-PCR analysis, gcC9 transcripts were significantly up-regulated in head kidney, spleen, hepatopancreas and down-regulated in intestine from inactivated fish bacterial pathogen Flavobacterium columnare-stimulated fish, demonstrating the role of C9 in immune response. (c) 2007 Elsevier B.V. All rights reserved.

Intelectin gene from the grass carp Ctenopharyngodon idella: cDNA cloning, tissue expression, and immunohistochemical localization

The cDNA encoding grass carp intelectin was isolated from a head kidney cDNA library, and termed gcIntL. The deduced amino acid sequence of gcIntL consists of 318 amino acids, and about 55% identical and 74% similar to human intelectin, which is a new type of lectin recognizing galactofuranose, and plays a role in the recognition of bacteria-specific components in animal hosts. The gcIntL gene consists of seven exons and six introns, spacing over approximately 3 kb of genomic sequence. Phylogenetic analysis clearly demonstrated that the gcIntL formed a clade with Danio rerio intelectin and 35 kDa serum lectin. By real-time quantitative RT-PCR analysis, gcIntL transcripts were significantly induced in head kidney, trunk kidney, spleen, and intestine from LPS-stimulated fish. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcIntL gene have the same expression pattern, and both were detected in brain, gill, intestine, head kidney, trunk kidney, spleen, and heart. Furthermore, gcIntL protein could be detected in gill, intestine, trunk kidney, head kidney, spleen, heart, and brain including medulla oblongata and optic lobe, as determined by immunohistochemistry. This is the first report of intelectin expression pattern in fish, and of recombinant gcIntL and polyclonal antibody against gcIntL. (c) 2006 Elsevier Ltd. All rights reserved.

Short and long peptidoglycan recognition proteins (PGRPs) in zebrafish, with findings of multiple PGRP homologs in teleost fish

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect and mammals. Using a database mining approach and RT-PCR, multiple peptidoglycan recognition protein (PGRP) like genes have been discovered in fish including zebrafish Danio rerio, Japanese pufferfish TakiFugu rubripes and spotted green pufferfish Tetraodon nigroviridis. They share the common features of those PGRPs in arthropod and mammals, by containing a conserved PGRP domain. Based on the predicted structures, the identified zebrafish PGRP homologs resemble short and long PGRP members in arthropod and mammals. The identified PGRP genes in T. nigroviridis and TakiFugu rubripes resemble the long PGRPs, and the short PGRP genes have not been found in T. nigroviridis and TakiFugu rubripes databases. Computer modelling of these molecules revealed the presence of three alpha-helices and five or six beta-strands in all fish PGRPs reported in the present study. The long PGRP in teleost fish have multiple alternatively spliced forms, and some of the identified spliced variants, e.g., tnPGRP-L3 and tnPGRP-L4 (in: Tetraodon nigroviridis), exhibited no characters present in the PGRP homologs domain. The coding regions of zfPGRP6 (zf: zebrafish), zfPGRP2-A, zfPGRP2-B and zfPGRP-L contain five exons and four introns; however, the other PGRP-like genes including zfPGRPSC1a, zfPGRPSC2, tnPGRP-L1-, tnPGRP-L2 and frPGRP-L (fr: Takifugu rubripes) contain four exons and three introns. In zebrafish, long and short PGRP genes identified are located in different chromosomes, and an unknown locus containing another long PGRP-like gene has also been found in zebrafish, demonstrating that multiple PGRP loci may be present in fish. In zebrafish, the constitutive expressions of zfPGRP-L, zfPGRP-6 and zfPGRP-SC during ontogeny from unfertilized eggs to larvae, in different organs of adult, and the inductive expression following stimulation by Flavobacterium columnare, were detected by real-time PCR, but the levels and patterns varied for different PGRP genes, implying that different short and long PGRPs may play different roles in innate immune response. (c) 2007 Elsevier Ltd. All rights reserved.