TRAIL in the mandarin fish Siniperca chuatsi: Gene and its apoptotic effect in HeLa cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the TNF superfamily members, participating in many biological processes including cell proliferation and apoptotic death. In this study, a TRAIL gene was cloned from a perciform fish, the mandarin fish Siniperca chuatsi, a major cultured fish in China's aquaculture, and is named as SCTRAIL for S. chuatsi TRAIL. The full-length cDNA of SCTRAIL is 1359 bp, encoding a 283-amino-acid protein. This deduced protein contains the CYS231, a 23-mer fragment of transmembrane region, a glycosylation site and a TNF family signature, all of which are conserved among TRAIL members. SCTRAIL gene consists of six exons, with five intervening introns, spaced over approximately 9 kb of genomic sequence. Southern blotting demonstrated that the SCTRAIL gene is present as a single copy in mandarin fish genome. A 620 bp promoter region obtained by genome walking contains a number of putative transcription factor binding sites, such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPaLp, NF-kappa B and AP-1. The SCTRAIL is constitutively expressed in all the analyzed tissues, as revealed by RT-PCR, which is confirmed by Western blotting analysis using polyclonal antibody against bacteria-derived recombinant SCTRAIL protein. As an apoptosis-inducing ligand, the overexpression of SCTRAIL but not the mutant SCTRAIL-C203S in HeLa cells induced changes characteristic of apoptosis, including chromatin condensation, nucleus fragmentation, DNA ladder, and increase of sub-G0/G1 cells in FACS analysis. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular characterization and subcellular localization of Carassius auratus interferon regulatory factor-1

Interferon (IFN)-regulatory transcription factor-1 (IRF-1) has been studied in mammals and fish but little is known about the relationship between its gene structure and nuclear 'ion of IRF-1 protein. In this study, a cDNA encoding Carassius auratus IRF-1 (CaIRF-1) was isolated from an interferon-producing cell line, C. ouratus blastulae embryonic (CAB) cells, exposed to UV-inactivated grass carp hemorrhagic virus (GCHV). The CaIRF-1 genomic locus exhibits exon-intron arrangements similar to those of other vertebrate IRF-1 loci, with nine exons and eight introns, although together with pufferfish IRF-1, CaIRF-1 distinguishes itself from other vertebrate IRF-1 genes by a relatively compact genomic size. Similar to the known IRF-1 genes, CaIRF-1 is ubiquitously expressed, and is upregulated in vitro and in vivo in response to virus, Poty I:C, or CAB INF-containing supernatant (ICS). Subcellular localization analysis confirms the nuclear distribution of CaIRF-1 protein, and reveals two nuclear localization signals (NILS), any one of which is sufficient for nuclear translocation of CaIRF-1. One NLS Locates to amino acids 117-146, and appears to be the structural and functional equivalent of the NLS in mammalian IRF-1. The second NLS (amino acids 73-115) is found within the DNA-binding domain (DBD) of CaIRF-1, and contains two regions rich in basic amino acids (''(KDKSINK101)-K-95" and ''(75)KTWKANFR(82)"). In comparison with mammalian IRF-1, in which the corresponding amino acid stretch does not seem to drive nuclear translocation, five conserved basic amino acids (K-75, K-78, R-82, K-95, and K-101) and one non-conserved basic amino acid (K-97) are present in this NLS from CaIRF-1. This observation suggests that K97 Of CaIRF-1 might be essential for the function of its second NLS, wherein the six basic aminoacids might cooperate to drive CaIRF-1 to the nucleus. Therefore, the current study has revealed a new nuclear localization motif in the DBD of a vertebrate IRF-1. (C) 2007 Elsevier Ltd. All rights reserved.

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.

Gene structure of an antimicrobial peptide from mandarin fish, Siniperca chuatsi (Basilewsky), suggests that moronecidins and pleurocidins belong in one family: the piscidins

The gene of piscidin, an antimicrobial peptide, has been cloned from the mandarin fish, Siniperca chuatsi. From the first transcription initiation site, the mandarin fish piscidin gene extends 1693 nucleotides to the end of the 3' untranslated region and contains four exons and three introns. A predicted 79-residue prepropeptide consists of three domains: a signal peptide (22 aa), a mature peptide (22 aa) and a C-terminal prodomain (35 aa). The shortage of XQQ motif in the prodomain of mandarin fish piscidin and the similar gene structure between moronecidins (piscidins) and pleurocidins may indicate that they are derived from the same ancestor gene. We thus suggest that piscidin should be used as a terminology for these antimicrobial peptides in the future. The mandarin fish piscidin mRNA was abundant in intestine, spleen, pronephros and kidney analysed by real-time polymerase chain reaction. After stimulation with lipopoly saccharides (LPS), a marked increase in transcripts was observed in most tissues, indicating that piscidin is not only a constitutively expressed molecule, but also has an increased response to bacterial infection. The synthetic, amidated mandarin fish piscidin exhibited different antimicrobial activity against different fish bacterial pathogens, especially against species of Aeromonas, which may to certain extent reflect the pathogenicity of these bacteria.

Cloning, characterization and promoter analysis of common carp hairy/Enhancer-of-split-related gene, her6

Some members of hairy/Enhancer-of-split-related gene (HES) family have important effects on axial mesoderm segmentation and the establishment and maintenance of the somite fringe. In fishes. the her6 gene, a member of the HES family, is the homologue Of heS1 in mammals and chicken. In this study, the her6 gene and its full-length cDNA from the common carp (Cyprinus carpio) were isolated and characterized. The genomic sequence of common carp her6 is approximately 1.7 kb. with four exons and three introns, and the full-length cDNA of 1314 bp encodes a Putative polypeptide of 271 amino acids. To analyse the promoter sequence of common carp her6. sequences of various lengths upstream from the transcription initiation site of her6 were fused to enhanced green fluorescent. protein gene (eGFP) and introduced into zebrafish embryos by microinjection to generate transgenic embryos. Our results show that the upstream sequence of 500 bp can direct highly efficient and tissue-specific expression of eGFP in zebrafish embryos. whereas a fragment of 200 bp containing the TATA box and a partial suppressor of hairless paired site sequence (SPS) is not sufficient to drive eGFP expression in zebrafish embryos.

Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers

DMRT1 has been suggested to play different roles in sex determination and gonad differentiation, because different expression patterns have been reported among different vertebrates. The groupers, since their gonads first develop as ovary and then reverse into testis, have been thought as good models to study sex differentiation and determination. In this study, we cloned the full-length cDNAs of DMRT] gene from orange-spotted grouper (Epinephelus coioides), and prepared corresponding anti-EcDMRT1] antiserum to study the relationship of DMRT] to sex reversal. One important finding is that the grouper DMRT] is not only differentially expressed in different stage gonads, but also restricted to specific stages and specific cells of spermatogenesis. Grouper DMRT1 protein exists only in spermatogonia, primary spermatocytes and secondary spermatocytes, but not in the supporting Sertoli cells. Moreover, we confirmed that EcSox3 is expressed not only in oogonia and different stage oocytes, but also in Sertoli cells and spermatogonia, and EcSox9 is expressed only in Sertoli cells. The data suggested that grouper DMRT1 might be a more specific sex differentiation gene for spermatogenesis, and play its role at the specific stages from spermatogonia to spermatocytes. In addition, no introns were found in the grouper DMRT1, and no duplicated DMRT1, genes were detected. The finding implicates that the intronless DMRT1 that is able to undergo rapid transcriptional turnover might be a significant gene for stimulating spermatogenesis in the protogynous hermaphroditic gonad. (c) 2006 Published by Elsevier Ireland Ltd.

Gene structure and transcription of IRF-2 in the mandarin fish Siniperca chuatsi with the finding of alternative transcripts and microsatellite in the coding region

The gene of interferon regulatory factor-2 (IRF-2) has been cloned from the mandarin fish (Siniperca chuatsi). The IRF-2 gene has 6,418 nucleotides (nt) and contains eight exons and seven introns, encoding two mRNAs. The two IRF-2 mRNAs each contained an open reading frame of 873 nt, which both translate into the same 291 amino acids but differed in their 5' untranslated region: one mRNA was transcribed initially from the exon 1 bypassing exon 2, while the other was transcribed from the exon 2. The microsatellites (CA repeats) could be found in the carboxyl terminal region of mandarin fish IRF-2, which result in the truncated form molecules. The microsatellites' polymorphism was investigated, and eight alleles were found in 16 individuals. The microsatellites were also examined in IRF-2 of several freshwater perciform fishes. The transcription of the IRF-2 in different tissues with or without poly inosine-cytidine stimulation was analyzed by real-time PCR, and the constitutive transcription of both molecules could be detected in all the tissues examined.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrame protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAlL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRFI sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen. (c) 2005 Elsevier B.V. All rights reserved.

Gene structure of goose-type lysozyme in the mandarin fish Siniperca chuatsi with analysis on the lytic activity of its recombinant in Escherichia coli

A goose-type lysozyme (g-lysozyme) gene has been cloned from the mandarin fish (Siniperca chuatsi), with its recombinant protein expressed in Escherichia coli. From the first transcription initiation site, the mandarin fish g-lysozyme gene extends 1307 nucleotides to the end of the 3' untranslated region, and it contains 5 exons and 4 introns. The open reading frame of the glysozyme transcript has 582 nucleotides which encode a 194 amino acid peptide. The 5' flanking region of mandarin fish glysozyme gene shows several common transcriptional factor binding sites when compared with that from Japanese flounder (Paralichthys olivaceus). The recombinant mandarin fish g-lysozyme was expressed in E. coli by using pET-32a vector, and the purified recombinant g-lysozyme shows lytic activity against Micrococcus lysodeikticus. (c) 2005 Elsevier B.V All rights reserved.

Inductive expression and characterization analysis of Paralichthys olivaceus pigment epithelium-derived factor in a virally infected cell line

Pigment epithelium-derived factor (PEDF) is acknowledged to be a non-inhibitory member of the serine protease inhibitor (serpin) superfamily, with antiangiogenesis, and neuroprotective and immumoregulatory function, mainly in the tissues of nervous system. Here, A PEDF gene homolog, Paralichthys olivaceus PEDF (PoPEDF), was isolated from flounder embryonic cells (FEC) treated with UV-inactivated Grass carp hemorrhage virus (GCHV) and subsequently identified as a differentially expressed gene. The full length of PoPEDF cDNA is 1803 bp with an open reading frame of 1212 bp encoding a 403-amino-acid protein. This deduced protein contains an N-terminal signal peptide, a glycosylation site, a consensus serpin motif, and a 34-mer and a 44-mer fragment, all of which are very conserved in the PEDF family. PoPEDF gene exhibits a conserved exon-intron arrangement with 8 exons and 7 introns. This conserved evolutionary relationship was further confirmed by a phylogenetic analysis, where fish PEDFs and mammalian members formed a well-supported clade. Constitutive expression of PoPEDF was widely detected in many tissues. In response to UV-inactivated GCHV or poly(I:C), PEDF mRNA was upregulated in FEC cells with time. This is the first report on the transcriptional induction of PEDF in virally infected cells. (C) 2005 Elsevier Inc. All rights reserved.

Molecular cloning of TRAF2 binding protein gene and its promoter region from the grass carp Ctenopharyngodon idellus

A tumor necrosis factor receptor-associated factor 2 binding protein (T2BP) gene was isolated from the grass carp (Ctenopharyngodon idellus) by utilizing suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The grass carp T2BP (GT2BP) gene contains an open reading frame of 579 nucleotide(s) (nt), encoding 193 amino acids, with 23 nt 5'-untranslated region and a long 3'-untranslated region of 434 nt including poly (A), 1 AUUUA motif and 4 AUUUUA motifs. No signal peptide has been detected in the predicted GT2BP, but a characteristic forkhead associated domain is present. The GT2BP mRNA shares 83% identity with the zebrafish DNA sequence, and they both have no introns in the genomic DNA. The putative transcription factor binding sites of GT2BP include two C/EBP alpha binding sites, and one c-Jun binding, one AP-1 binding, and one nuclear factor kappa B (NF kappa B) binding sites. Southern blot analysis revealed that the GT2BP was a single-copy gene. Individual difference was observed in GT2BP expression in examined organs of healthy grass carp. However, the expression of GT2BP in all examined organs in a fish with the highest copepod infection level and the significantly higher expression level in spleen and liver in infected fish may indicate its up-regulation with the parasite infection. (c) 2005 Elsevier B.V. All rights reserved.

Molecular cloning of the viperin gene and its promoter region from the mandarin fish Siniperca chuatsi

A viperin gene has been cloned from the mandarin fish (Siniperca chuatsi). From the first transcription initiation site, the mandarin fish viperin gene extends 3163 nucleotides to the end of the 3' untranslated region, and it contains six exons and five introns. The open reading frame of the viperin transcript has 1062 nucleotides which encode a 354 amino acid peptide. The amino acid sequence of mandarin fish viperin shows high identities with its homologues in teleosts and mammals except for the first 70 amino acids. A characteristic feature in the viperin promoter region was the presence of five putative ICSBP (IRF8) binding sites and one IRFI binding site. The viperin gene expressed mainly in lymphoid tissues before stimulation, but its expression can be examined in almost all the organs investigated after stimulation with virus or Poly I:C. The expression pattern and promoter sequence may be considered as the indirect evidence that the transcription of viperin is regulated by interferons or interferon induced genes. (C) 2004 Elsevier B.V. All rights reserved.

Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout

The sequences and gene organisation of two LEAP-2 molecules (LEAP-2A and LEAP-2B) from rainbow trout, Oncorhynchus mykiss are presented. Both genes consist of a 3 exon/2 intron structure, with exon sizes comparable to known mammalian genes. LEAP-2A notably differs from LEAP-2B in having larger introns and a larger 3'UTR. The predicted proteins contain a signal peptide and prodomain, followed by a mature peptide of 41 aa containing four conserved cysteines. The RXXR cleavage site to release the mature peptide was also conserved. Both genes were found to be constitutively expressed in the liver, with expression in the intestine, and to a lesser extent the skin, evident after bacterial challenge. (C) 2004 Elsevier B.V. All rights reserved.