Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.

Molecular cloning and characterization of the copper/zinc and manganese superoxide dismutase genes from the human parasite Clonorchis sinensis

A copper/zinc superoxide dismutase (Cu/ZnSOD) gene and a manganese superoxide dismutase (MnSOD) gene of the human parasite Clonorchis sinensis have been cloned and their gene products functionally characterized. Genes Cu/ZnSOD and MnSOD encode proteins of 16 kDa and 25.4 kDa, respectively. The deduced amino acid sequences of the two genes contained highly conserved residues required for activity and secondary structure formation of Cu/ZnSOD and MnSOD, respectively, and show up to 73.7% and 75.4% identities with their counterparts in other animals. The genomic DNA sequence analysis of Cu/ZnSOD gene revealed this as an intronless gene. Inhibitor studies with purified recombinant Cu/ ZnSOD and MnSOD, both of which were functionally expressed in Escherichia coli, confirmed that they are copper/zinc and manganese-containing SOD, respectively. Immunoblots showed that both C. sinensis Cu/ZnSOD and MnSOD should be antigenic for humans, and both, especially the C. sinensis MnSOD, exhibit extensive cross-reactions with sera of patients infected by other trematodes or cestodes. RT-PCR and SOD activity staining of parasite lysates indicate that there are no significant differences in mRNA level or SOD activity for both species of SOD, indicating cytosolic Cu/ZnSOD and MnSOD might play a comparatively important role in the C. sinensis antioxidant system.

Identification of three duplicated Spin genes in medaka (Oryzias latipes)

Gene and genomic duplications are very important and frequent events in fish evolution, and the divergence of duplicated genes in sequences and functions is a focus of research on gene evolution. Here, we report the identification and characterization of three duplicated Spindlin (Spin) genes from medaka (Oryzias latipes): OlSpinA, OlSpinB, and OlSpinC. Molecular cloning, genomic DNA Blast analysis and phylogenetic relationship analysis demonstrated that the three duplicated OlSpin genes should belong to gene duplication. Furthermore, Western blot analysis revealed significant expression differences of the three OlSpins among different tissues and during embryogenesis in medaka, and suggested that sequence and functional divergence might have occurred in evolution among them. © 2005 Elsevier B.V. All rights reserved.

Characterization of two genes encoding leucine-rich repeat-containing proteins in grass carp Ctenopharyngodon idellus

The cDNAs and genes of two different types of leucine- rich repeat-containing proteins from grass carp ( Ctenopharyngodon idellus) were cloned. Homology search revealed that the two genes, designated as GC-GARP and GC-LRG, have 37% and 32% deduced aminoacid sequence similarities with human glycoprotein A repetitions predominant precursor ( GARP) and leucine-rich alpha2-glycoprotein (LRG), respectively. The cDNAs of GC-GARP and GC-LRG encoded 664 and 339 amino acid residues, respectively. GC-GARP and GC-LRG contain many distinct structural and/or functional motifs of the leucine- rich repeat (LRR) subfamily, such as multiple conserved 11-residue segments with the consensus sequence LxxLxLxxN/CxL ( x can be any amino acid). The genes GC-GARP and GC-LRG consist of two exons, with 4,782 bp and 2,119 bp in total length, respectively. The first exon of each gene contains a small 5'-untranslated region and partial open reading frame. The putative promoter region of GC-GARP was found to contain transcription factor binding sites for GATA-1, IRF4, Oct-1, IRF-7, IRF-1, AP1, GATA-box and NFAT, and the promoter region of GC-LRG for MYC-MAX, MEIS1, ISRE, IK3, HOXA9 and C/EBP alpha. Phylogenetic analysis showed that GC-GARP and mammalian GARPs were clustered into one branch, while GC-LRG and mammalian LRGs were in another branch. The GC-GARP gene was only detected in head kidney, and GC-LRG in the liver, spleen and heart in the copepod ( Sinergasilus major)- infected grass carp, indicating the induction of gene expression by the parasite infection. The results obtained in the present study provide insight into the structure of fish LRR genes, and further study should be carried out to understand the importance of LRR proteins in host - pathogen interactions.

Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G(4)

In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper. The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa). Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH similar to 32 aa similar to E motif of the F. columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F. columnare G(4) represents a new family of zincins. The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa. Sequence homology analysis revealed that the Pop is also novel as it has <50% identity with other reported prolyl oligopeptidase family proteins. The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F. columnare.

Characterization of development-related genes for the cestode Bothriocephalus acheilognathi

Differential gene expression of mature and immature Bothriocephalus acheilognathi cestodes was analyzed using the suppression subtractive hybridization technique. Five mature-associated cDNAs were isolated and characterized. Virtual Northern blot and RT-PCR analyses confirmed that four of the five genes were up-regulated in mature parasites. The sequence analysis revealed that one gene encoded the structural protein chorion precursor, and that three encoded functional proteins homologous to yolk ferritin, sodium/hydrogen exchanger and muscin-like protein. Another gene appeared to be specific to B. acheilognathi, encoding a putative metal-bound protein. Although results obtained in the present study are preliminary, the information about the five genes may provide clues for further investigation on the decline in parasite numbers during the maturation of B. acheilognathi.

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.

Cloning and expression analysis in mature individuals of two chicken type-II GnRH (cGnRH-II) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide family, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II precursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplication hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the expression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.

Identification of two novel interferon-stimulated genes from cultured CAB cells induced by UV-inactivated grass carp hemorrhage virus

Interferon (IFN) exerts its antiviral effect by inducing the expression of a number of IFN-stimulated genes (ISGs) to establish a host antiviral state. Earlier studies identified some important fish IFN system genes from IFN-induced CAB cells (crucian carp Carassius auratus L. embryonic blastulae cells) after treatment with UV-inactivated GCHV (grass carp hemorrhage virus). Herein, the cloning of 2 novel IFN-stimulated genes, termed Gig1 and Gig2, is described for the same cell system. The complete cDNA sequences of Gig1 and Gig2 contain 1244 bp encoding for a 194-amino-acid protein and 693 bp for a 158-amino-acid protein, respectively. A search of public databases revealed that these are 2 novel IFN-stimulated genes, since neither significant homologous genes nor conserved motifs were identified. Active GCHV, UV-inactivated GCHV and CAB IFN-containing supernatant (ICS) induced transcription of these genes and distinct kinetics were observed. An analysis of differences in expression between the 2 genes and the IFN signal factors CaSTAT1 and CaIRF7 indicated that GCHV infection activated different signal pathways for their up-regulation. Upon virus infection, the transcription of Gig1 but not of Gig2 is strongly suppressed by cycloheximide (CHX). In contrast, following treatment with CAB IFN-containing supernatant, CHX does not inhibit either gene transcription. The results suggest that GCHV infection can induce expression of both Gig1 and Gig2 via newly synthesized CAB IFN, most probably through the JAK-STAT signal pathway, and can also directly activate Gig2 transcription without ongoing protein synthesis.

Residual plastids of bleached mutants of Euglena gracilis and their effects on the expression of nucleus-encoded genes

Bleached mutants of Euglena gracilis were obtained by treatment with ofloxacin (Ofl) and streptomycin (Sm) respectively. As shown by electron microscopy, the residual plastids contain prothylakoids in an Ofl mutant, and the highly developed and tightly stacked membranous structure found in cells of two Sm, mutants. Nine genes of the plastid genome were examined with PCR, showing that ribosomal protein genes and most other plastid genes were lost in all but one Sm mutant. Using differential display and RT-PCR, it was shown that chloroplast degeneration could cause changes in transcription of certain nucleus-encoded genes during heterotrophic growth in darkness.

Differential expression of two Carassius auratus Mx genes in cultured CAB cells induced by grass carp hemorrhage virus and interferon

UV-inactivated GCHV (grass carp hemorrhage virus) is able to induce an antiviral state in cultured CAB cells (crucian carp Carassius auratus blastulae embryonic cells) via the production of interferon (IFN). In the current work, the full-length cDNAs of two Mx genes, termed CaMx1 and CaMx2, have been cloned and sequenced from UV-inactivated GCHV-infected and still IFN-producing CAB cells by suppression subtractive hybridization. Their putative proteins show the characteristically structural features of mammalian IFN-induced Mx proteins, including GTP-binding motif, dynamin family signature and leucine zipper motif. CaMx1 exhibits 85% sequence identity to zebrafish MxA and 72-74% to three Atlantic salmon Mx proteins. CaMx2 is most similar to zebrafish MxE, with 80% identity, and then rainbow trout Mx3, with 52%. Constitutive expression was detected by RT-PCR for CaMx1, but not for CaMx2, in normal CAB cells, but their up-regulations could be induced after treatment with active GCHV, UV-inactivated GCHV and CAB IFN. Distinct kinetics of expression was observed for either CaMx1 or CaMx2 corresponding to the three stimuli, and even between CaMx1 and CaMx2, corresponding to the same stimulus. Upon virus infection, the transcriptional induction was strongly blocked for CaMx2 by cycloheximide (CHX), whereas almost nothing was observed for CaMx1. By contrast, following treatment with CAB IFN, CHX did not inhibit either gene transcription. Collectively, these results suggest that there are very distinct mechanisms for modulating the expression of both CaMx1 and CaMx2 in normal and GCHV-infected CAB cells.

Identification and expression analysis of two IFN-inducible genes in crucian carp (Carassius auratus L.)

Interferon (IFN) exerts its antiviral effects mainly through activation of a subset of IFN-stimulated genes (ISG), but relatively few of fish ISGs have been isolated and characterized so far. Here, we report two fish ISGs, termed CaIF158 and CaIF156, cloned from a subtractive cDNA library constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. Database search revealed that both ISGs had a high-level homology with all members of a well conserved gene family with multiple tetratricopeptide repeat (TPR) motifs, including human IF160, IF158, IF156, IFI54 and their homologues in some other mammalian species. The transcripts of CaIF158 and CaIF156 were undetectable in CAB cells but could be induced by active GCHV, UV-inactivated GCHV or CAB IFN. Analysis of expression difference between them and IFN signal factors, CaSTAT1 and CaIRF7, indicated that their transcriptions were mediated possibly through JAK-STAT signal pathway, which was further supported by the induction analysis in UV-inactivated GCHV infected, IFN-treated and untreated cells in the presence or absence of cycloheximide (CHX), a potent inhibitor of protein synthesis. In addition, a pufferfish (Fugu rubrides) DNA sequence representing putative FrIFI56 was also revealed when CalF158 and CalF156 were used to search the pufferfish genome database. Phylogenetic analysis showed that these fish ISGs form a unique clad independent of mammalian homologues, reflecting a distant evolutionary relationship from mammals. These studies identified the first teleost IFI56 and IFI58 orthologues. (C) 2003 Elsevier B.V All rights reserved.

Identification of antiviral-relevant genes in the cultured fish cells induced by UV-inactivated virus

UV-inactivated grass carp hemorrhage virus (GCHV) can induce high titer of interferon in cultured CAB (crucian carp (Carassius auratus L.) blastulae) cells, and thus defend host cells against the virus invasion. The mechanism is proposed that an antiviral state should be established in the host cells by activating expression of a set of antiviral-relevant genes. In this study, suppressive subtractive hybridization is applied to constructing a subtracted cDNA library with mRNAs isolated from UV-inactivated GCHV infected and mock-infected CAB cells. 272 differential cDNA fragments are identified by both PCR and dot blot from the subtractive cDNA library. Sequencing analysis reveals 69 genes, including 46 known gene homologues, and 23 unknown putative genes. The known genes include the genes involved in interferon signaling pathways, such as Stat1 and Jak1, the antiviral genes, such as Mx and Viperin, and a set of interferon-stimulated genes observed in mammalian cells. Most of the unknown putative genes contain AU-rich element in their sequences. Differential expressions of these genes are further confirmed by virtual Northern blot and RT-PCR. The data imply that UV-inactivated GCHV is not only able to induce production of interferon in the infected CAB cells, but also leads to the expression of a series of antiviral-relevant genes or immune-relevant genes, and therefore reveals that the signaling pathway of interferon system and antiviral mechanism in fish are similar to those in mammals.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.

A conserved family of doublesex-related genes from fishes

The sex-determining gene Mab-3 of C. elegans and the doublesex gene of Drosophila each contain a common DM domain and share a similar role. Human doublesex-related gene DMRT1 also encodes a conserved DM-related DNA-binding domain. We present here the amplification of a broad range of DM domain sequences from three fish species using degenerate PCR. Our results reveal unexpected complexity of the DM domain gene family in vertebrates. (C) 2002 Wiley-Liss, Inc.