Molecular characterisation and inductive expression of a fish protein arginine methyltransferase 1 gene in response to virus infection

Protein arginine methyltransferase 1 (PRMT1) is currently thought as an effector to regulate interferon (IFN) signalling. Here Paralichthys olivaceus PRMT1 (PoPRMT1) gene was identified as a vitally induced gene from UV-inactivated Scophthalmus maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). PoPMRT1 encodes a 341-amino-acid protein that shares the conserved domains including post-I, motif I, II and III. Homology comparisons show that the putative PoPMRT1 protein is the closest to zebrafish PMRT1 and belongs to type I PRMT family (including PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, PRMT8). Expression analyses revealed an extensive distribution of PoPMRT1 in all tested tissues of flounder. In vitro induction of PoPRMT1 was determined in UV-inactivated SMRV-infected FEC cells, and under the same conditions, flounder Mx wash also transcriptionally up-regulated, indicating that an IFN response might be triggered. Additionally, live SMRV infection of flounders induced an increased expression of PoPRMT1 mRNA and protein significantly in spleen, and to a lesser extent in head kidney and intestine. Immunofluorescence analysis revealed a major cyptoplasmic distribution of PoPRMT1 in normal FEC but an obvious increase occurred in nucleus in response to UV-inactivated SMRV. This is the first report on in vitro and in vivo expression of fish PRMT1 by virus infection, suggesting that PoPRMT1 might be implicated in flounder antiviral immune response. (c) 2006 Elsevier Ltd. All rights reserved.

Characterization of an early gene encoding for dUTPase in Rana grylio virus

dUTPase (DUT) is a ubiquitous and important enzyme responsible for regulating levels of dUTP. Here, an iridovirus DUT was identified and characterized from Rana grylio virus (RGV) which is a pathogen agent in pig frog. The DUT encodes a protein of 164aa with a predicted molecular mass of 17.4 kDa, and its transcriptional initiation site was determined by 5'RACE to start from the nucleotide A at 15 nt upstream of the initiation codon ATG. Sequence comparisons and multiple alignments suggested that RGV DUT was quite similar to other identified DUTs that function as homotrimers. Phylogenetic analysis implied that DUT horizontal transfers might have occurred between the vertebrate hosts and iridoviruses. Furthermore, its temporal expression pattern during RGV infection course was characterized by RT-PCR and Western blot analysis. It begins to transcribe and translate as early as 4 h postinfection (p.i.), and remains detectable at 48 h p.i. DUT-EGFP fusion protein was observed in the cytoplasm of pEGFP-N3-Dut transfected EPC cells. Immunofluorescence also confirmed DUT cytoplasm localization in RGV-infected cells. Using drug inhibition analysis by a de novo protein synthesis inhibitor (cycloheximide) and a viral DNA replication inhibitor (cytosine arabinofuranoside), RGV DUT was classified as an early (E) viral gene during the in vitro infection. Moreover, RGV DUT overexpression was shown that there was no effect on RGV replication by viral replication kinetics assay. (c) 2006 Published by Elsevier B.V.

Growth and energy budget of F-2 'all-fish' growth hormone gene transgenic common carp

The growth and energy budget for F-2 'all-fish' growth hormone gene transgenic common carp Cyprinus carpio of two body sizes were investigated at 29.2 degrees C for 21 days. Specific growth rate, feed intake, feed efficiency, digestibility coefficients of dry matter and protein, gross energy intake (I-E), and the proportion of I-E utilized for heat production (H-E) were significantly higher in the transgenics than in the controls. The proportion of I-E directed to waste products [faecal energy (F-E) and excretory energy loss (Z(E) + U-E) where Z(E) is through the gills and U-E through the kidney], and the proportion of metabolizable energy (M-E) for recovered energy (R-E) were significantly lower in the transgenics than in the controls. The average energy budget equation of transgenic fish was as follows: 100 I-E = 19.3 F-E + 6.0 (Z(E) + U-E) + 45.2 H-E + 29.5 R-E or 100 M-E = 60.5 H-E + 39.5 R-E. The average energy budget equation of the controls was: 100 I-E = 25.2 F-E + 7.4 (Z(E) + U-E) + 35.5 H-E + 31.9 R-E or 100 M-E = 52.7 H-E + 47.3 R-E. These findings indicate that the high growth rate of 'all-fish' transgenic common carp relative to their non-transgenic counterparts was due to their increased feed intake, reduced lose of waste productions and improved feed efficiency. The benefit of the increased energy intake by transgenic fish, however, was diminished by their increased metabolism.

Molecular evidence for the monophyly of East Asian groups of Cyprinidae (Teleostei : Cypriniformes) derived from the nuclear recombination activating gene 2 sequences

The family Cyprinidae is one of the largest families of fishes in the world and a well-known component of the East Asian freshwater fish fauna. However, the phylogenetic relationships among cyprinids are still poorly understood despite much effort paid on the cyprinid molecular phylogenetics. Original nucleotide sequence data of the nuclear recombination activating gene 2 were collected from 109 cyprinid species and four non-cyprinid cypriniform outgroup taxa and used to infer the cyprinid phylogenetic relationships and to estimate node divergence times. Phylogenetic reconstructions using maximum parsimony, maximum likelihood, and Bayesian analysis retrieved the same clades, only branching order within these clades varied slightly between trees. Although the morphological diversity is remarkable, the endemic cyprinid taxa in East Asia emerged as a monophyletic clade referred to as Xenocypridini. The monophyly for the subfamilies including Cyprininae and Leuciscinae, as well as the tribes including Labeonini, Gobionini, Acheilognathini, and Leuciscini, was also well resolved with high nodal support. Analysis of the RAG2 gene supported the following cyprinid molecular phylogeny: the Danioninae is the most basal subfamily within the family Cyprinidae and the Cyprininae is the sister group of the Leuciscinae. The divergence times were estimated for the nodes corresponding to the principal clades within the Cyprinidae. The family Cyprinidae appears to have originated in the mid-Eocene in Asia, with the cladogenic event of the key basal group Danioninae occurring in the early Oligocene (about 31-30 MYA), and the origins of the two subfamilies, Cyprininae and Leuciscinae, occurring in the mid-Oligocene (around 26 MYA). (c) 2006 Elsevier Inc. All rights reserved.

The first non-mammalian CXCR3 in a teleost fish: Gene and expression in blood cells and central nervous system in the grass carp (Ctenopharyngodon idella)

A novel fish chemokine receptor gene, chemokine (C-X-C motif) receptor 3 (CXCR3)-like was isolated from the grass carp Ctenopharyngodon idella , with its full-length genomic sequence. The cDNA of grass carp CXCR3-like (gcCXCR3-like) consists of 1261 bp with a 49bp 5'-UTR and a 189 bp 3'-UTR. An open reading frame of 1023 bp encodes a 341-amino acid peptide, with seven transmembrane helices. The deduced amino acid sequence showed the same sequence identities (37.8%) with its counterparts in goat and human. The gcCXCR3-like gene consists of two exons, with one intervening intron, spaced over approximately 2 kb of genomic sequence. Phylogenetic analyses clearly demonstrated that the gcCXCR3-like resembles the CXCR3s of other vertebrates. Real-time PCR analysis showed that gcCXCR3-like was expressed in all tested organs except heart and the expression level of gcCXCR3-like was highest in brain. Flow cytometric analyses showed the positive rate of labelled leukocytes from the healthy grass carp was 17.3%, and the labelled leukocytes were divided into three types by cell sorting. Immunohistochemical localization revealed that gcCXCR3-like expressed in whole brain regions including cerebel, diencephalon, medulla oblongata, optic lobe, and rhinencephalon, and that the labelled leukocytes are actually populations of monocyte and/or phagocyte, lymphocyte and the granulocyte. It is considered that fish CXCR expression and their function may need to be investigated in both nervous and immune systems. (c) 2006 Elsevier Ltd. All rights reserved.

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.

Cloning and characterization of a new multi-stress inducible metallothionein gene in Tetrahymena pyriformis

A new multi-stress-inducible metallothionein (MT) gene isoform has been cloned and characterized from the ciliate Tetrahymena pyriformis. Both the 5'- and 3'-UT regions of the Tp-MT2 gene are very different from the previously reported Tp-MT1 isoform in this organism and from other described MT genes in Tetrahymena pigmentosa and Tetrahymena thermophila. The putative protein sequence of Tp-MT2 contains cysteine clusters with characteristics of the typical Tetrahymena Cd-inducible MT genes. However, the sequence has a special feature of four intragenic tandem repeats within its first half, with a conserved structural pattern x(5/8)CCCx(6)CCx(6)CxCxNCxCCK. To investigate the transcriptional activities of both Tp-MT2 and Tp-MT1 genes toward heavy metals (Cd, Hg, Cu, Zn) and H2O2, the mRNA levels of these two isoforms were evaluated by means of real-time quantitative PCR. Results showed that Tp-MT2 had a higher basal expression level than Tp-MT1 and both genes were induced by Cd, Hg, Cu, and Zn ions after short exposure (I h), although to different extents. Cd was the most effective metal inducer of both two isoforms, but the relative expression level of Tp-MT2 was much lower than that of Tp-MT1. Different expression patterns were also shown between the two genes when treated with Cd over a period of 24 h. We suggest that TpMT-1 plays the role of a multi-inducible stress gene, while TpMT-2 may have a more specific function in basal metal homeostasis although it may have undergone a functional differentiation process. The putative functional significance and evolutionary mode of the TpMT-2 isoform are discussed. (c) 2006 Elsevier GmbH. All rights reserved.

Effects of age and dietary protein level on digestive enzyme activity and gene expression of Pelteobagrus fulvidraco larvae

The present research studied the effects of age and dietary protein level on pepsin, trypsin and amylase activity and their mRNA level in Petteobagrus fulvidraco larvae from 3 to 26 days after hatch (DAH). Three DAH larvae were fed three isoenergetic diets, containing 42.8% (CP 43), 47.3% (CP 47) and 52.8% (CP 53) crude protein. Live food (newly hatched Artemia, unenriched) was included as a control. The effects of age on enzyme activity and mRNA were as follows: pepsin and trypsin activity in all treatment groups showed a significant (P < 0.05) increase at the beginning and decrease later although the timing of decrease was not the same among treatment groups and between the digestive enzymes. Pepsin and trypsin mRNA level followed the pattern of their respective enzyme changes. Age significantly affected amylase activity (P < 0.05) while age had no effect on amylase mRNA during the experimental period. The four diets significantly (P < 0.05) affected activity and mRNA level of pepsin and trypsin. Diets did not affect amylase activity or mRNA level. These results suggest that the effects of age on pepsin and trypsin gene expressions are at the transcriptional level. Dietary protein level does affect pepsin and trypsin gene expression in the early life of P. fulvidraco. There were no transcriptional effects on amylase gene expression. (c) 2005 Elsevier B.V. All rights reserved.

Comparison of evolutionary rates in the mitochondrial DNA cytochrome b gene and control region and their implications for phylogeny of the Cobitoidea (Teleostei : Cypriniformes)

It is widely accepted that mitochondrial DNA (mtDNA) control region evolves faster than protein encoding genes with few exceptions. In the present study, we sequenced the mitochondrial cytochrome b gene (cyt b) and control region (CR) and compared their rates in 93 specimens representing 67 species of loaches and some related taxa in the Cobitoidea (Order Cypriniformes). The results showed that sequence divergences of the CR were broadly higher than those of the cyt b (about 1.83 times). However, in considering only closely related species, CR sequence evolution was slower than that of cyt b gene (ratio of CR/cyt b is 0.78), a pattern that is found to be very common in Cypriniformes. Combined data of the cyt b and CR were used to estimate the phylogenetic relationship of the Cobitoidea by maximum parsimony, neighbor-joining, and Bayesian methods. With Cyprinus carpio and Danio rerio as outgroups, three analyses identified the same four lineages representing four subfamilies of loaches, with Botiinae on the basal-most clade. The phylogenctic relationship of the Cobitoidea was ((Catostomidae + Gyrinocheilidae) + (Botiinae + (Balitorinae + (Cobitinae + Nemacheilinae)))), which indicated that Sawada's Cobitidae (including Cobitinae and Botiinae) was not monophyletic. Our molecular phylogenetic analyses are in very close agreement with the phylogenetic results based on the morphological data proposed by Nalbant and Bianco, wherein these four subfamilies were elevated to the family level as Botiidae, Balitoridae, Cobitidae, and Nemacheilidae. (c) 2005 Elsevier Inc. All rights reserved.

Genomic organization, gene duplication, and expression analysis of interleukin-1 beta in channel catfish (Ictalurus punctatus)

Interleukin-1 beta (IL-1 beta) is one of the pivotal early response pro-inflammatory cytokines that enables organisms to respond to infection and induces a cascade of reactions leading to inflammation. In spite of its importance and two decades of studies in the mammalian species, genes encoding IL-1 beta were not identified from non-mammalian species until recently. Recent research, particularly with genomic approaches, has led to sequencing of IL-1 beta from many species. Clinical studies also Suggested IL-1 beta as an immunoreagulatory molecule potentially useful for enhancing vaccination. However, no IL-1 beta genes have been identified from channel catfish, the primary aquaculture species from the United States. In this study, we identified two distinct cDNAs encoding catfish IL-1 beta. Their encoding genes were identified, sequenced, and characterized. The catfish IL-1 beta genes were assigned to bacterial artificial chromosome (BAC) clones. Genomic studies indicated that the IL-1 beta genes were tandemly duplicated on the same chromosome. Phylogenetic analysis of various IL-1 beta genes indicated the possibility of recent species-specific gene duplications in channel catfish, and perhaps also in swine and carp. Expression analysis indicated that both IL-1 beta genes were expressed, but exhibited distinct expression profiles in various catfish tissues, and after bacterial infection with Edwardsiella ictaluri. (c) 2005 Elsevier Ltd. All rights reserved.

NK-lysin of channel catfish: Gene triplication, sequence variation, and expression analysis

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In addition to the previously known four classes of antimicrobial peptides, a fifth class of antimicrobial peptides has been recently identified to include NK-lysins that have a globular three-dimensional structure and are larger with 74-78 amino acid residues. NK-lysin has been shown to harbor antimicrobial activities against a wide spectrum of microorganisms including bacteria, fungi, protozoa, and parasites. To date, NK-lysin genes have been reported from only a limited number of organisms. We previously identified a NK-lysin cDNA in channel catfish. Here we report the identification of two noveltypes of NK-lysin transcripts in channel catfish. Altogether, three distinct NK-lysin transcripts exist in channel catfish. In this work, their encoding genes were identified, sequenced, and characterized. We provide strong evidence that the catfish NK-lysin gene is tripled in the same genomic neighborhood. All three catfish NK-lysin genes are present in the same genomic region and are tightly linked on the same chromosome, as the same BAC clones harbor all three copies of the NK-lysin genes. All three NK-lysin genes are expressed, but exhibit distinct expression profiles in various tissues. In spite of the existence of a single copy of NK-lysin gene in the human genome, and only a single hit from the pufferfish,genome, there are two tripled clusters of NK-lysin genes on chromosome 17 of zebrafish in addition to one more copy on its chromosome 5. The similarity in the genomic arrangement of the tripled NK-lysin genes in channel catfish and zebrafish suggest similar evolution of NK-lysin genes. (c) 2005 Elsevier Ltd. All rights reserved.

Zebraflsh z-otu, a novel Otu and Tudor domain-containing gene, is expressed in early stages of oogenesis and embryogenesis

Several studies have suggested that Otu domain had de-ubiquitinating activity and Tudor domain was important for the formation of germ cells. Here, we reported a novel zebrafish ovary-specific gene containing Otu and Tudor domain, z-otu, which was expressed at stages I-III oocytes and embryonic stages from zygotes to early blastula during embryonic cells maintained their totipotency. Therefore, z-otu might link the ubiquitin signaling pathway to early oogenesis and maintaining the totipotency of embryonic cell. (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.

The phylogenetic relationships of the gobioninae (Teleostei : Cyprinidae) inferred from mitochondrial cytochrome b gene sequences

The Gobioninae are a group of morphologically and ecologically diverse Eurasian freshwater cyprinid fishes. The intergeneric relationships of this group are unresolved and the possible monophyly of this subfamily remains to be established. We used complete mitochondrial cytochrome b gene sequences from most genera within the gobionine group, in addition to a selection of cyprinid outgroups, to investigate the possible monophyly of this group and resolve the interrelationships within the group. Our results support the monophyly of the Gobioninae and identify four monophyletic groups within the subfamily; the Hemibarbus group, the Sarcocheilichthys group, the Gobio group, and the Pseudogobio group. The morphologically aberrant genera Gobiobotia, Xenophysogobio and Gobiocypris are included in the Gobioninae, with the latter a sister group of Gnathopogon.