Ikuo Hirono

Molecular Cloning, Characterization, and Expression of TNF cDNA and Gene from Japanese Flounder Paralychthys olivaceus

We cloned a cDNA and the gene for Japanese flounder TNF. The TNF cDNA consisted of 1217 bp, which encoded 225 amino acid residues. The identities between Japanese flounder TNF and members of the mammalian TNF family were ∼20–30%. The positions of cysteine residues that are important for disulfide bonds were conserved with respect to those in mammalian TNF-α. The Japanese flounder TNF gene has a length of ∼2 kbp and consists of four exons and three introns. The positions of the exon-intron junction positions of Japanese flounder TNF gene are similar to those of human TNF-α. However, the length of the first intron of Japanese flounder is much shorter than that of the human TNF-α gene. There are simple CA or AT dinucleotide repeats in the 5′-upstream and 3′-downstream regions of the Japanese flounder TNF gene. Southern blot hybridization indicted that Japanese flounder TNF exists as a single copy. Expression of Japanese flounder TNF mRNA is greatly induced after stimulation of PBLs with LPS, Con A, or PMA. These results indicated that Japanese flounder TNF is more like mammalian TNF-α than mammalian lymphotoxin-α, with respect to its gene structure, length of amino acid sequence, number and position of cysteine residues, and regulation of gene expression.

Genome Sequences of Three Koi Herpesvirus Isolates Representing the Expanding Distribution of an Emerging Disease Threatening Koi and Common Carp Worldwide

ABSTRACT Since the mid-1990s, lethal infections of koi herpesvirus (KHV) have been spreading, threatening the worldwide production of common carp and koi (both Cyprinus carpio). The complete genome sequences of three KHV strains from Japan, the United States, and Israel revealed a 295-kbp genome containing a 22-kbp terminal direct repeat. The finding that 15 KHV genes have clear homologs in the distantly related channel catfish virus (ictalurid herpesvirus 1) confirms the proposed place of KHV in the family Herpesviridae, specifically in the branch with fish and amphibian hosts. KHV thus has the largest genome reported to date for this family. The three strains were interpreted as having arisen from a wild-type parent encoding 156 unique protein-coding genes, 8 of which are duplicated in the terminal repeat. In each strain, four to seven genes from among a set of nine are fragmented by frameshifts likely to render the encoded proteins nonfunctional. Six of the affected genes encode predicted membrane glycoproteins. Frameshifts or other mutations close to the 3′ ends of coding sequences were identified in a further six genes. The conclusion that at least some of these mutations occurred in vivo prompts the hypothesis that loss of gene functions might be associated with emergence of the disease and provides a basis for further investigations into the molecular epidemiology of the virus.

Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species

Lysozyme cDNA was isolated from a kuruma shrimp, Marsupenaeus japonicus, hemocyte cDNA library. The cDNA consists of 1055 base pairs (bp) and encodes a chicken-type (c-type) lysozyme with a deduced amino acid sequence of 156 residues. The kuruma shrimp lysozyme has a high identity (79.7%) with pacific white shrimp lysozyme, and low to moderate identities (33.3-43.0%) with lysozymes of insects and vertebrates. Comparisons with other c-type lysozymes from invertebrates and vertebrates showed that the two catalytic residues (Glu58 and Asp75) and the eight cysteine residue motif were completely conserved. Two novel insertion sequences were also observed in the kuruma and pacific white shrimp lysozyme amino acid sequences. Interestingly, phylogenetic analysis revealed that the kuruma shrimp lysozyme was more closely related to vertebrate c-type lysozymes. Expression of the cDNA in insect cells, using a baculovirus expression system, yielded a recombinant lysozyme with optimum activity at pH 7.5 and 50 degrees C, as evaluated by a lysoplate assay. The kuruma shrimp lysozyme displayed lytic activities against several Vibrio species and fish pathogens, including Vibrio penaeicida (a pathogenic bacteria to the kuruma shrimp) and suggested that shrimp lysozyme affects a greater variety of pathogens.

Identification of immune-related genes in hemocytes of black tiger shrimp (Penaeus monodon).

An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 × 105. Of these, 615 clones having inserts larger than 500 by were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the clotting system and the prophenoloxidase-activating system, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.

Iron-regulated haemolysin gene from Edwardsiella tarda.

We have cloned and sequenced the haemolysin gene locus from Edwardsiella tarda (ETH). This region encoded two open reading frames, designated ethA and ethB. ethA is the haemolysin gene consisting of 4782 bp encoding a product of 165.3 kDa and ethB is an activation/secretion protein gene of 1677 bp that encodes a product of 61.9 kDa. There were two putative ferric uptake regulator (Fur) binding sites on the 5′ upstream region of the ethB gene overlapping the promoter region and ribosome‐binding site. The haemolysin produced by the cloned gene was secreted by Escherichia coli. The deduced amino acid sequences of the ethA and ethB genes were found to be homologous to those of the haemolysin and activation/secretion proteins of Haemophilus ducreyi, Proteus mirabilis, and Serratia marcescens. E. coli carrying the ethA gene but not the ethB gene completely lost haemolytic activity, although the ethA gene was transcribed. The protein expressed by E. coli carrying a recombinant plasmid which encoded the ethA gene had haemagglutination activity. The EthB protein was necessary for activation of EthA protein (haemolysin). The ethA and ethB genes were very prevalent in haemolytic E. tarda strains isolated from diseased fish. Transcription of the ethB gene was regulated by iron. The ethA and ethB genes were transcribed independently.

Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein.

In this study, we cloned the goose-type (g-type) lysozyme gene from the Japanese flounder genomic DNA library, the first such data in fish and only the second after the chicken g-type lysozyme gene. The Japanese flounder g-type lysozyme gene was 1252 bp in length from the transcription site to the polyadenylation site, coded for 758 bp of mRNA and 195 deduced amino acids, which contain five exons and four introns. A phylogenetic analysis based on amino acid sequences showed that the flounder gene was closer to g-type lysozyme, followed by phage-type lysozyme and then chicken-type (c-type) lysozyme. Although exon 1 of the flounder gene differs from exons 1 and 2 of the chicken g-type lysozyme gene, three catalytic residues, as well as their neighboring amino acids were conserved between the Japanese flounder and the four avian g-type lysozymes. In a Southern blot analysis using the genomic DNA of homo-cloned Japanese flounder, the flounder g-type lysozyme gene showed a simple pattern, suggesting that it is encoded by a single copy gene. A Northern blot analysis showed that this gene was expressed in all tissues of Japanese flounder that we examined in this study and showed major differences from those expressed tissues of the chicken g-type gene. Japanese flounder g-type lysozyme mRNA levels in the intestine, heart and whole blood increased after injecting the fish with Edwardsiella tarda. Recombinant flounder g-type lysozyme, which has an optimal pH and temperature of pH 6.0 and 25 degrees C, possessed lytic activity against Micrococcus lysodeikticus and several fish pathogenic bacteria. This is the first report of a g-type lysozyme gene other than for reported avian species.

Comparative analysis of differentially expressed genes in normal and white spot syndrome virus infected Penaeus monodon

BackgroundWhite spot syndrome (WSS) is a viral disease that affects most of the commercially important shrimps and causes serious economic losses to the shrimp farming industry worldwide. However, little information is available in terms of the molecular mechanisms of the host-virus interaction. In this study, we used an expressed sequence tag (EST) approach to observe global gene expression changes in white spot syndrome virus (WSSV)-infected postlarvae of Penaeus monodon.ResultsSequencing of the complementary DNA clones of two libraries constructed from normal and WSSV-infected postlarvae produced a total of 15,981 high-quality ESTs. Of these ESTs, 46% were successfully matched against annotated genes in National Center of Biotechnology Information (NCBI) non-redundant (nr) database and 44% were functionally classified using the Gene Ontology (GO) scheme. Comparative EST analyses suggested that, in postlarval shrimp, WSSV infection strongly modulates the gene expression patterns in several organs or tissues, including the hepatopancreas, muscle, eyestalk and cuticle. Our data suggest that several basic cellular metabolic processes are likely to be affected, including oxidative phosphorylation, protein synthesis, the glycolytic pathway, and calcium ion balance. A group of immune-related chitin-binding protein genes is also likely to be strongly up regulated after WSSV infection. A database containing all the sequence data and analysis results is accessible at http://xbio.lifescience.ntu.edu.tw/pm/.ConclusionThis study suggests that WSSV infection modulates expression of various kinds of genes. The predicted gene expression pattern changes not only reflect the possible responses of shrimp to the virus infection but also suggest how WSSV subverts cellular functions for virus multiplication. In addition, the ESTs reported in this study provide a rich source for identification of novel genes in shrimp.

Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster.

Background Despite its economic importance, we have a limited understanding of the molecular mechanisms underlying shell formation in pearl oysters, wherein the calcium carbonate crystals, nacre and prism, are formed in a highly controlled manner. We constructed comprehensive expressed gene profiles in the shell-forming tissues of the pearl oyster Pinctada fucata and identified novel shell formation-related genes candidates. Principal Findings We employed the GS FLX 454 system and constructed transcriptome data sets from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in P. fucata. We sequenced 260477 reads and obtained 29682 unique sequences. We also screened novel nacreous and prismatic gene candidates by a combined analysis of sequence and expression data sets, and identified various genes encoding lectin, protease, protease inhibitors, lysine-rich matrix protein, and secreting calcium-binding proteins. We also examined the expression of known nacreous and prismatic genes in our EST library and identified novel isoforms with tissue-specific expressions. Conclusions We constructed EST data sets from the nacre- and prism-producing tissues in P. fucata and found 29682 unique sequences containing novel gene candidates for nacreous and prismatic layer formation. This is the first report of deep sequencing of ESTs in the shell-forming tissues of P. fucata and our data provide a powerful tool for a comprehensive understanding of the molecular mechanisms of molluscan biomineralization.

Two different types of hepcidins from the Japanese flounder Paralichthys olivaceus

The cysteine‐rich peptide hepcidin is known to be an antimicrobial peptide and iron transport regulator that has been found in both fish and mammals. Recently, we found two different types (designated Hep‐JF1 and Hep‐JF2) of hepcidin cDNA in the Japanese flounder, Paralichthys olivaceus, by expressed sequence tag analysis. The identity of amino acid sequences between Hep‐JF1 and Hep‐JF2 was 51%. The Hep‐JF1 and Hep‐JF2 genes both consist of three exons and two introns, and both exist as single copies in the genome. The predicted mature regions of Hep‐JF1 and Hep‐JF2 have six and eight Cys residues, respectively. The first Cys residue of Hep‐JF1 was deleted and the second was replaced with Gly. The number and positions of Cys residues in Hep‐JF2 are the same as they are in human Hep. Hep‐JF1 is specifically expressed in liver while the expression of Hep‐JF2 was detected from gill, liver, heart, kidney, peripheral blood leucocytes, spleen and stomach. Gene expression of Hep‐JF1 in liver decreased during experimental iron (iron‐dextran) overload. Expression of Hep‐JF1 in liver was decreased by injecting fish with iron‐dextran and increased by injecting lipopolysaccharide. Iron overload did not significantly affect expression of Hep‐JF2 in liver but it did increase expression of Hep‐JF2 in kidney. Lipopolysaccharide injection increased expression of Hep‐JF2 in both liver and kidney. In liver, some cells expressed both Hep‐JF1 and Hep‐JF2 while some other cells expressed just one of them. Synthesized Hep‐JF2 peptide showed antimicrobial activity, while synthesized Hep‐JF1 peptide did not against several bacteria including fish‐pathogenic bacteria used in this study.

The Four TCR Genes of Teleost Fish: The cDNA and Genomic DNA Analysis of Japanese Flounder (Paralichthys olivaceus) TCR α-, β-, γ-, and δ-Chains

We have isolated and identified all four TCR α, β, γ, and δ cDNAs and genomic clones from a Japanese flounder leukocyte cDNA library and bacterial artificial chromosomal genomic library. Numerous TCR transcripts were sequenced to examine the variability against antigenic peptide, and were shown hypervariability on their complementarity-determining region 3 (CDR3) loops. Among CDR3s, CDR3δ showed a long and broad length distribution, indicating greater similarity to that of Ig. From cDNA sequences and genomic gene analysis of each chain, we found that flounder TCR β, γ, and δ have two different C gene segments, while the TCR α C region exists as a single segment. The flounder Cγs and Cδs showed different lengths in the connecting peptide (CP) region between the different types of polypeptides. The Cδ1 gene consists of two exons, one that encodes an extracellular Ig-like domain (exon 1) and the other that encodes either a very short or possibly a lacking CP region, a transmembrane region, and a cytoplasmic tail (exon 2); these are located within TCR α gene locus. Southern blot analysis, using the bacterial artificial chromosomal genomic DNA clones, revealed that the Cδ2 gene segment, which has a long CP region and different genomic organization to the Cδ1 gene, exists on same gene locus as the TCR γ-chain. This suggests that the flounder possesses very unique genomic DNA organization and gene loci for TCR, Cα/Cδ1, and Cγ/Cδ2.