Comparative analysis of two Nramp loci from rainbow trout.

Innate resistance to intracellular parasites is controlled in part by Nramp1 (Natural resistance-associated macrophage protein 1) in mammals and birds. To isolate Nramp homologs from rainbow trout, a combination of library screening and rapid amplification of cDNA ends was performed. Two closely related Nramp loci, designated OmNramp alpha and OmNramp beta, were cloned and characterized. OmNramp alpha and OmNramp beta encode two highly conserved proteins of 585 and 558 amino acids, respectively. Deduced amino acid seqences showed that the OmNramp alpha and OmNramp beta proteins share 90% of their residues and contain all of the signature features of the Nramp family of proteins: 12 transmembrane domains, two N-linked glycosylation sites, and a conserved transport motif. Phylogenetic analysis supported a close relation to Nramp2 proteins, a related member of the Nramp family. Despite this relation, juvenile trout expressed OmNramp alpha in a manner consistent with an Nramp1 homolog and OmNramp beta similar to an Nramp2 locus. Both trout loci were expressed at relatively high amounts in the ovaries of juveniles, a finding not reported in the investigations of previously characterized mammalian and avian homologs. These results suggest a role for Nramp loci in the follicular development of teleost fishes, as well as in mammals. Because salmonid fishes are ancestral tetraploids, fragments of OmNramp alpha and OmNramp beta were isolated from smelt, a diploid relative, to determine whether the trout loci represent duplicates of a single gene. Homologous sequences for both loci were found in smelt, supporting the hypothesis that OmNramp alpha and OmNramp beta are indeed independent loci that were present before the chromosomal duplication of salmonids. The isolation of Nramp loci from rainbow trout may eventually produce a genetic tool for the control of disease in aquaculture operations. Determining the involvement of trout homologs in innate immunity may also provide insight regarding the evolution of host resistance to pathogens.     

Putative prion protein from Fugu (Takifugu rubripes)

Prion proteins (PrP) of mammals, birds, reptiles and amphibians have been successfully cloned, expressed and purified in sufficient yields to enable 3D structure determination by NMR spectroscopy in solution. More recently, PrP ortholog genes have also been identified in several fish species, based on sequence relationships with tetrapod PrPs. Even though the sequence homology of fish PrPs to tetrapod PrPs is below 25%, structure prediction programs indicate a similar organization of the 3D structure. In this study, we generated recombinant polypeptide constructs that were expected to include the C-terminal folded domain of Fugu-PrP1 and analyzed these proteins using biochemical and biophysical methods. Because soluble expression could not be achieved, and refolding from guanidine-HCl did not result in a properly folded protein, we co-expressed Escherichia coli chaperone proteins in order to obtain the protein in a soluble form. Although CD spectroscopy indicated the presence of some regular secondary structure in the protein thus obtained, there was no evidence for a globular 3D fold in the NMR spectra. We thus conclude that the polypeptide products of the fish genes annotated as corresponding to bona fide prnp genes in non-fish species cannot be prepared for structural studies when using procedures similar to those that were successfully used with PrPs from mammals, birds, reptiles and amphibians.     

The plasmablast-like leukocyte in the kidney of fugu (Takifugu rubripes)

In teleosts, the kidney is the major immune organ. From the kidney of fugu (Takifugu rubripes), we isolated a unique leukocyte population. This population shows properties similar to those of mammalian plasmablasts. First, adherent cells expressing IgM protein on their surface were obtained from the fugu kidney. Flow cytometry (FCM) showed that these cells were mainly composed of two cell populations: IgM⁺CD8α^? cells and IgM⁺CD8α⁺ cells. Further characterization of the IgM⁺CD8α^? population by RT-PCR demonstrated that the cells expressed secretory-type IgM as well as Bcl-6 and Blimp-1, developmental marker genes for the B cell lineage. Western blotting also showed that the cells secreted IgM protein. These results indicate that the IgM⁺CD8α^? cells are similar to cells at the plasmablast stage in mammals. This is the first report isolating plasmablast-like leukocytes in fish species. Our data also suggests that the teleosts kidney is a organ where B cells terminally differentiate into the plasma cells.     

B-lymphocyte-induced maturation protein-1 (Blimp-1) gene of torafugu (Takifugu rubripes)

Homologous gene of B-lymphocyte-induced maturation protein-1 (Blimp-1) of torafugu (Takifugu rubripes) was identified by tblast search analysis. RT-PCR and 5'RACE clearly defined the sequence of the UTR and coding region which has been ambiguously determined by tblast analysis. Fugu Blimp-1 was mainly expressed in the lymphoid organs. These finding imply that Blimp-1 would take a major role in the terminal differentiation of B-cells to plasma cells in fish.     

The genome size evolution of medaka (Oryzias latipes) and fugu (Takifugu rubripes)

Evolution of the genome size in eukaryotes is often affected by changes in the noncoding sequences, for which insertions and deletions (indels) of small nucleotide sequences and amplification of repetitive elements are considered responsible. In this study, we compared the genomic DNA sequences of two kinds of fish, medaka (Oryzias latipes) and fugu (Takifugu rubripes), which show two-fold difference in the genome size (800 Mb vs. 400 Mb). We selected a contiguous DNA sequence of 790 kb from the medaka chromosome LG22 (linkage group 22), and made a precise comparison with the sequence (387 kb) of the corresponding region of Takifugu. The sequence of 178 kb in total was aligned common between two fishes, and the remaining sequences (612 kb for medaka and 209 kb for fugu) were found abundant in various repetitive elements including many types of unclassified low copy repeats, all of which accounted for more than a half (54%) of the genome size difference. Furthermore, we identified a significant difference in the length ratio of the unaligned sequences that locate between the aligned sequences (USBAS), particularly after eliminating known repetitive elements. These USBAS with no repetitive elements (USBAS-nr) located within the intron and intergenic region. These results strongly indicated that amplification of repetitive elements and compilation of indels are major driving forces to facilitate changes in the genome size.     

红鳍东方鲀特异性免疫相关基因的研究进展

红鳍东方(Takifugu rubripes)是我国北方重要的经济鱼类,但随着人工养殖规模的扩大,红鳍东方的病害越来越突出,给红鳍东方的生产造成了影响。因此,对红鳍东方免疫相关基因的研究意义十分重大。综述红鳍东方特异性免疫相关基因的研究进展。     

红鳍东方鲀不同生长时期脑和肌肉的转录组比较分析

红鳍东方鲀(Takifugu rubripes)是中国北方重要的海水养殖鱼类.本研究通过Illumina测序技术对13月龄和15月龄红鳍东方鲀的脑组织和肌肉组织共12个样本进行了转录组测序与分析.结果表明:在脑组织样本中平均共得到5 510万条原始reads,在肌肉组织样本中平均共得到5 482万条原始reads.在1...     

红鳍东方鲀(Takifugu rubripes)MC4R基因的多态性分析

采用PCR-SSCP(single strand conformation polymorphism)技术和DNA测序方法分析红鳍东方鲀MC4R(Melanocortin-4receptor)基因编码区多态性。在MC4R基因编码区48 nt和264 nt均发生了碱基的转换突变(G→A),两个突变位点分别位于M1和M2引物扩增产物中。引物M1扩增产物SSCP分析得到两种基因型:AA基因型和AB基因型,并且AA基因型和A等位基因频率明显高于AB基因型和B等位基因。引物M2扩增产物也得到两种基因型:CC基因型和CD基因型,CC基因型和C等位基因频率明显高于CD基因型和D等位基因。遗传变异结果分析表明,两个突变位点均属于低度多态性,而且群体遗传杂合度较低,反映了该群体的遗传一致性较高。     

Identification and comparative analysis of the RpL14 gene from Takifugu rubripes

The ribosomal protein RpL14 gene has been characterized in several species, including, human, rat and fruit fly. Haploinsufficiency for the gene causes the Minute phenotype in Drosophila, and it has been proposed as a regulator in the tumorigenic pathway in human. Several features concerning the gene structure have been studied, and some of these differ between human/rat and Drosophila. To address functional and evolutionary questions about these differences we have isolated and sequenced a cDNA and a genomic clone covering the RpL14 gene from the pufferfish Takifugu rubripes (Fugu). The Fugu RpL14 gene is approximately 2 Kb, with 5 introns, and encodes a protein of 137 amino acids. The protein contains a KOW-motif and a nuclear localization signal, which are conserved among a wide range of RPL14 proteins. On the other hand, a variable amino acid (alanine) repeat observed in human is missing in Takifugu rubripes, and the protein is shorter than its mammalian counterparts. Compared with human, the RpL14 gene in Fugu contains introns localized at identical positions in the gene, and most of them are shorter. A comparison of the RpL14 gene structure from a broad range of organisms indicates that both loss and gain of introns have occurred during the evolution of the gene.     

Carbohydrate-binding site of a novel mannose-specific lectin from fugu (Takifugu rubripes) skin mucus

Pufflectin-s, identified in the skin mucus of the fugu Takifugu rubripes, is a novel mannose-specific lectin with similar structure to monocotyledonous plant lectins. In the present study, mutational analysis was used to reveal the mannose-binding sites of pufflectin-s. Putative binding sites were mutated as follows: binding site 1; rPL-D32E (Asp³² → Glu³²), rPL-N34S (Asn³⁴ → Ser³⁴) and rPL-V36A (Val³⁶ → Ala³⁶) whereas binding site 2; rPL-D61E (Asp⁶¹ → Glu⁶¹), rPL-N63S (Asn⁶³ → Ser⁶³) and rPL-V65A (Val⁶⁵ → Ala⁶⁵). All recombinant proteins were expressed in Escherichia coli, purified with two chromatographic steps, and then subjected to mannose-binding assay by affinity chromatography. Recombinant wild-type pufflectin-s (rPL-wt) as well as three mutants with changes in binding site 2 could bind to mannose, in contrast to the three mutants with changes in binding site 1 in which mannose-binding activity was completely lost. These results clearly demonstrate that, at the least, binding site 1 is critical to mannose-binding activity in pufflectin-s.     

The sex-determining locus in the tiger pufferfish, Takifugu rubripes

The tiger pufferfish (fugu), Takifugu rubripes, is a model fish that has had its genome entirely sequenced. By performing genomewide linkage analyses, we show that the sex of fugu is determined by a single chromosomal region on linkage group 19 in an XX-XY system.     

Molecular cloning and expression of gelatinases (MMP-2 and MMP-9) in the pufferfish Takifugu rubripes

To determine the metabolism location of the extra-cellular matrix proteins in fugu (Takifugu rubripes), we cloned the cDNAs of the fugu gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, and examined their expressions in various adult tissues using a quantitative real-time PCR. The expression profiles of fugu gelatinases were different among tissues. FgMMP-9 mRNA was abundant in tissues that contain blood cells abundantly where fgMMP-2 mRNA was little expressed. We also examined the expression of these genes in fugu embryos during development using a whole mount in situ hybridization. Fugu MMP-2 mRNA was expressed in the pharyngeal area and mesenchyme in embryos at 80 hours post fertilization (hpf). While fugu MMP-9 mRNA was expressed in the vent at 140 hpf and the caudal end of the fin fold at 172 hpf. Although fugu MMP-2 mRNA was expressed in the pectoral fin bud at 120 hpf, fugu MMP-9 mRNA did not appear in this tissue until 10 days post fertilization (dpf). These data show expression profiles differ between the fugu gelatinases and suggest expressions of these genes are controlled at the matrix protein degradation site in fugu embryos during development.