The first completed genome sequence from a teleost fish (Fugu rubripes) adds significant diversity to the nuclear receptor superfamily

Defining complete sets of gene family members from diverse species provides the foundation for comparative studies. Using a bioinformatic approach, we have defined the entire nuclear receptor complement within the first available complete sequence of a non-human vertebrate (the teleost fish Fugu rubripes). In contrast to the human set (48 total nuclear receptors), we found 68 nuclear receptors in the Fugu genome. All 68 Fugu receptors had a clear human homolog, thus defining no new nuclear receptor subgroups. A reciprocal analysis showed that each human receptor had one or more Fugu orthologs, excepting CAR (NR1I3) and LXRβ (NR1H2). These 68 receptors add striking diversity to the known nuclear receptor superfamily and provide important comparators to human nuclear receptors. We have compared several pharmacologically relevant human nuclear receptors (FXR, LXRα/β, CAR, PXR, VDR and PPARα/γ/δ) to their Fugu orthologs. This comparison included expression analysis across five Fugu tissue types. All of the Fugu receptors that were analyzed by PCR in this study were expressed, indicating that the majority of the additional Fugu receptors are likely to be functional.     

Genomics of fish IL-17 ligand and receptors: a review

Interleukin-17 (IL-17) is a cytokine family composed of six ligands (A–F). Especially, the IL-17A and IL-17F are best characterized cytokines of IL-17 family cytokine. These are produced by Th17 cells and induce the expression of many mediators of inflammation properties. In addition, the five member of IL-17 receptor family (RA-RE) have been identified in mammals. Although the research on fish IL-17 is a little to date, this review discusses some of the recent advances in research on IL-17 ligand and receptor genes in fish. IL-17 family member was chosen from the fish genome database, and its structure and phylogeny is analyzed in detail. Moreover, invertebrate IL-17 genes are also discussed, and the isolation and current status of fish IL-17 receptor genes are summarized. Comparative genomic analysis of the IL-17 family among mammals, teleost and invertebrates provided new insights. Novel IL-17 ligand (IL-17N) was identified from teleost, moreover it was suggested that IL-17N may be a teleost specific ligand by synteny and phylogenetic analysis. On the other hand, IL-17 receptors are well conserved between mammal and teleost, the five member of IL-17 receptor family: IL-17RA-RE were found on the teleost genome. In addition, the IL-17RA gene was duplicated in tandem on the stickleback and medaka genome. Knowledge about the IL-17 ligand/receptor in fish is very limited. Therefore this review will hopefully encourage future studies of IL-17 in fish.     

The neural cell adhesion molecule L1: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu

The human gene for the neural cell adhesion molecule L1 is located on Xq28 between the ALD and MeCP2 loci. Mutations in the L1 gene are associated with four related neurological disorders, X-linked hydrocephalus, spastic paraplegia (SPG1), MASA syndrome, and X-linked corpus callosum agenesis. The clinical relevance of L1 has led us to sequence the L1 gene in human and to investigate its conservation in the vertebrate model genome of the pufferfish, Fugu rubripes (Fugu), a species with a compact genome of around 40 Mb. For this purpose we have sequenced a human and a Fugu cosmid clone containing the corresponding L1 genes. For comparison, we have also amplified and sequenced the complete Fugu L1 cDNA. We find that the genomic structure of L1 is conserved. The human and Fugu L1 gene both have 28 exons of nearly identical size. Differential splicing of exons 2 and 27 is conserved over 430 million years, the evolutionary time span between the teleost Fugu and the human L1 gene. In contrast to previously published Fugu genes, many introns are larger in the Fugu L1 gene, making it slightly larger in size despite the compact nature of the Fugu genome. Homology at the amino acid and the nucleotide level with 40% and 51%, respectively, is lower than that of any previously reported Fugu gene. At the level of protein structure, both human and Fugu L1 molecules are composed of six immunoglobulin (Ig)-like domains and five fibronectin (Fn) type III domains, followed by a transmembrane domain and a short cytoplasmic domain. Only the transmembrane and the cytoplasmic domains are significantly conserved in Fugu, supporting their proposed function in intracellular signalling and interaction with cytoskeletal elements in the process of neurite outgrowth and fascicle formation. Our results show that the cytoplasmic domain can be further subdivided into a conserved and a variable region, which may correspond to different functions. Most pathological missense mutations in human L1 affect conserved residues. Fifteen out of 22 reported missense mutations alter amino acids that are identical in both species.     

Genomic structure and sequence of the pufferfish (Fugu rubripes) growth hormone-encoding gene: a comparative analysis of teleost growth hormone genes

A nested polymerase chain reaction (PCR) technique for amplifying a fragment of the gene (GH) encoding teleost growth hormone has been developed. Using this technique, a fragment of the pufferfish, Fugu rubripes and Arothron maculatus; dwarf gourami, Colisa lalia; guppy, Poecilia reticulata; and goldfish, Carassius auratus GH genes were cloned. The Fugu rubripes (Fugu) gene fragment was used to isolate the GH gene from a Fugu genomic library. The complete nucleotide sequence of a 8.5-kb SacI genomic fragment containing the Fugu GH gene has been determined. The GH gene spans 2.5 kb from the first codon to polyadenylation signal, and contains six exons and five introns similar to the GH genes of salmonids, tilapia, barramundi, flounder and yellowtail. The GH introns contain microsatellite and satellite sequences. The microsatellites found in the fifth intron of the GH gene are also present in the corresponding introns of tilapia, barramundi and flounder GH genes. Southern analysis revealed that the GH gene is a single-copy gene in the Fugu. The promoter region of the Fugu GH gene contains conserved sequences that are likely to be involved in the pituitary-specific expression of the gene. A phylogenetic tree of nucleotide (nt) sequences of all known teleost GH genes has been inferred using the distance matrix method. The topology of this tree reflects the major phylogenetic groupings of teleosts. The intron patterns and repetitive sequences of GH genes can serve as useful natural markers for the classification and phylogenetic studies of teleosts.     

Antagonistic RNA aptamer specific to a heterodimeric form of human interleukin-17A/F

Interleukin-17 (IL-17) is a pro-inflammatory cytokine produced primarily by a subset of CD4⁺ T cells, called Th17 cells, that is involved in host defense, inflammation and autoimmune disorders. The two most structurally related IL-17 family members, IL-17A and IL-17F, form homodimeric (IL-17A/A, IL-17F/F) and heterodimeric (IL-17A/F) complexes. Although the biological significance of IL-17A and IL-17F have been investigated using respective antibodies or gene knockout mice, the functional study of IL-17A/F heterodimeric form has been hampered by the lack of an inhibitory tool specific to IL-17A/F. In this study, we aimed to develop an RNA aptamer that specifically inhibits IL-17A/F. Aptamers are short single-stranded nucleic acid sequences that are selected in vitro based on their high affinity to a target molecule. One selected aptamer against human IL-17A/F, AptAF42, was isolated by repeated cycles of selection and counterselection against heterodimeric and homodimeric complexes, respectively. Thus, AptAF42 bound IL-17A/F but not IL-17A/A or IL-17F/F. The optimized derivative, AptAF42dope1, blocked the binding of IL-17A/F, but not of IL-17A/A or IL-17F/F, to the IL-17 receptor in the surface plasmon resonance assay in vitro. Consistently, AptAF42dope1 blocked cytokine GRO-α production induced by IL-17A/F, but not by IL-17A/A or IL-17F/F, in human cells. An RNA footprinting assay using ribonucleases against AptAF42dope1 in the presence or absence of IL-17A/F revealed that part of the predicted secondary structure fluctuates between alternate forms and that AptAF42dope1 is globally protected from ribonuclease cleavage by IL-17A/F. These results suggest that the selected aptamer recognizes a global conformation specified by the heterodimeric surface of IL-17A/F.     

Cytokine Gene Expression in CD4 Positive Cells of the Japanese Pufferfish,Takifugu rubripes

CD4⁺ T (Th) cells are a central component of the adaptive immune response and are divided into distinct sets based on their specific cytokine production pattern. Several reports have suggested that fish possess Th subset activity similar to that of mammals. The aim of the present study was to isolate CD4⁺ T cells from the blood of Japanese pufferfish, Fugu rubripes, and to characterize their cytokine expression profile. We produced a specific antibody against Fugu CD4 and performed cell sorting with the magnetic activated cell sorting system. Sorted Fugu CD4⁺ cells were characterized by morphology and expression analysis of cell marker genes. Fugu CD4⁺ cells expressed T-cell marker genes but not macrophage or B-cell marker genes. In addition, peripheral blood lymphocytes were stimulated with lipopolysaccharide (LPS), polycytidylic acid (polyI:C), concanavalin A (ConA) prior to sorting, and then Multiplex RT-PCR was used to examine the expression of Th cytokines by the stimulated Fugu CD4⁺ cells. LPS and polyI:C stimulation upregulated the expression of Th1, Th17 and Treg cytokines and downregulated the expression of Th2 cytokines. ConA stimulation upregulated the expression of all Th cytokines. These results suggest that fish exhibit the same upregulation of Th-specific cytokine expression as in mammals.     

FRG1, a gene in the FSH muscular dystrophy region on human chromosome 4q35, is highly conserved in vertebrates and invertebrates

The human FRG1 gene maps to human chromosome 4q35 and was identified as a candidate for facioscapulohumeral muscular dystrophy. However, FRG1 is apparently not causally associated with the disease and as yet, its function remains unclear. We have cloned homologues of FRG1 from two additional vertebrates, the mouse and the Japanese puffer fish Fugu rubripes, and investigated the genomic organization of the genes in the two species. The intron/exon structure of the genes is identical throughout the protein coding region, although the Fugu gene is five times smaller than the mouse gene. We have also identified FRG1 homologues in two nematodes; Caenorhabditis elegans and Brugia malayi. The FRG1 protein is highly conserved and contains a lipocalin sequence motif, suggesting it may function as a transport protein.     

T-helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus?

Introduction

A hallmark of systemic autoimmune diseases like systemic lupus erythematosus (SLE) is the increased expression of interferon (IFN) type I inducible genes, so-called IFN type I signature. Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6⁺ memory T-helper cells producing IL-17A, IL-17F, IL-21, and/or IL-22 are increased in SLE patients and whether this increase is related to the presence of IFN type I signature.

Methods

In total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive (IFN⁺) (n = 16) and negative (IFN^-) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4⁺CD45RO⁺CCR6⁺ T cells (CCR6⁺ cells) was measured with flow cytometry and compared between IFN⁺, IFN^- patients and HCs.

Results

Increased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ cells were observed in IFN⁺ patients compared with IFN^- patients and HCs. IL-17A and IL-17F expression within CCR6⁺ cells correlated significantly with IFIG expression. In addition, we found significant correlation between B-cell activating factor of the tumor necrosis family (BAFF)–a factor strongly correlating with IFN type I - and IL-21 producing CCR6⁺ cells.

Conclusions

We show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ memory T-helper cells in IFN⁺ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis.     

Identification and characterization of<Emphasis Type="Italic"> Fugu</Emphasis> orthologues of mammalian interleukin-12 subunits

We have isolated and characterized cDNAs and genes for pufferfish, Fugu rubripes, (Fugu) orthologues of mammalian interleukin (IL)-12 subunits (IL-12 p35 and IL-12 p40). The deduced amino acid sequences of the Fugu IL-12 subunits showed homology with mammalian IL-12 subunits (p35: 50.4–58.0% similarity; p40: 51.2–55.4% similarity). Phylogenetic analysis confirmed that Fugu IL-12 p35 and p40 genes cluster with their mammalian counterpart lineages. The genomic organization of each of the Fugu IL-12 subunit genes is similar to that of the corresponding mouse IL-12 subunit genes, although the Fugu genes are very compact due to small intron size. Comparative genomic analysis showed conserved syntenies within the IL-12 p35 and p40 regions between Fugu and human, indicating that the Fugu IL-12 p35 and p40 genes are orthologues for mammalian IL-12 p35 and p40 encoding genes, respectively. Expression of IL-12 p35 mRNA was observed in lymphoid tissues and several non-lymphoid tissues, while expression of IL-12 p40 mRNA was constitutive and nearly ubiquitous. In the spleen and head kidney, expression of IL-12 p35 was induced by polyriboinosinic polyribocytidylic acid [poly(I:C)] and not by lipopolysaccharide (LPS), while expression of IL-12 p40 was constitutive and unresponsive to both poly(I:C) and LPS. These results indicate that IL-12 levels are regulated by production of IL-12 p35 mRNA and suggest that IL-12 in fish may be involved in antiviral defense. This is the first report of the identification and characterization of IL-12 subunit cDNAs and genes in a non-mammalian vertebrate.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AB096265, AB096266, AB096267, and AB096268.     

Faithful expression of a tagged Fugu WT1 protein from a genomic transgene in zebrafish: efficient splicing of pufferfish genes in zebrafish but not mice

The teleost fish are widely used as model organisms in vertebrate biology. The compact genome of the pufferfish, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identification of conserved regulatory elements, whilst the zebrafish is particularly suited to genetic and developmental studies. We demonstrate that a pufferfish WT1 transgene can be expressed and spliced appropriately in transgenic zebrafish, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebrafish with the same construct, we show that Fugu RNA is processed correctly in zebrafish but not in mice. Furthermore, we show for the first time that a Fugu genomic construct can produce protein in transgenic zebrafish: a full-length Fugu WT1 transgene with a C-terminal β-galactosidase fusion is spliced and translated correctly in zebrafish, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebrafish:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.     

Distribution and Evolution of the Xylanase Genes xynA and xynB and Their Homologues in Strains of Butyrivibrio fibrisolvens

The ruminal bacterium Butyrivibrio fibrisolvens is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants. However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of B. fibrisolvens. In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of Butyrivibrio fibrisolvens have been investigated. Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49 xynA gene. However, these four xynA-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to xynA (subfamily I). Homologues of xynB, a second previously described xylanase gene from B. fibrisolvens, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains. However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of xynA. The distribution of genes and the nucleotide sequence relationships between the members of the two xynA subfamilies are consistent with the progenitor of all strains of B. fibrisolvens having contained a xynA subfamily I gene. Since many xylanolytic strains of B. fibrisolvens did not contain members of either of the xynA subfamilies or of the xynB family, at least one additional xylanase gene family remains to be identified in B. fibrisolvens.     

High-mobility-group family genes from Lampetra japonica reveal their early origin and molecular evolution in the vertebrate lineage

High-mobility group family (HMG) genes are ubiquitous in vertebrates, including mammals, birds, amphibians and fishes. To elucidate the molecular phylogeny of the HMG genes in the primitive vertebrate, we have cloned three homologues of HMG-box genes, called Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX, from a cDNA library generated from lymphocyte-like cells of the Japanese lamprey (Lampetra japonica), an Agnathan that occupies a critical phylogenetic position between invertebrates and vertebrates. The open reading frames of Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX contained 627 bp, 585 bp and 678 bp, respectively. The analysis of the deduced amino acid sequences indicated that these three putative Lj-HMGB proteins contain four domains: HMG-box A, HMG-box B, an acidic carboxyl-terminal tail and a linker. A phylogenetic analysis revealed that the Lj-HMGB proteins fall outside the vertebrate clade; Lj-HMGBX is descended from a gene ancestral to the mammalian HMGB1/2/3. This discovery implies that there was a gene duplication event in the HMGB1/2/3 gene family that occurred after the divergence of the vertebrates (Cyclostomata) from the Cephalochordata and Urochordata at least 450 million years ago (MYA). The Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX genes were detected in most tissues of the lamprey by RT-PCR. Our findings provide insight into the phylogeny of the HMGB genes in vertebrates.     

Candidate plant gene homologues in grapevine involved in Agrobacterium transformation

The grapevine (Vitis vinifera) genome was analyzed in silico for homologues of plant genes involved in Agrobacterium transformation in Arabidopsis thaliana and Nicotiana spp. Grapevine homologues of the glucomannan 4-betamannosyltransferase 9 gene CslA-09 involved in bacterial attachment to the cell wall, homologues of reticulon-like proteins BTI1, 2, 3 and RAB8 GTPases, both involved in T-DNA transfer to the host cell, homologues of VirE2 interacting protein VIP1 that contributes to the targeting of T-DNA into the nucleus and to its integration, and homologues of the histone protein H2A, which promotes the expression of T-DNA encoded genes, were selected. Sequences homologous to the arabinogalactan-protein AtAGP17 were not found in the grape genome. Seventeen selected candidates were tested by semiquantitative RT-PCR analysis for changes in their expression levels upon inoculation with Agrobacterium tumefaciens C58. Of the tested homologues, the expression of VvRab8a, VvVip1a and two histone genes (VvHta2 and VvHta10) increased significantly, therefore we hypothesize that these might be involved in Agrobacterium transformation of V. vinifera.     

Genetic Analysis of IL-17 Gene Polymorphisms in Gout in a Male Chinese Han Population

Interleukin (IL)-17 is a proinflammatory cytokine mainly secreted by activated T helper 17 cells and involved in inflammatory immune responses. This study aimed to investigate the association between IL-17 variants as well as serum IL-17 levels with gout in male Chinese Han individuals. A total of 1,101 male gout patients and 1,239 ethic-matched controls were enrolled. Genetic distributions of three variants (rs2275913 in IL-17A, rs763780 in IL-17F, and rs4819554 in IL-17RA) were detected by real-time polymerase chain reaction using the Taqman probe method. The plasma concentrations of IL-17A and IL-17F were measured in 228 gout patients and 198 controls that came from above samples by an enzyme-linked immunosorbent assay. No significant differences were observed in the genetic distribution of these polymorphisms between cases and controls (rs2275913: χ² = 0.15, p = 0.928 by genotype, χ² = 0.14, p = 0.711 by allele; rs763780: χ² = 2.24, p = 0.326 by genotype, χ² = 0.26, p = 0.609 by allele; rs4819554: χ² = 1.79, p = 0.409 by genotype, χ² = 1.46, p = 0.227 by allele). Levels of serum IL-17A and IL-17F were significantly decreased in gout patients (both p<0.001). However, no difference was observed in acute gout patients between different genotypic carriers of rs2275913 and rs763780 regarding serum IL-17A and IL-17F levels (p>0.05). Although the genetic variants in IL-17 we studied in this research do not appear to be involved in the development of gout in male Chinese Han individuals, the IL-17 cytokine family may participate in gouty inflammation in an undefined way, which requires further validation.     

CD8 positive T cells express IL-17 in patients with chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is a progressive and irreversible chronic inflammatory disease of the lung. The nature of the immune reaction in COPD raises the possibility that IL-17 and related cytokines may contribute to this disorder. This study analyzed the expression of IL-17A and IL-17F as well as the phenotype of cells producing them in bronchial biopsies from COPD patients.

Methods

Bronchoscopic biopsies of the airway were obtained from 16 COPD subjects (GOLD stage 1-4) and 15 control subjects. Paraffin sections were used for the investigation of IL-17A and IL-17F expression in the airways by immunohistochemistry, and frozen sections were used for the immunofluorescence double staining of IL-17A or IL-17F paired with CD4 or CD8. In order to confirm the expression of IL-17A and IL-17F at the mRNA level, a quantitative RT-PCR was performed on the total mRNA extracted from entire section or CD8 positive cells selected by laser capture microdissection.

Results

IL-17F immunoreactivity was significantly higher in the bronchial biopsies of COPD patients compared to control subjects (P < 0.0001). In the submucosa, the absolute number of both IL-17A and IL-17F positive cells was higher in COPD patients (P < 0.0001). After adjusting for the total number of cells in the submucosa, we still found that more cells were positive for both IL-17A (P < 0.0001) and IL-17F (P < 0.0001) in COPD patients compared to controls. The mRNA expression of IL-17A and IL-17F in airways of COPD patients was confirmed by RT-PCR. The expression of IL-17A and IL-17F was co-localized with not only CD4 but also CD8, which was further confirmed by RT-PCR on laser capture microdissection selected CD8 positive cells.

Conclusion

These findings support the notion that Th17 cytokines could play important roles in the pathogenesis of COPD, raising the possibility of using this mechanism as the basis for novel therapeutic approaches.