Two inducible, functional cyclooxygenase-2 genes are present in the rainbow trout genome

The cyclooxygenases (Cox) catalyze the initial reactions in prostanoid biosynthesis, and produce the common prostanoids precursor, PGH(2). Mammalian species have two Cox isoforms; constitutively expressed cyclooxygenase-1 (Cox-1) and inducible cyclooxygenase-2 (Cox-2). Database searches suggest three Cox genes are present in many fish species. In this study, we cloned and characterized a second Cox-2 cDNA, Cox-2b, from the rainbow trout. Rainbow trout Cox-2b protein contains all the functionally important conserved amino acids for Cox enzyme activity. Moreover, the Cox-2b message contains AU-rich elements (AREs) in the 3' untranslated region (3'UTR) characteristic of inducible Cox-2 mRNAs. We took advantage of the existence of a rainbow trout cell line to demonstrate that expression from both the originally reported Cox-2 (Cox-2a) and Cox-2b genes is inducible. However, differential induction responses to alternative inducers are observed for rainbow trout Cox-2a and Cox-2b. Both Cox-2a and Cox-2b proteins expressed in COS cells are enzymatically active. Thus the rainbow trout has two functional, inducible Cox-2 genes. The zebrafish also contains two Cox-2 genes. However, genome structure analysis suggests diversion of the Cox-2a gene between zebrafish and rainbow trout.     

Comparative expression analysis of two paralogous Hsp70s in rainbow trout cells exposed to heat stress

Heat-shock protein 70 (Hsp70) is the major stress-inducible protein in vertebrates and highly conserved throughout evolution. To accurately investigate the mRNA expression profiles of multiple Hsp70s in rainbow trout Oncorhynchus mykiss, we isolated full-length cDNA clones encoding Hsp70 from the fish and investigated their mRNA expression profiles during heat stress. Consequently, two Hsp70s, Hsp70a and Hsp70b, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes in the genome. Northern blot analysis showed that each of Hsp70a and Hsp70b expressed two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells. The induction levels of total Hsp70b mRNAs were consistently higher than Hsp70a counterparts during heat stress, although the expression profiles of the two genes were similar to each other in temperature shift and time course experiments. Interestingly, an mRNA species with a larger molecular size was expressed only under severe heat stress not less than 28 degrees C irrespective of Hsp70a and Hsp70b. These results suggest that the comprehensive identification of duplicated genes is a prerequisite to examining the gene expression profiles for tetraploid species such as rainbow trout.     

The neuronal EGF-related genes NELL1 and NELL2 are expressed in hemopoietic cells and developmentally regulated in the B lineage

NELL1 and NELL2 (neural epidermal growth factor-like 1 and 2) are recently described members of the epidermal growth factor gene family that have previously been shown to be expressed almost exclusively in brain tissue. Here we demonstrate regulated expression of NELL1 and NELL2 in human hematopoietic cells. Mature NELL1 mRNA is not detected in any normal hemopoietic cell type, although the gene is transcribed during a narrow window of pre-B cell development, and cell lines at the same developmental stage express the NELL1 mRNA. The related NELL2 gene is expressed by all nucleated peripheral blood cells examined (B, T, monocyte, and natural killer cells), but not in any of the bone marrow B lineage cells at earlier stages of development. However, leukemic cell lines corresponding to the same early differentiation stages express abundant NELL2 mRNA. These results suggest normal and possible oncogenic roles for the NELL proteins in hemopoietic cells.     

CD4 homologues in Atlantic salmon

In mammals CD4 is a membrane glycoprotein on Th cells with four extracellular immunoglobulin-like (Ig-like) domains (D1-D4). It functions as a co-receptor during immune recognition between the TCR and the MHC II/peptide complex. The cytoplasmic domain binds p56lck, a protein kinase responsible for phosphorylating CD3 which is the first interaction in a cascade leading to T cell activation. We have previously reported a CD4-2 gene in rainbow trout (Oncorhynchus mykiss) which was found adjacent to the CD4-1 gene by synteny analysis. There are two subtypes (a and b) of CD4-2 in rainbow trout, with two Ig-like extracellular domains. Here we present the homologues of mammalian CD4 in Atlantic salmon (Salmo salar): CD4-1 with four extracellular domains and CD4-2a and CD4-2b with two extracellular domains. A Southern blot analysis shows two copies of the CD4-1 gene in the genomic DNA of the closely related rainbow trout. The genes for CD4-1 and CD4-2 have been sequenced and show typical traits for CD4 genes, such as the code for the first domain (D1) being divided between two exons and the other domains being largely coded for by single exons. The corresponding translated cDNAs show little (13-17%) identity to higher vertebrates and are approximately 37% similar to other translated, teleost sequences but are 89% identical to the closely related rainbow trout. However they exhibit conserved features such as the Lck binding motif in their cytoplasmic domains and the order of variable and constant type Ig-like domains. qRT-PCR data are presented describing the differential tissue expression of these genes together with other T cell markers (TCR and CD3) in several individuals.     

Altered gene expression patterns of innate and adaptive immunity pathways in transgenic rainbow trout harboring Cecropin P1 transgene

Background

We have recently developed several homozygous families of transgenic rainbow trout harbouring cecropin P1 transgene. These fish exhibit resistance characteristic to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). In our earlier studies we have reported that treatment of a rainbow trout macrophage cell line (RTS11) with a linear cationic α-helical antimicrobial peptide (e.g., cecropin B) resulted in elevated levels of expression of two pro-inflammatory relevant genes (e.g., IL-1β and COX-2). Therefore, we hypothesized that in addition to the direct antimicrobial activity of cecropin P1 in the disease resistant transgenic rainbow trout, this antimicrobial peptide may also affect the expression of immune relevant genes in the host. To confirm this hypothesis, we launched a study to determine the global gene expression profiles in three immune competent organs of cecropin P1 transgenic rainbow trout by using a 44k salmonid microarray.

Results

From the microarray data, a total of 2480 genes in the spleen, 3022 in the kidney, and 2102 in the liver were determined as differentially expressed genes (DEGs) in the cecropin P1 transgenic rainbow trout when compared to the non-transgenics. There were 478 DEGs in common among three tissues. Enrichment analyses conducted by two different bioinformatics tools revealed a tissue specific profile of functional pathway perturbation. Many of them were directly related to innate immune system such as phagocytosis, lysosomal processing, complement activation, antigen processing/presentation, and leukocyte migration. Perturbation of other biological functions that might contribute indirectly to host immunity was also observed.

Conclusions

The gene product of cecropin P1 transgene produced in the disease resistant transgenic rainbow trout not only can kill the pathogens directly but also exert multifaceted immunomodulatory properties to boost host immunity. The identified genes involved in different pathways related to immune function are valuable indicators associated with enhanced host immunity. These genes may serve as markers for selective breeding of rainbow trout or other aquaculture important fish species bearing traits of disease resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-887) contains supplementary material, which is available to authorized users.     

Transforming growth factor-β1b: A second TGF-β1 paralogue in the rainbow trout (Oncorhynchus mykiss) that has a lower constitutive expression but is more responsive to immune stimulation

The rainbow trout (Oncorhynchus mykiss) TGF-β1 sequence was one of the first fish cytokines described. Studies of its expression suggest it is constitutively expressed but displays refractory inducibility. Here we describe a second TGF-β1 (TGF-β1b) gene that is novel in several respects. TGF-β1b possesses typical TGF-β features, including a CXC motif and an integrin binding site, a tetrabasic cut site and a mature peptide of 112 amino acids (aa) containing nine conserved cysteine residues. The mature peptide is 83% identical to the first TGF-β1 sequence described in rainbow trout, that we designate TGF-β1a, and relative to TGF-β1a shows higher homology to Atlantic salmon TGF-β1b, zebrafish TGF-β1a, and sea bass and seabream TGF-β1. The gene organisation of salmonid TGF-β1b genes, as inferred from Atlantic salmon whole genome shotgun contigs, is a 6 exon/5 intron structure with exons 3 and 4 of salmonid TGF-β1a genes apparently fused together. The two trout TGF-β1 genes have a wide distribution in vivo, with highest expression found in immune tissues for both isoforms indicating that TGF-β1 has a predominant role in immunity of fish. Expression of both genes was also seen during the ontogeny of trout, with TGF-β1a relatively constant in expression level but TGF-β1b increasing over time. Immune responses in head kidney (HK) macrophages induced by pathogen associated molecular patterns (PAMPs), pro-inflammatory cytokines, mitogens and pathway activators highly elevated the expression level of TGF-β1b but not that of TGF-β1a. TGF-β1b expression was also increased by polyinosinic:polycytidylic acid (poly(I:C)) and/or lipopolysaccharide (LPS) stimulation in three different trout cell lines studied. Finally we show that TGF-β1b is potentially involved in defense against infection with viral haemorrhagic septicemia virus (VHSV), which had no effect on TGF-β1a expression. Thus, it is likely the TGF-β1b gene represents a copy which fulfils the major immune orchestrating functions of TGF-β1 as seen in other vertebrates.     

Temperature-dependent expression of immune-relevant genes in rainbow trout following Yersinia ruckeri vaccination

The gene expression of immune-relevant genes in rainbow trout Oncorhynchus mykiss following vaccination with a bacterin of Yersinia ruckeri, a bacterial pathogen causing enteric red mouth disease (ERM), was investigated at 5, 15, and 25 degrees C. Rainbow trout were immunized by i.p. injection of a water-based Y. ruckeri (serotype O1) bacterin, and gene expression profiles were compared to control groups injected with phosphate buffered saline (PBS). Blood and tissue samples (spleen and head kidney) were taken for subsequent analysis using solid phase enzyme-linked immunosorbent assay (ELISA) and real-time PCR, respectively. The up-regulation of cytokine genes was generally faster and higher at high water temperature, with major expression at 25 degrees C. The proinflammatory cytokine interleukin (IL)-1beta and interferon (IFN)-gamma were significantly up-regulated in all immunized groups, whereas the cytokine IL-10 was only up-regulated in fish kept at 15 and 25 degrees C. The gene encoding the C5a (anaphylatoxin) receptor was expressed at a significantly increased level in both head kidney and spleen of immunized fish. The secreted immunoglobulin M (IgM)-encoding gene was significantly up-regulated in the head kidney of immunized trout reared at 25 degrees C, and a positive correlation (r = 0.663) was found between gene expression of secreted IgM in the head kidney and Y. ruckeri-specific antibodies in plasma measured by ELISA. However, no regulation of the teleost specific immunoglobulin T (IgT), which was generally expressed at a much lower level than IgM, could be detected. The study indicated that expression of both innate and specific adaptive immune-response genes are highly temperature-dependent in rainbow trout.     

The beta 2-microglobulin locus of rainbow trout (Oncorhynchus mykiss) contains three polymorphic genes

Beta2-microglobulin (beta2m) associates with MHC and related class I H chains to form cell surface glycoproteins that mediate a variety of functions in defense. In humans, monomorphism of a single beta2m gene contrasts with the diversity and polymorphism of the class I H chain genes, and a similar picture was seen in almost all other species examined. In this regard, rainbow trout (Oncorhynchus mykiss) appeared unusual: trout beta2m genes gave a complicated and polymorphic pattern in Southern blots, and a minimum of 10 different mRNA encoding two distinct types of beta2m were expressed by a single fish. Characterization of genomic clones from the same fish now shows that the rainbow trout beta2m locus consists of two expressed genes and one partial gene that are closely linked. Four copies of the locus were identified and allelic variants of each gene defined, largely through comparison of the noncoding regions. A dramatic variation in the lengths of introns is caused by variable repetitive elements and accounts for the complex pattern seen in Southern blots. By comparison to noncoding sequences, the coding regions are conserved but the three loci differ within a cluster of codons that encode residues of beta2m that do not interact with class I H chains. Additional diversity in the trout beta2m genes appears to be due to somatic mutation that might be facilitated by the abundance of repetitive DNA elements within the 12 beta2m genes of an individual rainbow trout.     

Molecular characterization of the MuRF genes in rainbow trout: Potential role in muscle degradation

Muscle growth is determined primarily by the balance between protein synthesis and degradation. When rates of protein synthesis are similar between individuals, protein degradation is critical in explaining differences in growth efficiency. Studies in mammals showed that muscle atrophy results from increased protein breakdown, and is associated with activation of the ubiquitin proteasome pathway, including induction of the muscle-specific ubiquitin protein ligase, MuRF1. Animals lacking MuRF1 are resistant to muscle atrophy. In fish, little is known about the role of the proteasome/MuRF pathway in muscle degradation. The objectives of this study were to: 1) clone and characterize MuRF genes in rainbow trout; and 2) determine expression of MuRF genes in association with starvation- and vitellogenesis-induced muscle atrophy in rainbow trout. We have identified full-length cDNA sequences for three MuRF genes (MuRF1, MuRF2, and MuRF3). These genes encode proteins with typical MuRF structural domains, including a RING-finger, a B-box and a Leucine-rich coiled-coil domain. RT-PCR analysis showed that MuRF genes are predominantly expressed in muscle and heart tissues. Real time PCR analysis revealed that expression of all MuRF genes is up-regulated during starvation and MuRF3 is up-regulated in vitellogenesis-associated muscle degradation. These results suggest that MuRF genes have an important role in fish muscle protein degradation. Further studies are warranted to assess the potential use of MuRF genes as tools to monitor fish muscle growth and degradation.     

The molecular identification of factor H and factor I molecules in rainbow trout provides insights into complement C3 regulation

The complement system in vertebrates plays a crucial role in the elimination of pathogens. To regulate complement on self-tissue and to prevent spontaneous activation and systemic depletion, complement is controlled by both fluid-phase and membrane-bound inhibitors. One such inhibitor, complement factor I (CFI) regulates complement by proteolytic cleavage of components C3b and C4b in the presence of specific cofactors. Complement factor H (CFH), the main cofactor for CFI, regulates the alternative pathway of complement activation by acting in the breakdown of C3b to iC3b. To gain further insight into the origin of C3 regulation in bony fish we have cloned and characterized the CFI and CFH1 cDNAs in the rainbow trout (Oncorhynchus mykiss). In this study we report the primary sequence, the tissue expression profile, the polypeptide domain architecture and the phylogenetic analysis of trout CFI and CFH1 genes. The deduced amino acid sequences of trout CFI and CFH1 polypeptides exhibit 42% and 32% identity with human orthologs, respectively. RNA expression analysis showed that CFI is expressed differentially in trout tissues, while liver is the main source of CFH1 expression. Our data indicate that factor H and I genes have emerged during evolution as early as the divergence of teleost fish.     

Identification and expression analysis of rainbow trout <Emphasis Type="Italic">pumilio</Emphasis>-1 and <Emphasis Type="Italic">pumilio</Emphasis>-2

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.This work was in part supported by a grant from the Akiyama Foundation to E.I. Nucleotide sequence data for rainbow trout pumilio-1 and pumilio-2 have been deposited in the DDBJ/EMBL/GenBank databases.