A putative beta2-adrenoceptor from the rainbow trout (Oncorhynuchus mykiss). Molecular characterization and pharmacology.

Extensive molecular characterization of mammalian beta-adrenoceptors has revealed complex modes of regulation and interaction. Relatively little attention, however, has focused on adrenoceptors from early branching vertebrates such as fish. Using an RT-PCR approach we have cloned a rainbow trout beta2-adrenoceptor gene that codes for a 409-amino-acid protein with the same seven transmembrane domain structure as its mammalian counterparts. This rainbow trout beta2-adrenoceptor shares a high degree of amino-acid sequence conservation with other vertebrate beta2-adrenoceptors. The conclusion that this sequence is a rainbow trout beta2-adrenoceptor is further supported by phylogenetic analysis of vertebrate beta-adrenoceptor sequences and competitive pharmacological binding data. RNase protection assays demonstrate that the rainbow trout beta2-adrenoceptor gene is highly expressed in the liver and red and white muscle, with lower levels of expression in the gills, heart, kidney and spleen of the rainbow trout. The lack of regulatory phosphorylation sites within the G-protein-binding domain of the rainbow trout beta2-adrenoceptor sequence suggests that the in vivo control of trout beta2-adrenoceptor signaling differs substantially from that of mammals.     

Identification and molecular characterization of the rainbow trout calpains (Capn1 and Capn2): their expression in muscle wasting during starvation

Calpains are calcium regulated proteases involved in cellular functions that include muscle proteolysis both ante- and postmortem. Here, we describe the molecular characterization of the rainbow trout catalytic subunits of the mu- and m-calpains, respectively. The cDNA sequence for Capn1 encodes a protein of 704 amino acids with a calculated molecular mass of 79.9 kDa. The amino acid sequence shows 66% and 86% identity with the mouse and zebrafish Capn1, respectively. The Capn2 cDNA codes for a protein consisting of 701 amino acid residues with a calculated molecular mass of 78.2 kDa. The protein shows 65% amino acid sequence identity with the mouse and chicken Capn2. The two isozymes of rainbow trout have the characteristic domains: I (propeptide), II (cysteine catalytic site), III (electrostatic switch), and IV (contains five EF-hands). Because starvation induces muscle wasting, the hypothesis of this study was that starvation could affect regulation of the calpain system in muscle. Starvation of rainbow trout fingerlings (15-20 g) for 35 days stimulated the expression of Capn1 (2.2-fold increase, P < 0.01), Capn2 (6.0-fold increase, P < 0.01), and calpastatins (1.6-fold increase, P < 0.05) as measured by quantitative real-time RT-PCR. The mRNA changes led to a 1.23-fold increase in the calpain catalytic activity. The results suggest a potential role of calpains in protein mobilization as a source of energy under fasting condition.     

Cloning and functional expression of a cDNA encoding a metabolic acyl-CoA delta 9-desaturase of the cabbage looper moth, Trichoplusia ni.

Acyl-CoA delta 9-desaturases play essential roles in fatty acid metabolism and the regulation of cell membrane fluidity. In this research, a cDNA sequence was obtained from Trichoplusia ni adult fat body mRNA by using RT-PCR with degenerate primers based on other characterized delta 9-desaturase sequences. The remainder of the sequence was amplified using 3'- and 5'-RACE. A 1439 bp cDNA reconstructed from three overlapping PCR products contains an ORF encoding a 353-amino acids (aa) protein that shows clear homology (greater than 50% aa identity and greater than 65% aa similarity to characterized insect and vertebrate desaturases). The ORF of this cDNA was subcloned into an expression vector, which relieved the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient yeast strain following genetic transformation. The newly characterized desaturase from T. ni produced fatty acids delta 9-16 and delta 9-18 in a 1:6 ratio, compared to a 5:1 ratio, respectively, with the yeast delta 9 desaturase. A Northern blot hybridization and a RT-PCR experiment showed that temporal and tissue-specific patterns of expression of the corresponding mRNA are distinct from those of the delta 11-desaturase mRNA present in the pheromone glands of adult females. Based on its homology to other desaturases, the widespread distribution of its corresponding mRNA in various tissues, and its functional assay, we conclude that this cDNA encodes the apoprotein corresponding to the desaturase component of the metabolic delta 9-desaturase complex of T. ni.     

Cloning of a cDNA Coding for Active Tyrosine Hydroxylase in the Rainbow Trout (Oncorhynchus mykiss): Comparison with Other Hydroxylases and Enzymatic Expression

Abstract: Although catecholamines are of critical importance for neuroendocrine function in teleost fishes, there has been no tool to give access to pretranslational regulation of their synthesis enzymes. In this study, we undertook cloning of the cDNA coding for the tyrosine hydroxylase (TH) of the rainbow trout ( Oncorhynchus mykiss ). First, we looked for a tissue sufficiently rich in TH to make an expression library. The cDNA coding for the rainbow trout TH (rtTH) was then cloned and sequenced. The rtTH sequence encodes a protein of 489 amino acids. Several domains and amino acids required for enzyme activity, like cysteines or phosphorylation sites, are highly conserved between species. Northern blot analysis showed a single rtTH messenger RNA of 4.2 kb expressed in the anteroventral brain. The ability of rtTH to hydroxylate l -tyrosine was analyzed by transient expression of the rtTH cDNA in COS-1 cells. In vitro TH activity, using COS-1 cell extracts, demonstrated that TH activity in transfected cells was 40-fold higher than in untransfected cells. Western blot analysis revealed a single protein of ∼65 kDa in both COS-1 cells and in trout brain. This rtTH cDNA provides us with a tool for further studies on pretranslational regulation of the enzyme in salmonids.     

Characterization of a rainbow trout matrix metalloproteinase capable of degrading type I collagen.

Matrix metalloproteinases (MMPs) are widely distributed in vertebrate tissues and form a large family consisting of at least four distinct subfamilies. Higher vertebrate MMP-13 is well-known as collagenase-3, which represents the third member of a collagenase subfamily. In this study, we cloned cDNA coding for a unique fish homologue of human MMP-13 from a rainbow trout fibroblast cDNA library. The cDNA was 2.1 kb long and contained an open reading frame encoding a protein of 475 amino acids. The catalytic domain of the protein was 66% identical to the human counterpart with the greatest degree of identity occurring in the zinc binding site. In addition, it possessed three amino-acid residues (Tyr122, Asp233 and Gly235) characteristic of the collagenase subfamily, together with a six residue insertion which did not occur in the collagenase subfamily. Then the isolated cDNA was expressed in Escherichia coli and the recombinant protein was found to degrade gelatin and skin type I collagen. It is worth noting that rainbow trout type I collagen was more susceptible to proteolysis with the recombinant protein when compared with the calf one. It appeared that the recombinant protein also cleaved the nonhelical regions of rainbow trout muscle type V collagen. These results have revealed that the cDNA encodes a unique MMP-13 of rainbow trout. This is the first report of cDNA coding for fish MMP capable of degrading type I collagen.     

A CXC chemokine sequence isolated from the rainbow trout Oncorhynchus mykiss resembles the closely related interferon-gamma-inducible chemokines CXCL9, CXCL10 and CXCL11

A sequence encoding a CXC - type chemokine from rainbow trout was found to most resemble members of the CXCL9/CXCL10/CXCL11 sub-family. In mammals, all 3 chemokines are regulated by IFN-gamma and are chemotactic for activated T lymphocytes. The trout chemokine (gammaIP1), with a message of 787 nucleotides, contains 100 amino acids in a typical non-ELR CXC chemokine arrangement. A second sequence (gammaIP2), with 6 nucleotide differences in the coding region when compared to the first, was also identified although it is not known whether this is a second functional gene or a second allele. The gene is separated onto 4 exons, and the introns intervene in conserved positions according to the mammalian equivalents. The sequence encoded by the second exon shares the highest amino acid identity (37%) with CXCL10, with lower values of identity to other CXC chemokines (17-31%). Furthermore, phylogenetic analysis groups the trout chemokine with mammalian CXCL9, CXCL10 and CXCL11 peptides. Constitutive expression of gammaIP is seen in trout gill and low level expression in spleen, head kidney and liver. In RTS-11 cells, gammaIP expression can be induced with poly I:C, but not by LPS, suggesting virus-mediated regulation of gammaIP. Intraperitoneal injection of recombinant trout TNF-alpha caused elevation in gammaIP mRNA levels in trout head kidney.     

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.     

Comparative physiology and molecular analysis of carbonic anhydrase from the red blood cells of teleost fish

This study investigates the early evolution of vertebrate red blood cell (rbc) carbonic anhydrase (CA) by examining the physiological and molecular properties of rbc CA in teleost fish. When representatives of four different families of teleosts were compared, it was found that differences in overall rbc CA activity were due to different concentrations of CA, rather than differences in the enzymes kinetic properties. Additional molecular analysis of CA from the rbcs of rainbow trout provided further evidence that critical elements of the enzyme, such as the active site, have been highly conserved during vertebrate evolution. The active site of the trout CA differed from that of gar rbc CA at only two amino acid positions. The rainbow trout rbc CA sequence also showed high sequence homology with CA sequences from other fish tissues, and fits into an emerging group of fish CAs that are basal to mammalian CA I, II and III. Northern blot analysis of the tissue expression of the sequenced CA indicated that it is primarily found in the rbcs, but high amounts of cytosolic CA activity were also found in the gill, suggesting the presence of other cytosolic CA isozymes in this species.Abbreviations Az acetazolamide - CA carbonic anhydrase - MP maximum parsimony - NJ neighbour joining - RACE rapid amplification of cDNA ends - rbc red blood cellCommunicated by L.C.-H. Wang     

The vitronectin gene in rainbow trout: cloning, expression and phylogenetic analysis

Vitronectin is a major cell adhesion glycoprotein that is found in plasma and the extracellular matrix. Vitronectin consists of an N-terminal somatomedin B domain and two hemopexin-like domains and controls functions including cell adhesion, migration, haemostasis and immune defence. In order to study the molecular evolution of the complement lytic pathway regulation, we have cloned and characterized the vitronectin gene from rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequence of trout vitronectin exhibits 45%, 46%, 47% and 63% identity with human, chicken, Xenopus and zebrafish orthologs, respectively. The domain architecture of the trout vitronectin, consisting of a somatomedin B domain and two hemopexin-like domains, resembles that of mammalian vitronectins. Analysis of partial genomic clones shows that trout vitronectin gene exhibits the same exon-intron organization profile as the human ortholog gene. The trout vitronectin gene is probably present as a single copy in the trout genome, showing a differential expression pattern among tissues investigated.     

Molecular cloning and sequencing of coho salmon growth hormone cDNA

A cDNA library was constructed using mRNA isolated from coho salmon pituitaries. By employing rainbow trout growth hormone cDNA as a probe, the coho salmon cDNA was isolated and the complete nucleotide (nt) sequence determined. The coding region contains 630 nt while the 5'- and 3'-untranslated regions are 64 and 489 nt in length, respectively. Comparison of the noncoding regions of coho and chum salmon cDNAs reveal identity at the 5' end but significant variation in the 3' end. Chum salmon and rainbow trout have identical amino acid (aa) sequences, but coho salmon growth hormone has a sequence that differs by 6 of the 188 predicted aa. Since salmonids are tetraploid, this difference may be the result of either divergence of the same growth hormone locus or of variation between different loci. Comparisons of the cDNA restriction maps of these three fish species suggest the former possibility.     

Cloning and characterisation of tandem-repeat type galectin in rainbow trout (Oncorhynchus mykiss)

Fish beta-galactoside binding lectin (galectin) cDNA was cloned from the cDNA library of rainbow trout (Oncorhynchus mykiss) head kidney. The clone contained a single open reading frame encoding 341 amino acids (aa) (38 kDa protein), including the initiator methionine. Significant sequence homology to mammalian galectin-9 (40-55% identity) was observed. Its amino acid sequence showed two distinct N- and C-terminal domains (148 and 130 aa, respectively) connected by a peptide linker (63 aa). The galectin contains two consensus WG-E-R/K motifs thought to play an essential role in sugar-binding, indicating that this lectin is a member of the tandem-repeat type galectins which have not been identified in fish. The 1.6 kDa mRNA of the lectin was found by Northern blot analyses to be widely expressed in the spleen, head kidney, thymus, peritoneal exudate cells, ovary, gills and heart. Southern blot analyses with the probe for C-terminal of the lectin showed the existence of two hybridising genes. These results suggest that rainbow trout has at least one tandem-repeat type galectin as well as proto-type galectin.