Classical MHC class I genes composed of highly divergent sequence lineages share a single locus in rainbow trout (Oncorhynchus mykiss).

The classical MHC class I genes have been known to be highly polymorphic in various vertebrates. To date, putative allelic sequences of the classical MHC class I genes in teleost fish have been reported in several studies. However, the establishment of their allelic status has been hampered in most cases by the lack of appropriate genomic information. In the present study, using heterozygous and homozygous fish, we obtained classical-type MHC class I sequences of rainbow trout (Oncorhynchus mykiss) and investigated their allelic relationship by gene amplification and Southern and Northern hybridization analyses. The results indicated that all MHC class I sequences we obtained were derived from a single locus. Based on this, a unique polymorphic nature of the MHC class I locus of rainbow trout has been revealed. The mosaic combination of highly divergent ancient sequences in the peptide-binding domains is notable, and the variable nature around the boundary between the alpha3 and transmembrane domains is unprecedented.     

Major histocompatibility class II genes in rainbow trout (Oncorhynchus mykiss) exhibit temperature dependent downregulation

Major histocompatibility (MH) class II receptors are expressed on the surface of specialized antigen-presenting cells in vertebrate immune systems. Their function is to present peptides derived from exogenous pathogens to CD4+ T cells. Variation in the level of expression of these genes has been linked to pathogenesis in various diseases. Very little has been published on the function of MH class II receptors in teleost fish to date. In this study, we have produced polyclonal antibodies recognizing MH class II alpha and beta proteins of rainbow trout and employed them to characterize the expression pattern of these genes. Deglycosylation using N-glycosidase F and endoglycosidase H showed that MH class II alpha is glycosylated in rainbow trout. MH class II beta was also found to be glycosylated as reported previously. Results from Northern blotting revealed that the expression of these genes was not affected by exposure of rainbow trout to temperature of 5°C. However, at 2°C, downregulation of MH class II alpha and beta genes was evident at both the mRNA and protein levels as assessed by Northern and Western blotting, respectively. Because MH class II antigens play an important role in generating an immune response to bacterial and fungal pathogens, downregulation of these genes at low temperature could account for the susceptibility of fish to low temperature-related diseases such as bacterial cold-water disease and winter saprolegniosis.     

Organization and chromosomal location of the major histone cluster in brown trout,Atlantic salmon and rainbow trout

The major histone cluster (hisDNA) was mapped by fluorescent in situ hybridization (FISH) to mitotic chromosomes of Atlantic salmon, brown trout, and rainbow trout. The data reveal that in the three species hisDNA is tandemly repeated in a single locus. Southern blots of genomic DNA indicate that these clusters are representative of the vast majority of the histone genes in these species. Similar reiteration values were found among the three species. Genetic variability in the hisDNA was found only in brown trout for an EcoRI site.     

MHC class II invariant chain homologues in rainbow trout (Oncorhynchus mykiss)

The MHC class II invariant chain (Ii or CD74) in higher vertebrates is necessary for normal MHC class II loading in endosomal compartments. Detection of an Ii chain in fish would greatly support the idea that MHC class II function in fish and higher vertebrates is similar. Before this study only Ii homologues had been reported in fish that are unlikely to perform true Ii function. In the present study two Ii-like genes, Onmy-Iclp-1 and Onmy-Iclp-2, were detected in rainbow trout. Conservation of elements, particularly in Onmy-Iclp-1, suggests that the encoded proteins may be involved in MHC class II transport and peptide loading as is the Ii protein. The expression pattern of both rainbow trout genes was similar to that of the MHC class II beta chain, with strong expression in the lymphoid tissues, gills and intestine. Analysis of separated peripheral blood leucocyte fractions indicated that expression of Onmy-Iclp-1, Onmy-Iclp-2 and the MHC class II beta chain were all highest in B lymphocytes. This agrees with the expectation that the functions of the products of the new genes are closely associated with MHC class II. It is interesting why in rainbow trout there are two proteins that may function similar to Ii in higher vertebrates.     

The rainbow trout classical MHC class I molecule Onmy-UBA*501 is expressed in similar cell types as mammalian classical MHC class I molecules

Onmy-UBA is a polymorphic classical major histocompatibility (MHC) class I locus in rainbow trout (Oncorhynchus mykiss). A common allomorph is Onmy-UBA*501, which has been detected in several wildtype strains, in the clonal homozygous rainbow trout C25 and, in the current study, in the rainbow trout gonad cell line RTG-2. The extracellular domain of this allomorph was expressed in E. coli and a murine monoclonal antibody designated H9 was generated against the recombinant protein. In Western blot analysis Mab H9 specifically recognised an n-glycosylated protein of 45 kDa in leucocytes and erythrocytes of C25 fish and in RTG-2 cells. The level of Onmy-UBA*501 expression in erythrocytes was very low. Immunocytochemistry of isolated cells indicated expression in lymphocytes, macrophages, neutrophils, erythrocytes, RTG-2 cells and Onmy-UBA *501 transfected CHO cells, but not in untransfected CHO cells. Immunohistochemistry using frozen sections of C25 fish indicated that Onmy-UBA*501 expression is strong in the lymphoid organs (thymus, head kidney and spleen) and in the epithelia and endothelia of several organs. No significant expression was observed in muscle fibres, hepatocytes or neurons. These observations demonstrate that in jawed fish, the lowest phylogenetic group possessing an MHC system, the classical MHC class I molecules are expressed in similar cell types as in higher vertebrates.     

Structures of two major histocompatibility complex class I genes of the rainbow trout (Oncorhynchus mykiss)

Here we describe two rainbow trout major histocompatibility complex (MHC) class I genes characterized from lambda phage genomic clones prepared from a single fish. Clone GC71 contains all exons except a leader peptide-encoding exon. An open reading frame is maintained, and thus the gene MhcOnmy-U71 could be expressed in this individual. The class I gene found on clone GC41 lacks exons encoding the leader peptide and cytoplasmic domain. This gene, MhcOnmy-U41p, is a pseudogene due to a deletion in the alpha(2) domain-encoding exon causing premature termination. Both the Onmy-U71 and Onmy-U41p genes are distinguished by long introns between the exons encoding the alpha(1) and alpha(2) domains. Clone GC41 also contains the 3' exons of the LMP7/ PSMB8 gene encoding the gamma-interferon-induced proteosome subunit of rainbow trout.     

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.     

A novel, nonclassical MHC class I molecule specific to the common chimpanzee

All expressed human MHC class I genes (HLA-A, -B, -C, -E, -F, and -G) have functional orthologues in the MHC of the common chimpanzee (Pan troglodytes). In contrast, a nonclassical MHC class I gene discovered in the chimpanzee is not present in humans or the other African ape species. In exons and more so in introns, this Patr-AL gene is similar to the expressed A locus in the orangutan, Popy-A, suggesting they are orthologous. Patr-AL/Popy-A last shared a common ancestor with the classical MHC-A locus >20 million years ago. Population analysis revealed little Patr-AL polymorphism: just three allotypes differing only at residues 52 and 91. Patr-AL is expressed in PBMC and B cell lines, but at low level compared with classical MHC class I. The Patr-AL polypeptide is unusually basic, but its glycosylation, association with beta(2)-microglobulin, and antigenicity at the cell surface are like other MHC class I. No Patr-AL-mediated inhibition of polyclonal chimpanzee NK cells was detected. The Patr-AL gene is present in 50% of chimpanzee MHC haplotypes, correlating with presence of a 9.8-kb band in Southern blots. The flanking regions of Patr-AL contain repetitive/retroviral elements not flanking other class I genes. In sequenced HLA class I haplotypes, a similar element is present in the A*2901 haplotype but not the A*0201 or A*0301 haplotypes. This element, 6 kb downstream of A*2901, appears to be the relic of a human gene related to Patr-AL. Patr-AL has characteristics of a class I molecule of innate immunity with potential to provide common chimpanzees with responses unavailable to humans.     

Identification and Functional Prediction of Large Intergenic Noncoding RNAs (lincRNAs) in Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Long noncoding RNAs (lncRNAs) have been recognized in recent years as key regulators of diverse cellular processes. Genome-wide large-scale projects have uncovered thousands of lncRNAs in many model organisms. Large intergenic noncoding RNAs (lincRNAs) are lncRNAs that are transcribed from intergenic regions of genomes. To date, no lincRNAs in non-model teleost fish have been reported. In this report, we present the first reference catalog of 9674 rainbow trout lincRNAs based on analysis of RNA-Seq data from 15 tissues. Systematic analysis revealed that lincRNAs in rainbow trout share many characteristics with those in other mammalian species. They are shorter and lower in exon number and expression level compared with protein-coding genes. They show tissue-specific expression pattern and are typically co-expressed with their neighboring genes. Co-expression network analysis suggested that many lincRNAs are associated with immune response, muscle differentiation, and neural development. The study provides an opportunity for future experimental and computational studies to uncover the functions of lincRNAs in rainbow trout.     

Molecular cloning and characterization of cDNAs coding for apopolysialoglycoproteins in cherry salmon (Oncorhynchus masou) eggs

Recently we have cloned the cDNAs and genomic DNAs for apopolysialoglycoproteins (apoPSGPs) of Salmo gairdneri (rainbow trout) [Sorimachi, H., Emori, Y., Kawasaki, H., Kitajima, K., Inoue, S., Suzuki, K., & Inoue, Y. (1988) J. Biol. Chem. 262, 17678-17684], and the sequence analyses have indicated that the mRNAs for apoPSGPs vary in length and contain different numbers of identical 39-bp repeating units encoding the tridecapeptide (Asp-Asp-Ala-Thr-Ser-Glu-Ala-Ala-Thr-Gly-Pro-Ser-Gly) as well as highly conserved sequences encoding pre-, pro-, and telo-peptide regions. In this study we isolated cDNA clones for yamame (cherry salmon, river resident form; Oncorhynchus masou ishikawai) apoPSGP using a genomic DNA fragment for rainbow trout apoPSGP as a probe. The nucleotide sequence analyses revealed that the structures of mRNAs for yamame apoPSGP including the noncoding regions are essentially identical to those for rainbow trout, showing 90% sequence identity. Within the repeating region, 4 bp out of the 39 were replaced, producing a different tridecapeptide, Asp-Asp-Ala-Thr-Ser-Glu-Ala-Ala-Thr-Gly-Pro-Ser-Ser. This tridecapeptide is unique to yamame and common among all cDNAs obtained from yamame. Genomic Southern blot analysis showed that the yamame apoPSGP genes constituted a multiple gene family with a similar gene organization to that of rainbow trout. Oligodeoxynucleotide probes (18 bases) synthesized based on specific sequences for the yamame repeating unit hybridized only to the yamame DNA and not to the rainbow trout DNA, and vice versa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linkage arrangement of Na,K-ATPase genes in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss)

As part of our efforts to characterize Na,K-ATPase isoforms in salmonid fish, we investigated the linkage arrangement of genes coding for the alpha and beta-subunits of the enzyme complex in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss). Genetic markers were developed from four of five previously characterized alpha-subunit isoforms (alpha1b, alpha1c, alpha2 and alpha3) and four expressed sequence tags derived from yet undescribed beta-subunit isoforms (beta1a, beta1b, beta3a and beta3b). Sex-specific linkage analysis of polymorphic loci in a reference meiotic panel revealed that Na,K-ATPase genes are generally dispersed throughout the rainbow trout genome. A notable exception was the colocalization of two alpha-subunit genes and one beta-subunit gene on linkage group RT-12, which may thus share a conserved orthologous segment with linkage group 1 in zebrafish (Danio rerio). Consistent with previously reported homeologous relationships among the chromosomes of the rainbow trout, primers designed from the alpha3-isoform detected a pair of duplicated genes on linkage groups RT-27 and RT-31. Similarly, the evolutionary conservation of homeologous regions on linkage groups RT-12 and RT-16 was further supported by the map localization of gene duplicates for the beta1b isoform. The detection of homeologs within each gene family also raises the possibility that novel isoforms may be discovered as functional duplicates.     

A single major chromosomal region controls natural killer cell-like activity in rainbow trout

We report the identification of a single major chromosomal region controlling natural killer (NK) cell-like activity in rainbow trout (Oncorhynchus mykiss). A genetic map based on 484 AFLP and 39 microsatellite genotypes from 106 doubled haploid fish was constructed. These fish were produced by androgenesis from a hybrid of two clonal lines divergent in NK-like activity. NK-like activities for 75 of the doubled haploids were quantified by an in vitro chromium release assay utilizing ⁵¹Cr-labeled YAC-1 target cells. Composite interval mapping revealed a single major quantitative trait locus (QTL) associated with NK-like activity in this rainbow trout model. Genetic mapping revealed this QTL to also be unlinked to: fragmented MHC class I and MHC class II regions, the leukocyte receptor cluster, the natural killer cell enhancement factor (NKEF) gene, the RAG-1 gene, and two QTL associated with resistance to infectious pancreatic necrosis virus in rainbow trout. Collectively, these results extend the utility of rainbow trout as an immunological model and are consistent with the idea that a single chromosomal region homologous to the natural killer cell complex (NKC) located on syntenic portions of mouse chromosome (Chr) 6, human Chr 12, and rat Chr 4 may exist in a lower vertebrate model.     

Chromosome mapping of MHC class I in rainbow trout (Oncorhynchus mykiss)

The major histocompatibility complex (MHC) is well-studied in mammals. Much research has addressed the genomic organisation of MHC genes and it is well established that human MHC class I genes are located on chromosome 6. However, information on the organisation of the MHC complex in rainbow trout is only beginning to become available. In the present study it was determined that rainbow trout MHC class I sequences are located on chromosome 18. This is the first reported use of fluorescence in situ hybridisation (FISH) to identify the chromosomal location of genes involved in the immune system of fish.     

Expression, linkage, and polymorphism of MHC-related genes in rainbow trout, Oncorhynchus mykiss.

The architecture of the MHC in teleost fish, which display a lack of linkage between class I and II genes, differs from all other vertebrates. Because rainbow trout have been examined for a variety of immunologically relevant genes, they present a good teleost model for examining both the expression and organization of MHC-related genes. Full-length cDNA and partial gDNA clones for proteasome delta, low molecular mass polypeptide (LMP) 2, TAP1, TAP2A, TAP2B, class Ia, and class IIB were isolated for this study. Aside from the expected polymorphisms associated with class I genes, LMP2 and TAP2 are polygenic. More specifically, we found a unique lineage of LMP2 (LMP2/delta) that shares identity to both LMP2 and delta but is expressed like the standard LMP2. Additionally, two very different TAP2 loci were found, one of which encodes polymorphic alleles. In general, the class I pathway genes are expressed in most tissues, with highest levels in lymphoid tissue. We then analyzed the basic genomic organization of the trout MHC in an isogenic backcross. The main class Ia region does not cosegregate with the class IIB locus, but LMP2, LMP2/delta, TAP1A, and TAP2B are linked to the class Ia locus. Interestingly, TAP2A (second TAP2 locus) is a unique lineage in sequence composition that appears not to be linked to this cluster or to class IIB. These results support and extend the recent findings of nonlinkage between class I and II in a different teleost order (cyprinids), suggesting that this unique arrangement is common to all teleosts.     

Mx mRNA expression and RFLP analysis of rainbow trout Oncorhynchus mykiss genetic crosses selected for susceptibility or resistance to IHNV

Three interferon-inducible Mx genes have been identified in rainbow trout Oncorhynchus mykiss and their roles in virus resistance have yet to be determined. In mice, expression of the Mx1 protein is associated with resistance to influenza virus. We report a study to determine whether there was a correlation between the expression of Mx in rainbow trout and resistance to a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A comparison of Mx mRNA expression was made between different families of cultured rainbow trout selected for resistance or for susceptibility to IHNV. A trout-specific Mx cDNA gene probe was used to determine whether there was a correlation between Mx mRNA expression and resistance to the lethal effects of IHNV infection. Approximately 99% of trout injected with a highly virulent strain of the fish rhabdovirus, IHNV, were able to express full length Mx mRNA at 48 h post infection. This is markedly different from the expression of truncated, non-functional Mx mRNA found in most laboratory strains of mice, and the ability of only 25% of wild mice to express functional Mx protein. A restriction fragment length polymorphism (RFLP) assay was developed to compare the Mx locus between individual fish and between rainbow trout genetic crosses bred for IHNV resistance or susceptibility. The assay was able to discriminate 7 distinct RFLP patterns in the rainbow trout crosses. One cross was identified that showed a correlation between homozygosity at the Mx locus and greater susceptibility to IHN-caused mortality.