Comprehensive analysis of MHC class I genes from the U-, S-, and Z-lineages in Atlantic salmon

<p>Abstract</p> <p>Background</p> <p>We have previously sequenced more than 500 kb of the duplicated MHC class I regions in Atlantic salmon. In the IA region we identified the loci for the MHC class I gene <it>Sasa-UBA </it>in addition to a soluble MHC class I molecule, <it>Sasa-ULA</it>. A pseudolocus for <it>Sasa-UCA </it>was identified in the nonclassical IB region. Both regions contained genes for antigen presentation, as wells as orthologues to other genes residing in the human MHC region.</p> <p>Results</p> <p>The genomic localisation of two MHC class I lineages (Z and S) has been resolved. 7 BACs were sequenced using a combination of standard Sanger and 454 sequencing. The new sequence data extended the IA region with 150 kb identifying the location of one Z-lineage locus, <it>ZAA</it>. The IB region was extended with 350 kb including three new Z-lineage loci, <it>ZBA</it>, <it>ZCA </it>and <it>ZDA </it>in addition to a <it>UGA </it>locus. An allelic version of the IB region contained a functional <it>UDA </it>locus in addition to the <it>UCA </it>pseudolocus. Additionally a BAC harbouring two MHC class I genes (UHA) was placed on linkage group 14, while a BAC containing the S-lineage locus <it>SAA </it>(previously known as <it>UAA</it>) was placed on LG10. Gene expression studies showed limited expression range for all class I genes with exception of <it>UBA </it>being dominantly expressed in gut, spleen and gills, and <it>ZAA </it>with high expression in blood.</p> <p>Conclusion</p> <p>Here we describe the genomic organization of MHC class I loci from the U-, Z-, and S-lineages in Atlantic salmon. Nine of the described class I genes are located in the extension of the duplicated IA and IB regions, while three class I genes are found on two separate linkage groups. The gene organization of the two regions indicates that the IB region is evolving at a different pace than the IA region. Expression profiling, polymorphic content, peptide binding properties and phylogenetic relationship show that Atlantic salmon has only one MHC class Ia gene (<it>UBA</it>), in addition to a multitude of nonclassical MHC class I genes from the U-, S- and Z-lineages.</p

Comparative genomic analysis of Atlantic salmon, Salmo salar, from Europe and North America

Background: Several lines of evidence including allozyme analysis, restriction digest patterns and sequencing ofmtDNA as well as mini- and micro-satellite allele frequencies indicate that Atlantic salmon (Salmo salar) from NorthAmerica and Europe are genetically distinct. These observations are supported by karyotype analysis, whichrevealed that North American Atlantic salmon have 27 pairs of chromosomes whereas European salmon have 29pairs. We set out to construct a linkage map for a North American Atlantic salmon family and to compare this mapwith the well developed map for European Atlantic salmon.Results: We used microsatellite markers, which had previously been mapped in the two Atlantic salmon SALMAPmapping families from the River Tay, Scotland, to carry out linkage analysis in an Atlantic salmon family (NB1)whose parents were derived from the Saint John River stock in New Brunswick, Canada. As large differences inrecombination rates between female and male Atlantic salmon have been noted, separate genetic maps wereconstructed for each sex. The female linkage map comprises 218 markers in 37 linkage groups while the male maphas 226 markers in 28 linkage groups. We combined 280 markers from the female and male maps into 27composite linkage groups, which correspond to the haploid number of chromosomes in Atlantic salmon from theWestern Atlantic.Conclusions: A comparison of the composite NB1 and SALMAP linkage maps revealed the reason for thedifference in the chromosome numbers between European and North American Atlantic salmon: Linkage groupsAS-4 and AS-32 in the Scottish salmon, which correspond to chromosomes Ssa-6 and Ssa-22, are combined into asingle NB1 linkage group as are linkage groups AS-21 and AS-33 (corresponding to chromosomes Ssa-26 and Ssa-28). The comparison of the linkage maps also suggested some additional chromosomal rearrangements, but it willrequire finer mapping, potentially using SNPs, to test these predictions. Our results provide the first comparison ofthe genomic architecture of Atlantic salmon from North America and Europe with respect to chromosomeorganization

Detection of N-Glycolyl GM3 Ganglioside in Neuroectodermal Tumors by Immunohistochemistry: An Attractive Vaccine Target for Aggressive Pediatric Cancer

The N-glycolylated ganglioside NeuGc-GM3 has been described in solid tumors such as breast carcinoma, nonsmall cell lung cancer, and melanoma, but is usually not detected in normal human cells. Our aim was to evaluate the presence of NeuGc-GM3 in pediatric neuroectodermal tumors by immunohistochemistry. Twenty-seven archival cases of neuroblastoma and Ewing sarcoma family of tumors (ESFT) were analyzed. Formalin-fixed, paraffin-embedded tumor samples were cut into 5 μm sections. The monoclonal antibody 14F7, a mouse IgG1 that specifically recognizes NeuGc-GM3, and a peroxidase-labeled polymer conjugated to secondary antibodies were used. Presence of NeuGc-GM3 was evident in 23 of 27 cases (85%), with an average of about 70% of positive tumors cells. Immunoreactivity was moderate to intense in most tumors, showing a diffuse cytoplasmic and membranous staining, although cases of ESFT demonstrated a fine granular cytoplasmic pattern. No significant differences were observed between neuroblastoma with and without NMYC oncogene amplification, suggesting that expression of NeuGc-GM3 is preserved in more aggressive cancers. Until now, the expression of N-glycolylated gangliosides in pediatric neuroectodermal tumors has not been investigated. The present study evidenced the expression of NeuGc-GM3 in a high proportion of neuroectodermal tumors, suggesting its potential utility as a specific target of immunotherapy

Isolation, characterization and comparison of Atlantic and Chinook salmon growth hormone 1 and 2

<p>Abstract</p> <p>Background</p> <p>Growth hormone (GH) is an important regulator of skeletal growth, as well as other adapted processes in salmonids. The GH gene (<it>gh</it>) in salmonids is represented by duplicated, non-allelic isoforms designated as <it>gh1 </it>and <it>gh2</it>. We have isolated and characterized <it>gh</it>-containing bacterial artificial chromosomes (BACs) of both Atlantic and Chinook salmon (<it>Salmo salar </it>and <it>Oncorhynchus tshawytscha</it>) in order to further elucidate our understanding of the conservation and regulation of these loci.</p> <p>Results</p> <p>BACs containing <it>gh1 </it>and <it>gh2 </it>from both Atlantic and Chinook salmon were assembled, annotated, and compared to each other in their coding, intronic, regulatory, and flanking regions. These BACs also contain the genes for skeletal muscle sodium channel oriented in the same direction. The sequences of the genes for interferon alpha-1, myosin alkali light chain and microtubule associated protein Tau were also identified, and found in opposite orientations relative to <it>gh1 </it>and <it>gh2</it>. Viability of each of these genes was examined by PCR. We show that transposon insertions have occurred differently in the promoters of <it>gh</it>, within and between each species. Other differences within the promoters and intronic and 3'-flanking regions of the four <it>gh </it>genes provide evidence that they have distinct regulatory modes and possibly act to function differently and/or during different times of salmonid development.</p> <p>Conclusion</p> <p>A core proximal promoter for transcription of both <it>gh1 </it>and <it>gh2 </it>is conserved between the two species of salmon. Nevertheless, transposon integration and regulatory element differences do exist between the promoters of <it>gh1 </it>and <it>gh2</it>. Additionally, organization of transposon families into the BACs containing <it>gh1 </it>and for the BACs containing <it>gh2</it>, are very similar within orthologous regions, but much less clear conservation is apparent in comparisons between the <it>gh1</it>- and <it>gh2</it>-containing paralogous BACs for the two fish species. This is consistent with the hypothesis that a burst of transposition activity occurred during the speciation events which led to Atlantic and Pacific salmon. The Chinook and other <it>Oncorhynchus </it>GH1s are strikingly different in comparison to the other GHs and this change is not apparent in the surrounding non-coding sequences.</p

Distribution of ancestral proto-Actinopterygian chromosome arms within the genomes of 4R-derivative salmonid fishes (Rainbow trout and Atlantic salmon)

Comparative genomic studies suggest that the modern day assemblage of ray-finned fishes have descended from an ancestral grouping of fishes that possessed 12-13 linkage groups. All jawed vertebrates are postulated to have experienced two whole genome duplications (WGD) in their ancestry (2R duplication). Salmonids have experienced one additional WGD (4R duplication event) compared to most extant teleosts which underwent a further 3R WGD compared to other vertebrates. We describe the organization of the 4R chromosomal segments of the proto-rayfinned fish karyotype in Atlantic salmon and rainbow trout based upon their comparative syntenies with two model species of 3R ray-finned fishes. Results: Evidence is presented for the retention of large whole-arm affinities between the ancestral linkage groups of the ray-finned fishes, and the 50 homeologous chromosomal segments in Atlantic salmon and rainbow trout. In the comparisons between the two salmonid species, there is also evidence for the retention of large whole-arm homeologous affinities that are associated with the retention of duplicated markers. Five of the 7 pairs of chromosomal arm regions expressing the highest level of duplicate gene expression in rainbow trout share homologous synteny to the 5 pairs of homeologs with the greatest duplicate gene expression in Atlantic salmon. These regions are derived from proto-Actinopterygian linkage groups B, C, E, J and K. Conclusion: Two chromosome arms in Danio rerio and Oryzias latipes (descendants of the 3R duplication) can, in most instances be related to at least 4 whole or partial chromosomal arms in the salmonid species. Multiple arm assignments in the two salmonid species do not clearly support a 13 proto-linkage group model, and suggest that a 12 proto-linkage group arrangement (i.e., a separate single chromosome duplication and ancestral fusion/fissions/recombination within the putative G/H/I groupings) may have occurred in the more basal soft-rayed fishes. We also found evidence supporting the model that ancestral linkage group M underwent a single chromosome duplication following the 3R duplication. In the salmonids, the M ancestral linkage groups are localized to 5 whole arm, and 3 partial arm regions (i.e., 6 whole arm regions expected). Thus, 3 distinct ancestral linkage groups are postulated to have existed in the G/H and M lineage chromosomes in the ancestor of the salmonids

Assessing the feasibility of GS FLX Pyrosequencing for sequencing the Atlantic salmon genome

<p>Abstract</p> <p>Background</p> <p>With a whole genome duplication event and wealth of biological data, salmonids are excellent model organisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processes associated with complex behavioral phenotypes. It is surprising therefore, that no salmonid genome has been sequenced. Atlantic salmon (<it>Salmo salar</it>) is a good representative salmonid for sequencing given its importance in aquaculture and the genomic resources available. However, the size and complexity of the genome combined with the lack of a sequenced reference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sanger sequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility of using the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eight pooled BACs belonging to a minimum tiling path covering ~1 Mb of the Atlantic salmon genome were sequenced by GS FLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of a shotgun library.</p> <p>Results</p> <p>An initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with ~30× coverage allowed gene identification, but was incomplete even when 126 Sanger-generated BAC-end sequences (~0.09× coverage) were incorporated. The addition of paired end sequencing reads (additional ~26× coverage) produced a final assembly comprising 175 contigs assembled into four scaffolds with 171 gaps. Sanger sequencing of the ninth BAC (~10.5× coverage) produced nine contigs and two scaffolds. The number of scaffolds produced by the GS FLX assembly was comparable to Sanger-generated sequencing; however, the number of gaps was much higher in the GS FLX assembly.</p> <p>Conclusion</p> <p>These results represent the first use of GS FLX paired end reads for <it>de novo </it>sequence assembly. Our data demonstrated that this improved the GS FLX assemblies; however, with respect to <it>de novo </it>sequencing of complex genomes, the GS FLX technology is limited to gene mining and establishing a set of ordered sequence contigs. Currently, for a salmonid reference sequence, it appears that a substantial portion of sequencing should be done using Sanger technology.</p

Dusty core disease (DuCD): expanding morphological spectrum of RYR1 recessive myopathies

Several morphological phenotypes have been associated to RYR1-recessive myopathies. We recharacterized the RYR1-recessive morphological spectrum by a large monocentric study performed on 54 muscle biopsies from a large cohort of 48 genetically confirmed patients, using histoenzymology, immunohistochemistry, and ultrastructural studies. We also analysed the level of RyR1 expression in patients' muscle biopsies. We defined "dusty cores" the irregular areas of myofibrillar disorganisation characterised by a reddish-purple granular material deposition with uneven oxidative stain and devoid of ATPase activity, which represent the characteristic lesion in muscle biopsy in 54% of patients. We named Dusty Core Disease (DuCD) the corresponding entity of congenital myopathy. Dusty cores had peculiar histological and ultrastructural characteristics compared to the other core diseases. DuCD muscle biopsies also showed nuclear centralization and type1 fibre predominance. Dusty cores were not observed in other core myopathies and centronuclear myopathies. The other morphological groups in our cohort of patients were: Central Core (CCD: 21%), Core-Rod (C&amp;R:15%) and Type1 predominance "plus" (T1P+:10%). DuCD group was associated to an earlier disease onset, a more severe clinical phenotype and a lowest level of RyR1 expression in muscle, compared to the other groups. Variants located in the bridge solenoid and the pore domains were more frequent in DuCD patients. In conclusion, DuCD is the most frequent histopathological presentation of RYR1-recessive myopathies. Dusty cores represent the unifying morphological lesion among the DuCD pathology spectrum and are the morphological hallmark for the recessive form of disease

Genomic Organization and Evolution of the Vomeronasal Type 2 Receptor-Like (OlfC) Gene Clusters in Atlantic Salmon, Salmo salar

There are three major multigene superfamilies of olfactory receptors (OR, V1R, and V2R) in mammals. The ORs are expressed in the main olfactory organ, whereas the V1Rs and V2Rs are located in the vomeronasal organ. Fish only possess one olfactory organ in each nasal cavity, the olfactory rosette; therefore, it has been proposed that their V2R-like genes be classified as olfactory C family G protein-coupled receptors (OlfC). There are large variations in the sizes of OR gene repertoires. Previous studies have shown that fish have between 12 and 46 functional V2R-like genes, whereas humans have lost all functional V2Rs, and frog sp. have more than 240. Pseudogenization of V2R genes is a prevalent event across species. In the mouse and frog genomes, there are approximately double the number of pseudogenes compared with functional genes. An oligonucleotide probe was designed from a conserved sequence from four Atlantic salmon OlfC genes and used to screen the Atlantic salmon bacterial artificial chromosome (BAC) library. Hybridization-positive BACs were matched to fingerprint contigs, and representative BACs were shotgun cloned and sequenced. We identified 55 OlfC genes. Twenty-nine of the OlfC genes are classified as putatively functional genes and 26 as pseudogenes. The OlfC genes are found in two genomic clusters on chromosomes 9 and 20. Phylogenetic analysis revealed that the OlfC genes could be divided into 10 subfamilies, with nine of these subfamilies corresponding to subfamilies found in other teleosts and one being salmon specific. There is also a large expansion in the number of OlfC genes in one subfamily in Atlantic salmon. Subfamily gene expansions have been identified in other teleosts, and these differences in gene number reflect species-specific evolutionary requirements for olfaction. Total RNA was isolated from the olfactory epithelium and other tissues from a presmolt to examine the expression of the odorant genes. Several of the putative OlfC genes that we identified are expressed only in the olfactory epithelium, consistent with these genes encoding odorant receptors

Pathogenic Variants in the Myosin Chaperone UNC-45B Cause Progressive Myopathy with Eccentric Cores

The myosin-directed chaperone UNC-45B is essential for sarcomeric organization and muscle function from Caenorhabditis elegans to humans. The pathological impact of UNC-45B in muscle disease remained elusive. We report ten individuals with bi-allelic variants in UNC45B who exhibit childhood-onset progressive muscle weakness. We identified a common UNC45B variant that acts as a complex hypomorph splice variant. Purified UNC-45B mutants showed changes in folding and solubility. In situ localization studies further demonstrated reduced expression of mutant UNC-45B in muscle combined with abnormal localization away from the A-band towards the Z-disk of the sarcomere. The physiological relevance of these observations was investigated in C. elegans by transgenic expression of conserved UNC-45 missense variants, which showed impaired myosin binding for one and defective muscle function for three. Together, our results demonstrate that UNC-45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown conserved role of UNC-45B in myofibrillar organization

Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire

<p>Abstract</p> <p>Background</p> <p>The genomes of salmonids are considered pseudo-tetraploid undergoing reversion to a stable diploid state. Given the genome duplication and extensive biological data available for salmonids, they are excellent model organisms for studying comparative genomics, evolutionary processes, fates of duplicated genes and the genetic and physiological processes associated with complex behavioral phenotypes. The evolution of the tetrapod hemoglobin genes is well studied; however, little is known about the genomic organization and evolution of teleost hemoglobin genes, particularly those of salmonids. The Atlantic salmon serves as a representative salmonid species for genomics studies. Given the well documented role of hemoglobin in adaptation to varied environmental conditions as well as its use as a model protein for evolutionary analyses, an understanding of the genomic structure and organization of the Atlantic salmon α and β hemoglobin genes is of great interest.</p> <p>Results</p> <p>We identified four bacterial artificial chromosomes (BACs) comprising two hemoglobin gene clusters spanning the entire α and β hemoglobin gene repertoire of the Atlantic salmon genome. Their chromosomal locations were established using fluorescence <it>in situ </it>hybridization (FISH) analysis and linkage mapping, demonstrating that the two clusters are located on separate chromosomes. The BACs were sequenced and assembled into scaffolds, which were annotated for putatively functional and pseudogenized hemoglobin-like genes. This revealed that the tail-to-tail organization and alternating pattern of the α and β hemoglobin genes are well conserved in both clusters, as well as that the Atlantic salmon genome houses substantially more hemoglobin genes, including non-Bohr β globin genes, than the genomes of other teleosts that have been sequenced.</p> <p>Conclusions</p> <p>We suggest that the most parsimonious evolutionary path leading to the present organization of the Atlantic salmon hemoglobin genes involves the loss of a single hemoglobin gene cluster after the whole genome duplication (WGD) at the base of the teleost radiation but prior to the salmonid-specific WGD, which then produced the duplicated copies seen today. We also propose that the relatively high number of hemoglobin genes as well as the presence of non-Bohr β hemoglobin genes may be due to the dynamic life history of salmon and the diverse environmental conditions that the species encounters.</p> <p>Data deposition: BACs S0155C07 and S0079J05 (fps135): GenBank <ext-link ext-link-id="GQ898924" ext-link-type="gen">GQ898924</ext-link>; BACs S0055H05 and S0014B03 (fps1046): GenBank <ext-link ext-link-id="GQ898925" ext-link-type="gen">GQ898925</ext-link></p