RESUMEN
BACKGROUND: The Atlantic salmon (Salmo salar) immunoglobulin heavy chain (IgH) locus possesses two parallel IgH isoloci (IGH-A and IGH-B), that are related to the genomic duplication event in the family Salmonidae. These duplicated IgH loci in Atlantic salmon provide a unique opportunity to examine the mechanisms of genome diversity and genome evolution of the IgH loci in vertebrates. In this study, we defined the structure of these loci in Atlantic salmon, and sequenced 24 bacterial artificial chromosome (BAC) clones that were assembled into the IGH-A (1.1 Mb) and IGH-B (0.9 Mb) loci. In addition, over 7,000 cDNA clones from the IgH variable (VH) region have been sequenced and analyzed. RESULTS: The present study shows that the genomic organization of the duplicated IgH loci in Atlantic salmon differs from that in other teleosts and other vertebrates. The loci possess multiple Cτ genes upstream of the Cµ region, with three of the Cτ genes being functional. Moreover, the duplicated loci possess over 300 VH segments which could be classified into 18 families. This is the largest number of VH families currently defined in any vertebrate. There were significant structural differences between the two loci, indicating that both IGH-A and -B loci have evolved independently in the short time after the recent genome duplication approximately 60 mya. CONCLUSIONS: Our results indicate that the duplication of the IgH loci in Atlantic salmon significantly contributes to the increased diversity of the antibody repertoire, as compared with the single IgH locus in other vertebrates.
Asunto(s)
Evolución Molecular , Duplicación de Gen/genética , Genes de las Cadenas Pesadas de las Inmunoglobulinas/genética , Sitios Genéticos/genética , Salmo salar/genética , Animales , Océano Atlántico , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Reordenamiento Génico de Cadena Pesada de Linfocito B/genética , Variación Genética , Regiones Constantes de Inmunoglobulina/química , Regiones Constantes de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
BACKGROUND: 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 Sasa-UBA in addition to a soluble MHC class I molecule, Sasa-ULA. A pseudolocus for Sasa-UCA 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. RESULTS: 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, ZAA. The IB region was extended with 350 kb including three new Z-lineage loci, ZBA, ZCA and ZDA in addition to a UGA locus. An allelic version of the IB region contained a functional UDA locus in addition to the UCA 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 SAA (previously known as UAA) was placed on LG10. Gene expression studies showed limited expression range for all class I genes with exception of UBA being dominantly expressed in gut, spleen and gills, and ZAA with high expression in blood. CONCLUSION: 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 (UBA), in addition to a multitude of nonclassical MHC class I genes from the U-, S- and Z-lineages.
Asunto(s)
Proteínas de Peces/genética , Genes MHC Clase I , Salmo salar/genética , Alelos , Secuencia de Aminoácidos , Animales , Betahistina , Mapeo Cromosómico , Cromosomas Artificiales Bacterianos , Biología Computacional , Perfilación de la Expresión Génica , Orden Génico , Ligamiento Genético , Biblioteca Genómica , Genómica/métodos , Datos de Secuencia Molecular , Filogenia , Salmo salar/inmunología , Análisis de Secuencia de ADNRESUMEN
The Atlantic salmon (Salmo salar) T-cell receptor gamma (TCRgamma) gene has extensive diversity in its capacity for antigen recognition due to the V-J-C gene segments recombinational possibilities, and N-region diversity. This is the first report completely characterizing and annotating a TCRgamma gene locus in teleosts. We identified two different TCRgamma loci in Atlantic salmon. The first locus, TCRgamma 1, spans 260 kbp and contains four tandemly repeated clusters each of which consists of 1-4 variable (V) segments, 1-2 sets of a joining (J) segment and a constant (C) region. In total, 10 V segments, 5 J segments and 5 C regions were found in locus 1. In the second locus, TCRgamma 2, a single non-expressed V-J-C cluster was found. Surprisingly, the Atlantic salmon TCRgamma loci have a larger number of C regions relative to other teleosts, mouse and human. In addition, each Atlantic salmon TCRgamma C region has a different connecting peptide region that may result in distinct cellular responses. Expression data confirm the diverse repertoire found at the genomic level. At least 6 out of 7 functional V segments, all 5 J segments and all 5 C regions found in TCRgamma 1 were identified in TCRgamma transcription analysis. The identification and characterization of the functional TCRgamma 1 along with the associated TCR alpha/delta locus suggest that Atlantic salmon have a functional gammadelta T-cell immune component.
Asunto(s)
Variación Genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Salmo salar/genética , Salmo salar/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Clonales , ADN Complementario/análisis , ADN Complementario/genética , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Genoma , Datos de Secuencia Molecular , Filogenia , Receptores de Antígenos de Linfocitos T gamma-delta/química , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Análisis de Secuencia de ADNRESUMEN
The complete TCR alpha/delta locus of Atlantic salmon (Salmo salar) has been characterized and annotated. In the 900 kb TCR alpha/delta locus, 292 Valpha/delta segments and 123 Jalpha/delta segments were identified. Of these, 128 Valpha/delta, 113 Jalpha, and a Jdelta segment appeared to be functional as they lacked frame shifts or stop codons. This represents the largest repertoire of Valpha/delta and Jalpha segments of any organism to date. The 128 functional Valpha/delta segments could be grouped into 29 subgroups based upon 70% nucleotide similarity. Expression data confirmed the usage of the diverse repertoire found at the genomic level. At least 99 Valpha, 13 Vdelta 86 Jalpha, 1 Jdelta, and 2 Ddelta segments were used in TCR alpha or delta transcription, and 652 unique genes were identified from a sample of 759 TCRalpha cDNA clones. Cumulatively, the genomic and expression data suggest that the Atlantic salmon T-cell receptor has enormous capacity to recognize a wide diversity of antigens.