RESUMEN
Recent large-scale cDNA cloning studies have shown that a significant proportion of the transcripts expressed from vertebrate genomes do not appear to encode protein. Moreover, it was reported in mammals (human and mice) that these non-coding transcripts are expressed and regulated by mechanisms similar to those involved in the control of protein-coding genes. We have produced a collection of cDNA sequences from immunologically active tissues with the aim of discovering chicken genes involved in immune mechanisms, and we decided to explore the non-coding component of these immune-related libraries. After finding known non-coding RNAs (miRNA, snRNA, snoRNA), we identified new putative mRNA-like non-coding RNAs. We characterised their expression profiles in immune-related samples. Some of them showed changes in expression following viral infections. As they exhibit patterns of expression that parallel the behaviour of protein-coding RNAs in immune tissues, our study suggests that they could play an active role in the immune response.
Asunto(s)
Pollos/genética , ADN Complementario/genética , ARN no Traducido/genética , Animales , Infecciones por Birnaviridae/genética , Infecciones por Birnaviridae/inmunología , Pollos/inmunología , Etiquetas de Secuencia Expresada , Femenino , Perfilación de la Expresión Génica , Biblioteca de Genes , Activación de Linfocitos , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Masculino , Enfermedad de Marek/genética , Enfermedad de Marek/inmunología , MicroARNs/genética , MicroARNs/aislamiento & purificación , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos , ARN no Traducido/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Linfocitos T/inmunologíaRESUMEN
We have identified and characterised a cluster of six TRIM-B30.2 genes flanking the chicken BF/BL region of the B complex. The TRIM-B30.2 proteins are a subgroup of the TRIM protein family containing the tripartite motif (TRIM), consisting of a RING domain, a B-box and a coiled coil region, and a B30.2-like domain. In humans, a cluster of seven TRIM-B30.2 genes has been characterised within the MHC on Chromosome 6p21.33. Among the six chicken TRIM-B30.2 genes two are orthologous to those of the human MHC, and two (TRIM41 and TRIM7) are orthologous to human genes located on Chromosome 5. In humans, these last two genes are adjacent to GNB2L1, a guanine nucleotide-binding protein gene, the ortholog of the chicken c12.3 gene situated in the vicinity of the TRIM-B30.2 genes. This suggests that breakpoints specific to mammals have occurred and led to the remodelling of their MHC structure. In terms of structure, like their mammalian counterparts, each chicken gene consists of five coding exons; exon 1 encodes the RING domain and the B-box, exons 2, 3 and 4 form the coiled-coil region, and the last exon represents the B30.2-like domain. Phylogenetic analysis led us to assume that this extended BF/BL region may be similar to the human extended class I region, because it contains a cluster of BG genes sharing an Ig-V like domain with the BTN genes (Henry et al. 1997a) and six TRIM-B30.2 genes containing the B30.2-like domain, shared with the TRIM-B30.2 members and the BTN genes.
Asunto(s)
Pollos/genética , Genes MHC Clase I , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Datos de Secuencia Molecular , Filogenia , Estructura Secundaria de Proteína , Alineación de SecuenciaRESUMEN
Aryl hydrocarbon receptor (AhR) genes encode proteins involved in mediating the toxic responses induced by several environmental pollutants. Here, we describe the identification of the first two AhR1 (alpha and beta) genes and two additional AhR2 (alpha and beta) genes in the tetraploid species Atlantic salmon (Salmo salar L.) from a cosmid library screening. Cosmid clones containing genomic salmon AhR sequences were isolated using a cDNA clone containing the coding region of the Atlantic salmon AhR2gamma as a probe. Screening revealed 14 positive clones, from which four were chosen for further analyses. One of the cosmids contained genomic AhR sequences that were highly similar to the rainbow trout (Oncorhynchus mykiss) AhR2alpha and beta genes. SMART RACE amplified two complete, highly similar but not identical AhR type 2 sequences from salmon cDNA, which from phylogenetic analyses were determined as the rainbow trout AhR2alpha and beta orthologs. The salmon AhR2alpha and beta encode proteins of 1071 and 1058 residues, respectively, and encompass characteristic AhR sequence elements like a basic-helix-loop-helix (bHLH) and two PER-ARNT-SIM (PAS) domains. Both genes are transcribed in liver, spleen and muscle tissues of adult salmon. A second cosmid contained partial sequences, which were identical to the previously characterized AhR2gamma gene. The last two cosmids contained partial genomic AhR sequences, which were more similar to other AhR type 1 fish genes than the four characterized salmon AhR2 genes. However, attempts to amplify the corresponding complete cDNA sequences of the inserts proved very difficult, suggesting that these genes are non-functional or very weakly transcribed in the examined tissues. Phylogenetic analyses of the conserved regions did, however, clearly indicate that these two AhRs belong to the AhR type 1 clade and have been assigned as the Atlantic salmon AhR1alpha and AhR1beta genes. Taken together, these findings demonstrate that multiple AhR genes are present in Atlantic salmon genome, which likely is a consequence of previous genome duplications in the evolutionary past of salmonids. Plausible explanations for the high incidence of AhR genes in fish and more specifically in salmonids, like rapid divergences in specialized functions, are discussed.
Asunto(s)
Biblioteca de Genes , Variación Genética , Filogenia , Receptores de Hidrocarburo de Aril/genética , Salmo salar/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Análisis por Conglomerados , Cósmidos/genética , Cartilla de ADN , ADN Complementario/genética , Componentes del Gen , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
In order to understand the expression and evolution of host resistance to pathogens, we need to examine the links between genetic variability at the major histocompatibility complex ( Mhc), phenotypic expression of the immune response and parasite resistance in natural populations. To do so, we characterized the Mhc class I and IIB genes of house sparrows with the goal of designing a PCR-based genotyping method for the Mhc genes using denaturing gradient gel electrophoresis (DGGE). The incredible success of house sparrows in colonizing habitats worldwide allows us to assess the importance of the variability of Mhc genes in the face of various pathogenic pressures. Isolation and sequencing of Mhc class I and IIB alleles revealed that house sparrows have fewer loci and fewer alleles than great reed warblers. In addition, the Mhc class I genes divided in two distinct lineages with different levels of polymorphism, possibly indicating different functional roles for each gene family. This organization is reminiscent of the chicken B complex and Rfp-Y system. The house sparrow Mhc hence appears to be intermediate between the great reed warbler and the chicken Mhc, both in terms of numbers of alleles and existence of within-class lineages. We specifically amplified one Mhc class I gene family and ran the PCR products on DGGE gels. The individuals screened displayed between one and ten DGGE bands, indicating that this method can be used in future studies to explore the ecological impacts of Mhc diversity.
Asunto(s)
Genes MHC Clase II/genética , Genes MHC Clase I/genética , Variación Genética , Pájaros Cantores/genética , Alelos , Animales , Southern Blotting , Electroforesis en Gel de Poliacrilamida/métodos , Exones/genética , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , Pájaros Cantores/inmunologíaRESUMEN
The aryl hydrocarbon receptor (AhR) mediates the toxicity of several environmental contaminants, e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin, and other halogenated hydrocarbons in vertebrates. This receptor initiates the transcription of several biotransformation enzymes, which in turn are responsible for causing severe harm to biological tissue. Here we describe the isolation and complete characterization of the first two AhR genes from the teleost fish Atlantic salmon (Salmo salar). The predicted amino acid sequences contain regions characteristic of other vertebrate AhRs including basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains but show little similarity to other vertebrate AhRs across the C-terminal half. Furthermore, they do not contain distinct Q-rich domains as found in the mammalian AhR, which is in line with previously described fish AhR genes. The salmon cDNAs encode 1106 and 1107 putative residues, respectively, approximately 50 amino acids longer than previously characterized AhR genes. Phylogenetic analyses demonstrated that the two salmon AhR sequences cluster within the AhR subfamily of the bHLH-PAS family, in a clade containing fish AhR2 genes. Although the two AhR2 forms are 92% identical at the amino acid level, the distribution of sequence differences and the presence of both forms in 30 tested individuals suggest that they are not allelic but derived from separate loci. Interestingly, they are not orthologs of the rainbow trout (Oncorhynchus mykiss) AhR2 alpha and beta genes and the new salmon loci are therefore here designated AhR2 gamma and AhR2 delta. In line with this, PCR with DNA from rainbow trout revealed a new trout AhR locus that was more similar to the two salmon genes than to the trout AhR2 alpha and beta genes, suggesting that the rainbow trout possesses at least three distinct AhR2 genes. The presence of multiple AhR genes in these species is probably a consequence of the genome duplications that occurred in the early evolution of fish and later also specifically in the salmonid lineage. Reverse transcription-PCR analyses revealed that both AhR2 gamma and AhR2 delta are transcribed in the liver, spleen and muscles of adult salmon.