RESUMEN
Uranium mining and milling activities raise environmental concerns due to the release of radioactive and other toxic elements. Their long-term management thus requires a knowledge of past events coupled with a good understanding of the geochemical mechanisms regulating the mobility of residual radionuclides. This article presents the results on the traces of anthropic activity linked to previous uranium (U) mining activities in the vicinity of the Rophin tailings storage site (Puy de Dôme, France). Several complementary approaches were developed based on a study of the site's history and records, as well as on a radiological and chemical characterization of soil cores and a dendrochronology. Gamma survey measurements of the wetland downstream of the Rophin site revealed a level of 1050 nSv.h-1. Soil cores extracted in the wetland showed U concentrations of up to 1855 mg.kg-1, which appears to be associated with the presence of a whitish silt loam (WSL) soil layer located below an organic topsoil layer. Records, corroborated by prior aerial photographs and analyses of 137Cs and 14C activities, suggest the discharge of U mineral particles while the site was being operated. Moreover, lead isotope ratios indicate that contamination in the WSL layer can be discriminated by a larger contribution of radiogenic lead to total lead. The dendroanalysis correlate U emissions from Rophin with the site's history. Oak tree rings located downstream of the site contain uranium concentrations ten times higher than values measured on unaffected trees. Moreover, the highest U concentrations were recorded not only for the operating period, but more surprisingly for the recent site renovations as well. This integrated approach corroborates that U mineral particles were initially transported as mineral particles in Rophin's watershed and that a majority of the deposited uranium appears to have been trapped in the topsoil layer, with high organic matter content.
Asunto(s)
Monitoreo de Radiación , Uranio , Francia , Minería , Suelo , Uranio/análisisRESUMEN
Uranium mining activities expose uranium ore and mine tailings to the surface environment, where the release of radionuclides is facilitated by weathering at rates exceeding those typically found in nature. Therefore, close to former uranium mining sites, radionuclides and especially uranium concentrations in water may surpass local background levels. The methodology proposed herein, entails coupling, gamma-ray mapping, water sampling and chemical analyses including DGT (Diffusive Gradient in Thin Film) measurements, provides new insights into describing the environment of the La Commanderie site (France). Gamma-ray mapping allows identifying water seepage, output from a waste rock pile, as a potential pathway for radionuclides into the environment. Water seepage monitoring has shown: a low pH value (4.2), high sulfate content (179â¯mg.L-1) and high uranium concentrations of up to 436⯵g.L-1. These recordings indicate that an acid mining drainage (AMD) process is occurring inside or under the oxidized parts of the waste rock pile. Monitoring data over three flow periods revealed the release of the highest uranium concentrations during a high-flow period downstream of the site, which is compliant with local regulations. The AMD process is also responsible for the release of significant amounts of Fe, Mn and As within the immediate environment in both dissolved and particulate forms. Changes in dissolved oxygen concentration and redox potential during low flow periods, modify the speciation of Fe (in AMD waters) which acts as a scavenger for other elements such as As, Mn and U. The use of DGT under environmental conditions, and specifically AMD waters, seems to be relevant in comparison to filtered spot water sampling strategies. Moreover, based on DGT measurements, the dissolved part of the released uranium is considered as labile with concentrations above the environmental standards for freshwater organisms.
Asunto(s)
Monitoreo de Radiación , Oligoelementos/análisis , Contaminantes Radiactivos del Agua/análisis , Francia , Minería , Uranio/análisisRESUMEN
The pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for the study of virus-host cell dialog. As PrV has a strong tropism for mucous epithelial cells, we chose to follow in vitro the PrV time course-infection of porcine PK15 cells. The viral and cellular transcriptome modifications were simultaneously analysed using a combined SLA/PrV cDNA microarray, the porcine Qiagen-NRSP8 oligonucleotides microarray and real time quantitative PCR.Ahigh increase in viral gene expression was found from 4 h post-infection (PI), concomitantly to the first viral progeny and most viral genes were differentially expressed 12 h PI. No early global cellular shutoff was observed but many cellular genes were downregulated between 8 and 12 h PI, when UL41 transcripts encoding the virion shutoff protein, were first detected. Several genes involved in the MHC class I mediated antigenic pathway were downregulated including SLA-la, TAP1, TAP2, PSMB8 and PSMB9 genes. These results suggested that PrV prevents the viral antigen presentation by epithelial cells to cytotoxic T lymphocytes by decreasing transcription levels of SLA Ia mediated antigenic pathway genes. Other genes involved in the immune response, the apoptosis pathway, nucleic acid metabolism and cytoskeleton also appeared to be regulated during PrV infection. The combined approach will help to decipher host response evasion strategies developed by PrV and to study early cellular modifications.
Asunto(s)
Genómica , Herpesvirus Suido 1/fisiología , ARN Mensajero/genética , Animales , Línea Celular , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , PorcinosRESUMEN
Cardiac contusion is frequently found in patients with blunt chest trauma. It is important to note that even if there is a low incidence of pericardial effusion, iterative echocardiography should be used to provide essential information for the diagnosis of cardiac tamponade which can be life-threatening during hospitalisation. The case has been reported of a 17-year-old patient with blunt thoracic trauma in whom the introduction of anticoagulant treatment induced a delayed cardiac tamponade with myocardiac failure 3 weeks after a cardiac contusion. Thoracic computed tomography confirmed the diagnosis and moreover, revealed a pleural effusion with pulmonary embolism. The drainage of the pericardial effusion (700 ml) rapidly restored haemodynamic stability and as such has been proved to be life-saving.
Asunto(s)
Anticoagulantes/efectos adversos , Taponamiento Cardíaco/inducido químicamente , Lesiones Cardíacas/complicaciones , Heridas no Penetrantes/complicaciones , Adolescente , Anticoagulantes/uso terapéutico , Femenino , Lesiones Cardíacas/diagnóstico por imagen , Humanos , Derrame Pleural/diagnóstico por imagen , Derrame Pleural/etiología , Derrame Pleural/terapia , Embolia Pulmonar/diagnóstico por imagen , Embolia Pulmonar/tratamiento farmacológico , Embolia Pulmonar/etiología , Factores de Tiempo , Tomografía Computarizada por Rayos X , Heridas no Penetrantes/diagnóstico por imagenRESUMEN
Although the European rabbit (Oryctolagus cuniculus) is used both in agronomics and in research, genomic resources for this species are still limited and no microsatellite-based genetic map has been reported. Our aim was to construct a rabbit genetic map with cytogenetically mapped microsatellites so as to build an integrated genetic and cytogenetic map. A reference population of 187 rabbits comprising eight three-generation families with 10-25 offspring per family was produced. One hundred and ninety-four of 305 previously identified microsatellites were included in this study. Of these, 158 were polymorphic with two to seven alleles. The map reported here comprises 111 markers, including 104 INRA microsatellites, five microsatellites from another source and two phenotypic markers (angora and albino). Ninety markers were integrated into 20 linkage groups. The remaining 21 microsatellites mapped to separate linkage groups, 19 with a precise cytogenetic position and two with only a chromosomal assignment. The genetic map spans 2766.6 cM and covers 20 rabbit chromosomes, excluding chromosomes 20, 21 and X. The density of this map is limited, but we used it to verify the location of angora and albino on chromosomes 15q and 1q, respectively, in agreement with previously published data. This first generation genetic/cytogenetic map will help gene identification and quantitative trait loci mapping projects in rabbit.
Asunto(s)
Mapeo Cromosómico , Repeticiones de Microsatélite , Conejos/genética , Alelos , Animales , Genes , Ligamiento Genético , Marcadores Genéticos , Fenotipo , Polimorfismo GenéticoRESUMEN
We describe the generation and analysis of an integrated sequence map of a 2.4-Mb region of pig chromosome 7, comprising the classical class I region, the extended and classical class II regions, and the class III region of the major histocompatibility complex (MHC), also known as swine leukocyte antigen (SLA) complex. We have identified and manually annotated 151 loci, of which 121 are known genes (predicted to be functional), 18 are pseudogenes, 8 are novel CDS loci, 3 are novel transcripts, and 1 is a putative gene. Nearly all of these loci have homologues in other mammalian genomes but orthologues could be identified with confidence for only 123 genes. The 28 genes (including all the SLA class I genes) for which unambiguous orthology to genes within the human reference MHC could not be established are of particular interest with respect to porcine-specific MHC function and evolution. We have compared the porcine MHC to other mammalian MHC regions and identified the differences between them. In comparison to the human MHC, the main differences include the absence of HLA-A and other class I-like loci, the absence of HLA-DP-like loci, and the separation of the extended and classical class II regions from the rest of the MHC by insertion of the centromere. We show that the centromere insertion has occurred within a cluster of BTNL genes located at the boundary of the class II and III regions, which might have resulted in the loss of an orthologue to human C6orf10 from this region.
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Antígenos de Histocompatibilidad Clase I/genética , Complejo Mayor de Histocompatibilidad/genética , Porcinos/genética , Animales , Centrómero , Cromosomas Artificiales Bacterianos , Mapeo Contig , Genoma , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase II , Humanos , Masculino , FilogeniaRESUMEN
BACKGROUND AND AIM: A role for the intestinal microbial community (microbiota) in the onset and chronicity of Crohn's disease (CD) is strongly suspected. However, investigation of such a complex ecosystem is difficult, even with culture independent molecular approaches. METHODS: We used, for the first time, a comprehensive metagenomic approach to investigate the full range of intestinal microbial diversity. We used a fosmid vector to construct two libraries of genomic DNA isolated directly from faecal samples of six healthy donors and six patients with CD. Bacterial diversity was analysed by screening the two DNA libraries, each composed of 25,000 clones, for the 16S rRNA gene by DNA hybridisation. RESULTS: Among 1190 selected clones, we identified 125 non-redundant ribotypes mainly represented by the phyla Bacteroidetes and Firmicutes. Among the Firmicutes, 43 distinct ribotypes were identified in the healthy microbiota, compared with only 13 in CD (p<0.025). Fluorescent in situ hybridisation directly targeting 16S rRNA in faecal samples analysed individually (n=12) confirmed the significant reduction in the proportion of bacteria belonging to this phylum in CD patients (p<0.02). CONCLUSION: The metagenomic approach allowed us to detect a reduced complexity of the bacterial phylum Firmicutes as a signature of the faecal microbiota in patients with CD. It also indicated the presence of new bacterial species.
Asunto(s)
Bacterias/clasificación , Enfermedad de Crohn/microbiología , Heces/microbiología , Adolescente , Adulto , Bacterias/genética , Bacterias/aislamiento & purificación , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Ecosistema , Femenino , Biblioteca de Genes , Humanos , Hibridación Fluorescente in Situ , Masculino , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genéticaRESUMEN
A systematic nomenclature for the genes and alleles of the swine major histocompatibility complex (MHC) is essential to the development and communication of research in swine immunology. The Swine Leukocyte Antigen (SLA) Nomenclature Committee of the International Society for Animal Genetics (ISAG) has reviewed all of the DNA-sequence information for MHC class II genes, available in GenBank/EMBL/DDBJ databases, and the associated published reports to develop such a systematic nomenclature. This article summarizes the proposed nomenclature, which parallels the World Health Organization's nomenclature for factors of the human MHC. The SLA class II genes expressed on the cell membrane will be noted as SLA-DRA, SLA-DRB1, SLA-DQA, and SLA-DQB1. Nomenclature assignments for all SLA class II GenBank sequences are now noted. The committee will add new SLA class II allele designations, as they are discovered, and will maintain a publicly available list of all recognized genes and alleles using the Immuno Polymorphism Database (IPD). The sequences will be available from the IPD-MHC section of the database which contains non-human MHC sequences (http://www.ebi.ac.uk/ipd/mhc/sla/).
Asunto(s)
Antígenos de Histocompatibilidad Clase II/clasificación , Antígenos de Histocompatibilidad Clase II/genética , Porcinos/genética , Terminología como Asunto , Animales , Filogenia , Análisis de SecuenciaRESUMEN
Combinatorial diversity is highly restricted during formation of the pre-immune heavy chain repertoire of swine, raising the question of whether the same is true for the pre-immune light chain repertoire. Before addressing this question, we first used competitive PCR to show that kappa and lambda light chains in swine are equally expressed in mature B cells similar to the situation in humans but alike that in other studied Ungulates. This justified efforts to examine the repertoire of both light chain types. These studies also revealed that lambda is preferentially expressed at sites of B cells lymphogenesis, perhaps because of the use of a surrogate light chain containing lambda5. Data are presented here on >100 VkappaJkappa-containing transcripts and approximately 180 genomic Vkappa genes to show that >90% of the pre-immune repertoire is generated from three subfamilies of IGKV2 genes and one of five Jkappa segments. The kappa locus contains >or=50 IGKV2 genes belonging to at least five subfamilies and an undetermined but perhaps equal number of IGKV1 genes. The porcine IGKV1 and IGKV2 genes share 87% sequence similarity with their human counterparts and Jkappa1 through Jkappa5 share sequence and organizational homology with those in sheep, horse, human and mouse. Swine have a single Ckappa gene. These findings contrast with those from rodents and primates but are reminiscent of those on the pre-immune heavy chain repertoire of swine in that it is generated using a relatively restricted number of gene segments. These restricted pre-immune repertoires may reflect the minimal exposure of the fetus to maternal factors and environmental antigens. The significance for swine immunology of characterizing the pre-immune repertoire is discussed.
Asunto(s)
Genes de Inmunoglobulinas , Porcinos/genética , Porcinos/inmunología , Secuencia de Aminoácidos , Animales , Feto/inmunología , Regulación del Desarrollo de la Expresión Génica , Humanos , Cadenas J de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Tejido Linfoide/embriología , Tejido Linfoide/inmunología , Ratones , Familia de Multigenes , Homología de Secuencia de Aminoácido , Porcinos/embriologíaRESUMEN
A systematic nomenclature for the genes and alleles of the swine major histocompatibility complex (MHC) is essential to the development and communication of research in swine immunology. The Swine Leucocyte Antigen (SLA) Nomenclature Committee of the International Society for Animal Genetics has reviewed all of the DNA sequence information for MHC class-I genes, available in GenBank/EMBL/DDBJ databases, and the associated published reports in order to develop such a systematic nomenclature. This report summarizes the proposed nomenclature, which parallels the World Health Organization's nomenclature for factors of the human MHC. The classical class-I SLA genes are designated as SLA-1, SLA-2 and SLA-3; the non-classical as SLA-6, SLA-7 and SLA-8. Nomenclature assignments for all SLA class-I GenBank sequences are now noted. The Committee will add new SLA class-I allele designations, as they are discovered, and will maintain a publicly available list of all recognized genes and alleles by using the International ImMunoGeneTics Project and its Immuno Polymorphism Database/MHC (IPD/MHC) sequence database for MHC sequences in veterinary species.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/clasificación , Antígenos de Histocompatibilidad Clase I/genética , Porcinos/genética , Terminología como Asunto , Alelos , Animales , Antígenos de Histocompatibilidad Clase II , Filogenia , Análisis de SecuenciaRESUMEN
Rabbit (Oryctolagus cuniculus), besides its interest for medical research and biotechnological applications, has a small agronomic production in southern European countries. However, it is still a "map-poor" species with about 80 genes mapped. Recently, useful tools for research on this species have been developed, such as heterologous human-rabbit chromosome painting data and a rabbit BAC library. In this study, our aim is to enrich the rabbit cytogenetic map using the FISH technique. Towards this, we have used cDNAs (rabbit and non rabbit) present in the public databases to determine intra-exon primers used to screen our three-genome equivalent BAC library, by standard PCR directly on DNA pools, and by hybridization of high-density filters. 133 BAC clones containing the genes of interest were isolated and FISH-mapped to the rabbit chromosomes. We present the localization of new genes on all rabbit chromosomes except OCU20 and OCUY and some preliminary data on the rabbit/human comparative map. In addition, this set of BAC clones quite regularly distributed on the rabbit genome will be useful to isolate microsatellites, in order to construct a first generation genetic map.
Asunto(s)
Mapeo Cromosómico , Conejos/genética , Animales , Cromosomas Artificiales Bacterianos , Análisis Citogenético , Cartilla de ADN , Humanos , Hibridación Fluorescente in Situ , SinteníaRESUMEN
We report here the localisation of BAIAP1 (13q24), HTR1F (13q45), PTPRG (13q23) and UBE1C (13q24) by fluorescence in situ hybridisation (FISH), and BAIAP1 (Swr2114; 21 cR; LOD = 11.03), GATA2 (Sw2448; 37 cR; LOD = 8.26), IL5RA (Swr2114; 64 cR; LOD = 3.85), LMCD1 (Sw2450; 61 cR; LOD = 4.73), MME (CP; 50 cR; LOD = 7.75), RYK (Swc22; 12 cR; LOD = 18.62) and SGU003 (Sw1876; 6 cR; LOD = 16.99) by radiation hybrid (RH) mapping to porcine chromosome 13 (SSC13). The mapping of these 10 different loci (all mapped to human chromosome 3; HSA3) not only confirms the extended conservation of synteny between HSA3 and SSC13, but also defines more precisely the regions with conserved linkage. The syntenic region of the centromeric part of SSC13 was determined by isolating porcine bacterial artificial chromosome (BAC) clones (842D4 and 1031H1) using primers amplifying porcine microsatellite markers S0219 and S0076 (mapped to this region). Sequence comparison of the BAC end sequences with the human genome sequence showed that the centromeric part of SSC13 is homologous with HSA3p24.
Asunto(s)
Cromosomas Humanos Par 3/genética , Cromosomas/genética , Porcinos/genética , Animales , Cromosomas Artificiales Bacterianos/genética , Análisis Citogenético/métodos , Análisis Citogenético/veterinaria , Biblioteca de Genes , Orden Génico/genética , Ligamiento Genético/genética , Humanos , Hibridación Fluorescente in Situ/métodos , Hibridación Fluorescente in Situ/veterinaria , Metafase/genética , Repeticiones de Microsatélite/genética , Datos de Secuencia Molecular , Mapeo Físico de Cromosoma/métodos , Mapeo Físico de Cromosoma/veterinaria , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Mapeo de Híbrido por Radiación/métodos , Mapeo de Híbrido por Radiación/veterinariaRESUMEN
A porcine bacterial artificial chromosome (BAC) clone, containing the melanocortin 2 receptor gene (MC2R) was isolated. The complete coding sequence of the MC2R gene, contained in 1 exon, was determined. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) was performed on a 241-bp coding fragment. An AluI polymorphism, detecting a silent mutation, was found and typed on unrelated animals of five different pig breeds. The Meishan, Piétrain and Large White breeds differ significantly in allele frequencies from the Landrace and Czech Meat Pig breeds. The melanocortin 5 receptor gene (MC5R) was detected by PCR in the same BAC clone, as could be expected from the human and porcine mapping data. PCR-SSCP was performed on a 200-bp coding of MC5R, but no polymorphisms were detected. The BAC clone was mapped to Sscr6q27 by fluorescent in situ hybridization (FISH). A (CA)n microsatellite (SGU0002), isolated from the BAC, was localized on chromosome 6 by RH mapping near marker SW1473 and by linkage mapping on the MARC reference family at the same position as the marker SW2173 (97 cM). Allele frequencies, heterozygosity and polymorphism information contents (PIC) values were calculated for the five different pig breeds examined. The transcription of both genes in porcine liver, heart, kidney, fat, brain, pancreas, stomach, bladder, ovaries, lung, spleen, skin, adrenal gland and muscle tissues was examined by reverse transcriptase-polymerase chain reaction. Transcription was detected in skin and adrenal gland tissues for MC2R, while a positive signal was detected for MC5R in kidney, fat, pancreas, skin, adrenal gland and spleen tissues.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Receptores de Corticotropina/genética , Porcinos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico/veterinaria , Clonación Molecular , Ligamiento Genético/genética , Hibridación Fluorescente in Situ/veterinaria , Repeticiones de Microsatélite/genética , Polimorfismo Genético/genética , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/química , ARN Mensajero/genética , Receptor de Melanocortina Tipo 2 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Previous comparative mapping suggested that at least five pig chromosomes (Sscr4, 6, 9, 10 and 14) share homology with human chromosome 1 (Hsap1). A significant quantitative trait loci (QTL) for fat deposition has been identified on Sscr4 that appears to be near the junction region between Sscr4 and Sscr9 relative to Hsap1. It is of interest to define the boundaries of conserved synteny between pig chromosomes and Hsap1 to use human map information to identify putative comparative positional candidates for this QTL. Eleven genes, including Janus kinase 1 (JAK1), Prostaglandin E receptor3 (PTGER3), urate oxidase (UOX), coagulation factor 3 (F3), vascular cell adhesion molecule 1 (VCAM1), ribosomal protein L5 (RPL5), POU domain, class 2, transcription factor 1 (POU2F1), coagulation factor 5 (F5), Prostaglandin endoperoxide synthase-2 (PTGS2), myosin binding protein H (MYBPH) and Antithrombin III (SERPINC1), were selected to refine the boundaries of the blocks of conserved synteny between Hsap1 and pig chromosomes. Pig sequence tagged sites (STSs) were developed and used to physically map these 11 genes using a somatic cell hybrid panel. Eight loci have been mapped by using fluorescent in situ hybridization (FISH) to improve map resolution. Heterologous FISH was used to refine the location of VCAM1 on human chromosomes. In addition, human yeast artificial chromosomes (YACs) were mapped by heterologous FISH on pig metaphases to refine the boundaries of the regions of homology between Sscr4 and Sscr9 on Hsap1. Results from this study suggest the precise break in conserved synteny on Hsap1 corresponding to the Sscr4/6 and Sscr4/9 transitions are most likely on the Hsap1p22 and Hsap1q24-25 regions, respectively. Further, our data predict that Hsap1q21-24 is a candidate region for the backfat QTL localized to Sscr4.
Asunto(s)
Cromosomas Humanos Par 1 , Porcinos/genética , Sintenía , Animales , Mapeo Cromosómico , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Lugares Marcados de Secuencia , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
A comparative map of human chromosome 12 (HSA 12) and pig chromosome 5 (SSC 5) was constructed using ten pig expressed sequence tags (ESTs). These ESTs were isolated from primary granulosa cell cultures by differential display (EST b10b), or from a granulosa cDNA library (VIIIE1, DRIM, N*9, RIIID2 and RVIC1) or from a small intestine cDNA library (ATPSB, ITGB7, MYH9, and STAT2). Also used were two Traced Orthologous Amplified Sequence Tags (TOASTs) (LALBA, TRA1), one microsatellite-associated gene (IGF1) and finally five human YACs selected for their cytogenetic position, with a view to increasing the number of informative markers for the comparison. Large-insert clones were obtained by screening a pig bacterial artificial chromosome (BAC) library with specific primers for each EST and TOAST and for IGF1. These BACs were used as probes for fluorescent in situ hybridisation (FISH) both on porcine and human metaphases. In addition, the human YACs were FISH mapped on pig chromosomes. This allowed us to refine and, in some cases, to correct the previous mapping obtained with a somatic cell hybrid panel. While these data confirm chromosome painting results showing that the distal part of SSC 5p arm is conserved on HSA 22, while the rest of the chromosome corresponds to HSA 12, they also demonstrate gene-order differences between human and pig. In addition, it was also possible to determine the position of the synteny breakpoint.
Asunto(s)
Cromosomas Humanos Par 12/genética , Secuencia Conservada/genética , Orden Génico/genética , Hibridación Fluorescente in Situ , Porcinos/genética , Sintenía/genética , Animales , Gatos , Bovinos , Cromosomas Artificiales Bacterianos , Cromosomas Artificiales de Levadura , Evolución Molecular , Etiquetas de Secuencia Expresada , Marcadores Genéticos , Humanos , Células Híbridas/metabolismoRESUMEN
To improve the comparative map for pig chromosome 2 and increase the gene density on this chromosome, a porcine bacterial artificial chromosome (BAC) library was screened with 17 microsatellite markers and 18 genes previously assigned to pig chromosome 2. Fifty-one BAC clones located in the region of a maternally imprinted quantitative trait locus for backfat thickness (BFT) were identified. From these BACs 372 kb were sample sequenced. The average read length of a subclone was 442 basepair (bp). Contig assembly analysis showed that every bp was sequenced 1.28 times. Subsequently, sequences were compared with sequences in the nucleotide databases to identify homology with other mammalian sequences. Sequence identity was observed with sequences derived from 35 BACs. The average percentage identity with human sequences was 87.6%, with an average length of 143 bp. In total, sample sequencing of all BACs resulted in sequence identity with 29 human genes, 13 human expressed sequence tags (ESTs), 17 human genomic clones, one rat gene, one porcine gene and nine porcine ESTs. Eighteen genes located on human chromosome 11 and 19, and seven genes from other human locations, one rat gene and one porcine gene were assigned to pig chromosome 2 for the first time. The new genes were added to the radiation hybrid map at the same position as the locus from which the BAC that was sequenced was derived. In total 57 genes were placed on the radiation hybrid map of SSC2p-q13.
Asunto(s)
Mapeo Cromosómico , Porcinos/genética , Animales , Mapeo Cromosómico/veterinaria , Cromosomas Artificiales Bacterianos , Bases de Datos Factuales , Etiquetas de Secuencia Expresada , Humanos , Repeticiones de Microsatélite , Carácter Cuantitativo HeredableRESUMEN
A segment comprising 307,078 nucleotides of the pig major histocompatibility complex (SLA) was completely sequenced. The segment corresponded to the entire SLA classical class I-containing region of the serologically defined SLA H01 haplotype. In all, 11 genes were characterized, comprising 7 class I genes located on the centromeric part of the sequence (SLA-1, 2, 3, 4, 5, 9, and 11) and 4 ring finger-related family genes located on its telomeric part. No member of one family was intermingled with a member of the other or with any third-party gene. All class I genes except SLA-11 were similarly orientated. The SLA-1, 2, and 3 genes displayed both promoter and overall coding regions compatible with normal functions. The SLA-4, 11, and 9 genes were considered pseudogenes because they exhibited marked anomalies. Although the SLA-5 gene had a complete coding region, it displayed mutations in promoter elements which could modify its expression. The great molecular similarity observed among the class I genes extended far outside them, and resulted from segmental duplications. The ring finger genes exhibited great homology with their human counterparts. In pig, one of these genes appeared to correspond to a complete gene which in humans is probably a pseudogene. In all, the 11 genes characterized span about 20% of the total sequence. The remaining 80% consists of interspersed repeat elements. The present results, together with the sequence previously reported involving the SLA class I-related genes, open the way for a better understanding of pig MHC organization.
Asunto(s)
Genes MHC Clase I , Porcinos/genética , Alelos , Animales , Secuencia de Bases , Centrómero/genética , Proteínas de Unión al ADN/genética , Haplotipos , Antígenos de Histocompatibilidad Clase I/inmunología , Prueba de Histocompatibilidad , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Telómero/genética , Regiones no Traducidas , Dedos de ZincRESUMEN
The poorly developed transcript maps and the limited resources for genome analysis hamper positional cloning of trait loci in farm animals. This study demonstrates that this will now be easier by the combined use of BAC contigs and the import of the near complete human transcript map. The conclusion was obtained by a comparative analysis of a 2.4-Mb BAC contig of the RN region in pigs. The contig was constructed as part of a successful positional cloning project, which identified PRKAG3 as the causative gene for the RN phenotype. A comparative map including the corresponding regions on human chromosome 2q35 and mouse chromosome 1 (region 36-44 cM) is reported. Sixteen coding sequences were mapped on the BAC contig. The majority of these were identified by BLAST searches of BAC end sequences and BAC shotgun sequences generated during the positional cloning project. Map data for the orthologues in humans were available for 12 of the 16 coding sequences, and all 12 have been assigned to 2q35. Furthermore, no evidence for any rearrangement in gene order was obtained. The extensive linkage conservation indicates that the near complete human transcript map will be an invaluable resource for positional cloning projects in pigs and other domestic animals.
Asunto(s)
Mapeo Contig , ADN/genética , Animales , Proteínas Portadoras/genética , Proteínas Cromosómicas no Histona/genética , Cromosomas/genética , Cromosomas Artificiales Bacterianos , Cromosomas Humanos Par 2/genética , ADN/química , Humanos , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Filogenia , Receptores de Interleucina-8A/genética , Análisis de Secuencia de ADN , Lugares Marcados de Secuencia , Porcinos , Transcripción GenéticaRESUMEN
This article describes the construction of a high-resolution radiation hybrid map of Hsap 2q35 by using the TNG RH panel generated by irradiation with 50,000 rads. We were able to build a framework map of 1300 cR(50,000) including 34 markers ordered with odds higher than 1000:1. The comprehensive map includes 77 loci and describes a region of 3 Mb around the SLC11A1 gene. Because of the very small size of the fragments retained and a reduced retention frequency, it was difficult to build a high-resolution multi-point map of this region by using the TNG RH panel. Nevertheless, this study confirmed the very high potential of this RH panel for constructing a human, high-resolution physical map (2.3 kb/cR(50,000)). Moreover, human ESTs from Hsap 2q35 were hybridized with porcine BAC contigs to establish a porcine transcript map of the homologous region Sscr 15q25 (greater than 2.5 Mb). We identified 17 new genes in this porcine chromosomal region. We were able to compare the location of 26 genes mapped in both species. The gene order was similar except for two possible minor discrepancies in the Desmin sub-region, suggesting the existence of a porcine micro-region between TNP1 and IL8RB with an unknown origin.