RESUMEN
The variable region of the immunoglobulin heavy chain is created by a somatic rearrangement of a limited number of germline genes. This mechanism of gene assembly [V(D)J recombination] has been found to take place only in jawed vertebrates (gnathostomes). To understand how this mechanism evolved and diversified it is necessary to study the genomic organization of the heavy-chain gene in different vertebrate lineages. Since there is scant sequence information on the VH locus in fish, shotgun sequencing of a cosmid clone containing part of the VH genomic region of the Japanese pufferfish, Fugu rubripes, was undertaken. Eight full-length VH genes were isolated and characterized. They have higher homology to trout genes, but show the same structural features as VHs found in other vertebrates. Two VH subgroups have been identified whose members are interspersed. The frequency of synonymous and nonsynonymous substitution for VH comparisons between family members was found to be higher in the complementarity-determining regions than in the framework regions. Finally, there are four other genes interspersed with the VH genes, one of which is the first full-length retrotransposon element characterized in vertebrates.
Asunto(s)
Genes de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cósmidos/inmunología , Peces Venenosos , Reordenamiento Génico de Cadena Pesada de Linfocito B , Marcadores Genéticos/inmunología , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Culture filtrates derived from a Mycobacterium bovis cosmid library in Mycobacterium smegmatis were screened for bovine lymphocyte stimulatory antigens using peripheral blood mononuclear cells (PBMC) from cattle vaccinated with a low dose of Mycobacterium bovis BCG. Lymphocyte proliferation and interferon-gamma (IFN-gamma) production were used as cellular response markers for antigen recognition. In the primary screen, approximately 28% of all culture filtrates (CF) stimulated responses by PBMC from at least two out of four vaccinated cattle. In one of these CF, the M. bovis Ag85-B antigen was detected by Western-blot analysis. Despite heterogeneous lymphocyte responses of the animals, twenty-four of the culture filtrates stimulated lymphocyte proliferation and IFN-gamma production from at least six out of eight vaccinated animals in a secondary screen. Analysis of the cosmid DNA associated with these positive CF demonstrated that several contained homologous DNA sequences. It appears that the lymphocyte screening has detected M. bovis antigens that are immuno-dominant in cattle vaccinated with M. bovis BCG.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Antígenos de Diferenciación/aislamiento & purificación , Cósmidos/inmunología , Activación de Linfocitos/inmunología , Mycobacterium bovis/genética , Mycobacterium bovis/inmunología , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/inmunología , Adyuvantes Inmunológicos/uso terapéutico , Animales , Antígenos Bacterianos/inmunología , Antígenos de Diferenciación/inmunología , Vacuna BCG/uso terapéutico , Bovinos , División Celular/inmunología , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/metabolismo , Femenino , Biblioteca de Genes , Interferón gamma/biosíntesis , Mycobacterium bovis/química , Mycobacterium smegmatis/crecimiento & desarrollo , Tuberculosis Bovina/prevención & controlRESUMEN
Although numerous solitary germ-line V kappa genes and two small V kappa contiguously cloned gene regions (contigs) are known, no attempts to systematically elucidate the structure of the kappa locus of the mouse have been reported so far. As a first step to this aim we screened a cosmid library of C57BL/6J mouse DNA with 18 probes that are more or less specific for the different V kappa gene families. Ninety-one V kappa gene-containing cosmid clones were characterized by detailed restriction mapping and hybridizations. Several contigs were constructed from overlapping clones. The contigs and the still unlinked cosmid clones cover 1.6 Mb. Many of the cosmid clones were localized on chromosome 6 where the kappa locus is known to reside; no evidence for the existence of dispersed V kappa genes (orphons) was obtained. Eighty-five strong hybridization signals were assigned to distinct V kappa gene families, while for 11 weak signals the assignment was less definite. As to the distribution of gene families within the locus the following situation emerged: there are both, groups of genes which belong to one V kappa gene family ("clusters") and groups in which genes of different families are interspersed. The interspersion of gene families seems to be more pronounced than has been assumed so far. Additional V kappa genes which are known to exist will have to be isolated from other gene libraries of the same mouse Ig kappa haplotype.