RESUMEN
Short-lived peripheral blood eosinophils are recruited to the lungs of asthmatics after allergen challenge, where they become long-lived effector cells central to disease pathophysiology. GM-CSF is an important cytokine which promotes eosinophil differentiation, function, and survival after transit into the lung. In human eosinophils, GM-CSF production is controlled by regulated mRNA stability mediated by the 3' untranslated region, AU-rich elements (ARE). We identified human Y box-binding factor 1 (YB-1) as a GM-CSF mRNA ARE-specific binding protein that is capable of enhancing GM-CSF-dependent survival of eosinophils. Using a transfection system that mimics GM-CSF metabolism in eosinophils, we have shown that transduced YB-1 stabilized GM-CSF mRNA in an ARE-dependent mechanism, causing increased GM-CSF production and enhanced in vitro survival. RNA EMSAs indicate that YB-1 interacts with the GM-CSF mRNA through its 3' untranslated region ARE. In addition, endogenous GM-CSF mRNA coimmunoprecipitates with endogenous YB-1 protein in activated eosinophils but not resting cells. Thus, we propose a model whereby activation of eosinophils leads to YB-1 binding to and stabilization of GM-CSF mRNA, ultimately resulting in GM-CSF release and prolonged eosinophil survival.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/fisiología , Proteínas de Unión al ADN , Eosinófilos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Estabilidad del ARN , Factores de Transcripción , Regiones no Traducidas 3' , Asma/inmunología , Proteínas Potenciadoras de Unión a CCAAT/genética , Supervivencia Celular , Productos del Gen tat/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Hipersensibilidad Inmediata/inmunología , Factores de Transcripción NFI , Proteínas Nucleares , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta , Transducción Genética , Proteína 1 de Unión a la Caja YRESUMEN
The DPY-26 protein is required in the nematode Caenorhabditis elegans for X-chromosome dosage compensation as well as for proper meiotic chromosome segregation. DPY-26 was shown to mediate both processes through its association with chromosomes. In somatic cells, DPY-26 associates specifically with hermaphrodite X chromosomes to reduce their transcript levels. In germ cells, DPY-26 associates with all meiotic chromosomes to mediate its role in chromosome segregation. The X-specific localization of DPY-26 requires two dosage compensation proteins (DPY-27 and DPY-30) and two proteins that coordinately control both sex determination and dosage compensation (SDC-2 and SDC-3).
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiología , Cromosomas/fisiología , Compensación de Dosificación (Genética) , Proteínas del Helminto/fisiología , Meiosis , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas Portadoras/fisiología , Núcleo Celular/química , Trastornos del Desarrollo Sexual , Desarrollo Embrionario , Femenino , Expresión Génica , Genes de Helminto , Células Germinativas/fisiología , Proteínas del Helminto/análisis , Proteínas del Helminto/genética , Masculino , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/fisiología , Cromosoma X/fisiologíaRESUMEN
mes-3 is one of four maternal-effect sterile genes that encode maternal components required for normal postembryonic development of the germ line in Caenorhabditis elegans. mes-3 mutant mothers produce sterile progeny, which contain few germ cells and no gametes. This terminal phenotype reflects two problems: reduced proliferation of the germ line and germ cell death. Both the appearance of the dying germ cells and the results of genetic tests indicate that germ cells in mes-3 animals undergo a necrotic-like death, not programmed cell death. The few germ cells that appear healthy in mes-3 worms do not differentiate into gametes, even after elimination of the signaling pathway that normally maintains the undifferentiated population of germ cells. Thus, mes-3 encodes a maternally supplied product that is required both for proliferation of the germ line and for maintenance of viable germ cells that are competent to differentiate into gametes. Cloning and molecular characterization of mes-3 revealed that it is the upstream gene in an operon. The genes in the operon display parallel expression patterns; transcripts are present throughout development and are not restricted to germ-line tissue. Both mes-3 and the downstream gene in the operon encode novel proteins.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Impresión Genómica , Células Germinativas , Proteínas del Helminto/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , Diferenciación Celular , Clonación Molecular , ADN Complementario , Femenino , Expresión Génica , Células Germinativas/citología , Larva , Masculino , Datos de Secuencia Molecular , Operón , Fenotipo , ARN Mensajero/genéticaRESUMEN
The macronucleus of the ciliate Tetrahymena contains about 45 copies of the genome. The fraction of the molecules on which an adenine residue is modified to N6-methyladenine is characteristic of the specific site, and consistent between clones. A fragment of DNA containing a site that is uniformly methylated on the macronuclear chromosome was moved to a new location on the extrachromosomal rDNA. The methylation pattern of the fragment on the rDNA was different from that on the chromosome. The data suggest that DNA sequence is not sufficient to determine the level of methylation.
Asunto(s)
Adenina , ADN Protozoario/metabolismo , ADN Ribosómico/metabolismo , Tetrahymena/genética , Tetrahymena/metabolismo , Adenina/análogos & derivados , Adenina/análisis , Animales , Secuencia de Bases , ADN Ribosómico/genética , Vectores Genéticos , Metilación , Datos de Secuencia Molecular , Plásmidos , Mapeo RestrictivoRESUMEN
To identify genes that encode maternal components required for development of the germ line in the nematode Caenorhabditis elegans, we have screened for mutations that confer a maternal-effect sterile or "grandchildless" phenotype: homozygous mutant hermaphrodites produced by heterozygous mothers are themselves fertile, but produce sterile progeny. Our screens have identified six loci, defined by 21 mutations. This paper presents genetic and phenotypic characterization of four of the loci. The majority of mutations, those in mes-2, mes-3 and mes-4, affect postembryonic germ-line development; the progeny of mutant mothers undergo apparently normal embryogenesis but develop into agametic adults with 10-1000-fold reductions in number of germ cells. In contrast, mutations in mes-1 cause defects in cytoplasmic partitioning during embryogenesis, and the resulting larvae lack germ-line progenitor cells. Mutations in all of the mes loci primarily affect the germ line, and none disrupt the structural integrity of germ granules. This is in contrast to grandchildless mutations in Drosophila melanogaster, all of which disrupt germ granules and affect abdominal as well as germ-line development.
Asunto(s)
Caenorhabditis/genética , Células Germinativas/crecimiento & desarrollo , Animales , Caenorhabditis/embriología , Caenorhabditis/crecimiento & desarrollo , Mapeo Cromosómico , Cruzamientos Genéticos , Prueba de Complementación Genética , Genotipo , Mutación , FenotipoRESUMEN
We have cloned two DNA fragments containing 5'-GATC-3' sites at which the adenine is methylated in the macronucleus of the ciliate Tetrahymena thermophila. Using these cloned fragments as molecular probes, we analyzed the maintenance of methylation patterns at two partially and two uniformly methylated sites. Our results suggest that a semiconservative copying model for maintenance of methylation is not sufficient to account for the methylation patterns we found during somatic growth of Tetrahymena. Although we detected hemimethylated molecules in macronuclear DNA, they were present in both replicating and nonreplicating DNA. In addition, we observed that a complex methylation pattern including partially methylated sites was maintained during vegetative growth. This required the activity of a methylase capable of recognizing and modifying sites specified by something other than hemimethylation. We suggest that a eucaryotic maintenance methylase may be capable of discriminating between potential methylation sites to ensure the inheritance of methylation patterns.