{"id":116018,"date":"2014-07-07T00:00:00","date_gmt":"2014-07-07T00:00:00","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/regulacion-de-la-expresion-de-genes-endogenos-por-la-maquinaria-de-silenciamiento-genico-mediado-por-rna-en-mucor-circinelloides\/"},"modified":"2014-07-07T00:00:00","modified_gmt":"2014-07-07T00:00:00","slug":"regulacion-de-la-expresion-de-genes-endogenos-por-la-maquinaria-de-silenciamiento-genico-mediado-por-rna-en-mucor-circinelloides","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/acidos-nucleicos\/regulacion-de-la-expresion-de-genes-endogenos-por-la-maquinaria-de-silenciamiento-genico-mediado-por-rna-en-mucor-circinelloides\/","title":{"rendered":"Regulaci\u00f3n de la expresi\u00f3n de genes end\u00f3genos por la maquinaria de silenciamiento g\u00e9nico mediado por rna en mucor circinelloides."},"content":{"rendered":"<h2>Tesis doctoral de <strong> Ana Vila Martinez <\/strong><\/h2>\n<p>Regulaci\u00f3n de la expresi\u00f3n de genes end\u00f3genos por la maquinaria de silenciamiento g\u00e9nico mediado por rna en mucor circinelloides.  mucor circinelloides es un hongo filamentoso utilizado como modelo para el estudio del mecanismo de silenciamiento g\u00e9nico mediado por rna. Una vez caracterizado dicho mecanismo y los principales genes implicados, se pretende determinar el papel de la ruta de silenciamiento en la regulaci\u00f3n de genes end\u00f3genos, as\u00ed como identificar otros posibles elementos gen\u00e9ticos que participen en dicha ruta. Para ello, se plantean los siguientes objetivos concretos: 1. Identificaci\u00f3n del gen implicado en la eliminaci\u00f3n de la cadena pasajera de los sirnas y estudio de la existencia de interacciones entre las prote\u00ednas de la maquinaria de silenciamiento. 2. Estudio de la participaci\u00f3n de ago-1 en la biog\u00e9nesis de los distintos tipos de ex-sirnas de m. Circinelloides. 3. Identificaci\u00f3n de los genes regulados por el mecanismo de silenciamiento mediante el an\u00e1lisis de los perfiles transcript\u00f3micos de la estirpe silvestre y de mutantes en genes de silenciamiento.  4. Caracterizaci\u00f3n fenot\u00edpica detallada de los mutantes afectados en la ruta de silenciamiento, para identificar los procesos celulares regulados mediante dicho mecanismo. para desarrollar estos objetivos se ha utilizado la siguiente metodolog\u00eda: 1. La identificaci\u00f3n de otros genes implicados en el mecanismo de silenciamiento se llev\u00f3 a cabo mediante la disrupci\u00f3n de los genes candidatos por recombinaci\u00f3n hom\u00f3loga y su posterior an\u00e1lisis fenot\u00edpico. Para ello, se utilizaron t\u00e9cnicas de amplificaci\u00f3n de dna mediante pcr, clonaci\u00f3n, transformaci\u00f3n de protoplastos e hibridaciones tipo southern y northern. Las interacciones entre prote\u00ednas de la maquinaria de silenciamiento se analizaron mediante el sistema de doble h\u00edbrido de levaduras. 2. La participaci\u00f3n de ago-1 en la biog\u00e9nesis de los ex-sirnas se analiz\u00f3 mediante la secuenciaci\u00f3n de librer\u00edas de srnas. Ago-1 fue purificada mediante flpc para el aislamiento y secuenciaci\u00f3n de los srnas unidos a ella. Los datos obtenidos fueron validados experimentalmente mediante hibridaciones tipo northern. 3. El an\u00e1lisis transcript\u00f3mico de las distintas estirpes se realiz\u00f3 mediante hibridaciones con micromatrices y secuenciaci\u00f3n de rna (rna-seq). 4. El an\u00e1lisis fenot\u00edpico de los mutantes en genes de silenciamiento incluy\u00f3 la cuantificaci\u00f3n de la producci\u00f3n de esporas vegetativas y zigosporas, el estudio de la respuesta frente a diferentes situaciones de estr\u00e9s, la cuantificaci\u00f3n del proceso de autolisis y el an\u00e1lisis de la virulencia en galleria mellonella. los resultados obtenidos permitieron alcanzar las siguientes conclusiones: 1. El  gen qip  es esencial para el eficaz funcionamiento del mecanismo de silenciamiento g\u00e9nico inducido por transgenes, sugiriendo un papel en la activaci\u00f3n del complejo risc. No se detectaron interacciones entre prote\u00ednas de la maquinaria de silenciamiento de m. Circinelloides, lo que podr\u00eda deberse a la necesidad de modificaciones post-traduccionales espec\u00edficas de las prote\u00ednas de silenciamiento o a la formaci\u00f3n de complejos multiproteicos. 2. El gen ago-1 est\u00e1 implicado en el mecanismo de silenciamiento end\u00f3geno de m. Circinelloides, siendo necesario para la biog\u00e9nesis y\/o estabilidad de las cuatro clases de ex-sirnas. Las clases i y ii de ex-sirnas se unen espec\u00edficamente a ago-1 para llevar a cabo la identificaci\u00f3n y degradaci\u00f3n de sus mensajeros diana. Las clases iii y iv son generadas por una ruta de silenciamiento no can\u00f3nica, en la que se requiere la participaci\u00f3n de ago-1, pero no la uni\u00f3n espec\u00edfica de estos ex-sirnas a dicha prote\u00edna.  3. Los an\u00e1lisis transcript\u00f3micos ponen de manifiesto que el mecanismo de silenciamiento posee un papel modulador de la expresi\u00f3n de un gran n\u00famero de genes end\u00f3genos a lo largo del crecimiento vegetativo. Las funciones de las prote\u00ednas cifradas por estos genes est\u00e1n relacionadas con la biog\u00e9nesis y modificaci\u00f3n de la pared celular, el control de la divisi\u00f3n celular y el crecimiento, metabolismo de carbohidratos, envejecimiento celular o respuesta a estr\u00e9s.  4. La ruta can\u00f3nica de silenciamiento est\u00e1 implicada en la regulaci\u00f3n del proceso de esporulaci\u00f3n vegetativa y en la activaci\u00f3n del programa de autolisis inducido por estr\u00e9s nutricional. La regulaci\u00f3n del desarrollo sexual debe ser llevada a cabo por una ruta no can\u00f3nica independiente de dicer. Los experimentos de patog\u00e9nesis no revelan una clara participaci\u00f3n del mecanismo de silenciamiento en el proceso de virulencia, aunque no se descarta una respuesta diferencial en otros organismos modelo, como el rat\u00f3n.   regulation of endogenous gene expression by the rna silencing mechanism in mucor circinelloides.  mucor circinelloides is a filamentous fungus used as a model for studying the rna silencing mechanism. Once the mechanism and main genes involved were characterized, the next goal is to determine the role of the rna silencing pathway in the regulation of endogenous genes, and to identify other possible genetic elements that participate in the pathway. To do this, the following specific objectives were proposed: 1. Identification of the gene involved in the removal of the passenger strand of sirnas duplex, and analysis of the interactions between silencing proteins. 2. Study of the participation of ago-1 in the biogenesis of different classes of ex-sirnas in m. Circinelloides. 3. Identification the genes regulated by the rna silencing mechanism by analyzing the transcriptome of the wild type and silencing mutants. 4. Phenotypic characterization of mutants affected in the silencing pathway to identify the cellular processes regulated by this mechanism. to develop these objectives the following methodology has been used: 1. Identification of other genes involved in the silencing pathway was addressed by disrupting candidate genes by homologous recombination and subsequent phenotypic analysis. To this end, pcr amplification of dna fragments, cloning, transformation of protoplasts and southern and northern blot hybridizations were carry out. The study of interaction between rna silencing proteins was performed using the yeast two-hybrid system. 2. Cdna libraries of  srnas were generated and sequenced to analyze the participation of ago-1 in the biogenesis of ex-sirnas. Ago-1 was purified by fplc for the isolation and sequencing of ago-1 bound srnas. The sequencing data were validated by northern-blot experiments. 3. Transcriptomic analysis of the different strains was carried out by hybridization methods using microarrays and rna sequencing (rna-seq). 4. Phenotypic analysis of silencing mutants included quantification of vegetative spore and zygospores, study of the response to different stresses, quantification of the autolysis process and virulence analysis of the different silencing mutants in galleria mellonella.   the results allowed reaching the following conclusions: 1. The qip gene is essential for transgene-induced gene silencing, suggesting a role in the activation of the risc complex. No interactions were detected between proteins involved in the rna silencing pathway of m. Circinelloides. This could be due to the need for specific post-translational modifications of silencing proteins or the formation of multiprotein complexes. 2. Ago-1 is involved in endogenous rna silencing in m. Circinelloides, and it is necessary for the biogenesis and \/ or stability of all ex-sirnas classes. Classes i and ii of ex-sirnas specifically bind to ago-1 for the identification and degradation of their mrna targets. Classes iii and iv are generated by a non-canonical silencing mechanism that requires the participation of ago-1, but there is not specific binding of these ex-sirnas to ago-1 protein. 3. Transcriptomic analysis showed that the silencing mechanism has a modulatory role in the expression of a large number of endogenous genes during vegetative growth. The functions of the proteins coded by these genes are linked to the biogenesis and modification of the cell wall, the control of cell division and growth, carbohydrate metabolism, cell aging or stress response. 4. The canonical rna silencing pathway is involved in regulation of vegetative sporulation and autolysis induced by nutritional stress. Sexual development should be regulated by a non-canonical dicer-independent rna pathway. Pathogenesis experiments did not reveal a role of rna silencing in the virulence process, but a differential response can&apos;t be ruled out when using other model organisms, such as mouse.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Regulaci\u00f3n de la expresi\u00f3n de genes end\u00f3genos por la maquinaria de silenciamiento g\u00e9nico mediado por rna en mucor circinelloides.<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Regulaci\u00f3n de la expresi\u00f3n de genes end\u00f3genos por la maquinaria de silenciamiento g\u00e9nico mediado por rna en mucor circinelloides. <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Ana Vila Martinez <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Murcia<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 07\/07\/2014<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>Rosa Mar\u00eda Ruiz Vazquez<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: arturo P\u00e9rez eslava <\/li>\n<li>Miguel angel Aranda regules (vocal)<\/li>\n<li>Javier Avalos cordero (vocal)<\/li>\n<li>Jos\u00e9 Cansado vizoso (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Ana Vila Martinez Regulaci\u00f3n de la expresi\u00f3n de genes end\u00f3genos por la maquinaria de silenciamiento g\u00e9nico mediado [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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