Escrutinios genéticos con mutantes de fosfatasas implicadas en transcripción en la levadura saccharomyces cerevisiae
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2014-11-18
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Jaén: Universidad de Jaén
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[ES]En las levaduras, así como en todos los eucariotas en general, la transcripción se realiza mediante 3 ARN polimerasas, la ARN pol I, II y III, encargadas de sintetizar distintos tipos de transcritos. Concretamente la ARN polimerasa II, formada por 12 subunidades, es la responsable de la síntesis de todos los ARNm de la célula. Una de estas subunidades, Rpb1, contiene un dominio carboxilo terminal (CTD) indispensable para el procesamiento del ARN, siendo su deleción letal en levaduras y causando defectos de crecimiento por mutaciones. Una mutación en el pie de la ARN pol II, concretamente rpo21-4, resulta letal para la levadura en conjunto con la deleción para el gen de la fosfatasa del CTD, Rtr1. En este trabajo, con el fin de buscar genes que supriman la letalidad sintética provocada por la combinación de la mutación rpo21-4 y la falta de expresión del gen para la fosfatasa Rtr1 en la levadura Saccharomyces cerevisiae, se llevó a cabo un escrutinio genético masivo con la cepa YFN498, que combina la mutación rpo21-4 y la falta de expresión del gen RTR1. Para dicho escrutinio se utilizó una genoteca del genoma completo de S. cerevisiae, en plásmido multicopia. Nuestros resultados han permitido identificar 14 posibles supresores de dicha letalidad sintética, capaces de conferir crecimiento a la cepa YFN498.
[EN]In yeasts, as well as in all eukaryotes in general, transcription is mediated by three RNA polymerases, RNA pol I, II and III, responsible for the synthesis of different types of transcripts. Specifically, RNA pol II, consisting of 12 subunits, is responsible for the synthesis of every mRNA in cells. One of these subunits, Rpb1, contains a C-terminal domain (CTD), which is essential for RNA processing, being lethal when deleting it in yeasts and causing growth defects when is mutated. One mutation in the foot domain of RNA pol II, rpo21-4, is lethal when combined with a deletion for the gene coding the CTD phosphatase Rtr1. In this work, in an attempt to look for genes that suppress the synthetic lethality caused by the combination of the rpo21-4 mutation and the lack of 4 expression of RTR1 gene in S. cerevisiae, we performed a genetic screening with the YFN498 strain, which combines the rpo21-4 mutation and the lack of expression of the RTR1 gene. For this screening, a library containing the whole S. cerevisiae genome in multicopy plasmid was used. Our results have identified 14 possible suppressors able to recover growth to the YFN498 strain.
[EN]In yeasts, as well as in all eukaryotes in general, transcription is mediated by three RNA polymerases, RNA pol I, II and III, responsible for the synthesis of different types of transcripts. Specifically, RNA pol II, consisting of 12 subunits, is responsible for the synthesis of every mRNA in cells. One of these subunits, Rpb1, contains a C-terminal domain (CTD), which is essential for RNA processing, being lethal when deleting it in yeasts and causing growth defects when is mutated. One mutation in the foot domain of RNA pol II, rpo21-4, is lethal when combined with a deletion for the gene coding the CTD phosphatase Rtr1. In this work, in an attempt to look for genes that suppress the synthetic lethality caused by the combination of the rpo21-4 mutation and the lack of 4 expression of RTR1 gene in S. cerevisiae, we performed a genetic screening with the YFN498 strain, which combines the rpo21-4 mutation and the lack of expression of the RTR1 gene. For this screening, a library containing the whole S. cerevisiae genome in multicopy plasmid was used. Our results have identified 14 possible suppressors able to recover growth to the YFN498 strain.
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