La investigación en el laboratorio de CJN con el apoyo de subvenciones de funcionamiento de NSERC, el Instituto Canadiense del Cáncer Research Society (CCSRI) y la Canadian Breast Cancer Foundation (BC-Yukon Branch).

NOTA: El flujo de trabajo general de este procedimiento se describe en la Figura 2. 1. Determinación de la dosis inhibidora del crecimiento <ol><li> Preparación de la levadura cultivo durante la noche NOTA: Los medios de crecimiento de la levadura-extracto-peptona-dextrosa (YEPD) utilizado en este protocolo es una receta estándar 13. <ol><li> Streak a las células en una placa de agar YEPD BY4741 (Mata his3 Δ1 leu2Δ0 met15Δ0 ura3Δ0) y se incuba durante 48 horas a 30 ° C o hasta que se formen colonias visibles. </li><li> Preparar cultivos de una noche mediante la inoculación de 5 ml de medio líquido YEPD en un recipiente de cultivo estéril con una sola colonia. Incubar durante la noche a 30 ° C con rotación continua o agitación. La cultura se suele llegar a la saturación por la mañana (~ 2 × 10 8 células / ml). </li></ol></li><li> Preparación de medios de agar sólido que contiene dosis químicas diferentes <ol><li> Prepare varias poblaciones deel producto químico a ensayar en 100x concentración final deseada en un disolvente apropiado. NOTA: Las concentraciones eficaces dependerán de la química, y deben ser determinado empíricamente. Por tanto, es aconsejable probar inicialmente un amplio rango de concentración final; es decir, de baja M a alta mM. </li><li> Para cada concentración de producto químico a ensayar, alícuota de 3 ml de agar YEPD fundida a un tubo de cultivo estéril varios. Lugar en el 55 ° C baño de agua para mantener la solidificación. </li><li> Para cada concentración de producto químico a ensayar añadir 30 l de compuesto 100x a los medios fundidos, vórtice 2-3 segundos para mezclar, y pipeta de 1 ml en cada par de pocillos duplicados en una placa de 12 pocillos. Asegúrese de incluir un vehículo único control. Permitir a las placas reposar durante la noche a temperatura ambiente. Deseche cualquier medio extra. </li></ol></li><li> Células Spread chapado <ol><li> Inocular 10 ml de medio líquido YEPD con 2 l de un cultivo de levadura saturado, cultivadas durante la noche como en el paso 1.1.2. </li><li> Placa de 25 l de cultivo de levadura diluida por pocillo, distribuidos uniformemente usando perlas de vidrio estériles o varilla, y permiten que la placa se seque bajo una llama. Incubar la placa a 30 ° C durante 48 horas. </li><li> Evaluar el crecimiento de la levadura (tanto en número total y el tamaño de las colonias) en las placas. Seleccione una concentración subletal de compuesto que no inhibe el crecimiento en más de un 10% -15% para realizar la pantalla. </li></ol></li></ol> 2. Sistemática Pantalla Química Genética <ol><li> Mantenimiento de eliminación-mutante-array (DMA) y Preparación de Fuente Plate <ol><li> Para una descripción detallada de la construcción de matrices de mutantes de deleción de las existencias de glicerol consulte Baryshnikova et al. 14. Una vez mutantes de deleción han sido dispuestos a una densidad de 384 colonias por placa, la DMA se puede almacenar durante varios meses a 4 ° C. Replicar en agar YEPD que contiene 200 mg / ml de G418 si es necesario para prevenir el crecimiento de colonias en cadaotra. NOTA: Varias réplicas de serie se deben evitar para prevenir la pérdida de estirpes de crecimiento lento y minimizar la aparición de mutantes supresores. Esto es particularmente relevante si el mantenimiento de las colecciones como haploides. </li><li> Realice todos los pasos réplica de galvanoplastia utilizando un microbiana arraying sistema robótico. Alternativamente, manipular colonias utilizando herramientas manuales de colocación de clavos dispuestos. NOTA: La colección de deleción de levadura está disponible como haploides y diploides a partir de varias fuentes comerciales y se envía típicamente como stocks de glicerol en placas de 96 pocillos. </li></ol></li><li> Condensar el array mutante de deleción <ol><li> Preparar 250 ml de medio YEPD agar que contiene 200 mg / ml de G418. La concentración efectiva de G418 puede variar y cada lote debe ser probado empíricamente. </li><li> Preparar cinco placas de agar YEPD vertiendo 50 ml de agar YEPD que contiene 200 mg / ml de G418 por placa. Haga esto un día antes de condensar la matriz y permita que las placas se enfríen a temperatura ambientebibliografía durante la noche en una superficie plana. NOTA: Se requieren cuatro placas de la colección en la densidad de 1536-deformación, la quinta placa se vierte como extra. </li><li> Retire las 16 placas de Petri que contienen la colección mutante de deleción a una densidad de 384 colonias por placa y se deja llegar a temperatura ambiente (~ 1 h). </li><li> Limpiar cualquier condensación que se ha formado en la tapa de cada placa utilizando una tarea delicada toallita. El no hacerlo puede resultar en las gotas de agua que se deposita en la matriz y la contaminación cruzada de los mutantes dispuestos. </li><li> El uso de una matriz fijando robot microbiana, condensar el DMA de una densidad de 384 colonias por placa (16 platos de Petri) para 1536 colonias por placa (4 platos de Petri). Realice este modo que el 1 de colonia de la placa 1 pines a la fila 1 columna 1 de la matriz condensada, el 1 de colonia de la placa 2 a la fila 1 columna 2, el 1 de colonia de la placa 3 de fila 2 y la columna 1, y la colonia 1 de placa de 4 a la fila 2columna 2. </li><li> Incubar la matriz consolidada a 30 ° C durante la noche. </li><li> Examine la matriz para asegurar la transferencia uniforme de colonias de las placas de origen. Habrá varias posiciones en blanco, a propósito incorporados en la matriz, que sirven como una guía para asegurar la DMA se ha condensado correctamente (Figura 4A). NOTA: Estos serán los platos de origen de réplica en placa en medios que contienen el compuesto de ensayo. Una placa de una sola fuente puede ser utilizado para múltiples apuntalamientos. También muchos robots tendrán una función de compensación, lo que garantizará un número similar de levadura se están transfiriendo cada vez. </li></ol></li><li> Replica placa matriz mutante de deleción <ol><li> Preparar 700 ml de control (vehículo solo) y 700 ml de medio experimental (que contienen productos químicos). Esto es suficiente para realizar el ensayo por triplicado (12 placas por condición, además de dos extras). </li><li> Verter 50 ml de agar YEPD contengan la sustancia problema o control por placa. Permitir los platos to enfriar a temperatura ambiente durante la noche en una superficie plana. </li><li> Utilizando el robot para réplica de la placa, inocular el DMA en tres conjuntos de placas que contienen los productos químicos en la concentración determinado experimentalmente, así como tres conjuntos de placas que contienen el control del vehículo. Incubar las placas a temperatura ambiente durante 24-48 horas. </li></ol></li></ol> 3. Las placas de imagen y análisis de datos <ol><li> Retire las placas de la incubadora y permitir que alcancen la temperatura ambiente (~ 1 hora) antes de la imagen. Esto evitará que se forme condensación mientras que las imágenes de las placas. </li><li> Las placas radiográficas a las 24 y 48 horas por retirar la tapa y colocar boca abajo en un escáner de cama plana. Captura imágenes a una resolución de al menos 300 dpi. También puede utilizar una cámara digital para capturar imágenes. Si al hacerlo, placas lugar boca arriba sobre un fondo oscuro y quitar las tapas de imagen. NOTA: El formato de archivo y la colocación de placas dependerán del programa de cuantificación utilizado. </li&gt; <li> Realizar la cuantificación y la comparación de tamaños de colonia de matriz utilizando uno de varios programas de código abierto, incluyendo Balony 15, SGAtools 16 y ScreenMill 17. </li></ol> 4. Validación de Pantalla NOTA: En esta etapa varias supresiones levaduras mutantes puntuarán como hipersensibilidad a la sustancia química de interés. Estas interacciones químicas genética próximo deben ser validados en dos maneras. En primer lugar la identidad de las cepas sensibles debe ser confirmada por PCR. En segundo lugar, la sensibilidad química debe ser puntuado de forma independiente. A continuación ofrecemos una breve protocolo para la realización de ensayos de manchado para validar hipersensibilidad cepa, una técnica común en la biología de la levadura. <ol><li> Streak cabo deseada (hipersensible) mutantes de la colección de levadura supresión en agar YEPD que contiene 200 mg / ml de G418. Se incuba a 30 ° C hasta que se formen colonias. Verificar el genotipo de la cepa por PCR con cebadores de diagnóstico segúnel protocolo del fabricante. </li><li> Inocular 5 ml del líquido YEPD para cada cepa e incubar durante la noche a 30 ° C con rotación o agitación. </li><li> Mide 600 OD y diluir cada cepa a OD 600 = 1 en DH estéril 2 O. Estos son ahora los cultivos de levaduras normalizados. </li><li> Prescindir de 100 l de normalizado levadura de tipo salvaje (BY4741) cultura para así A1 de una placa de 96 pocillos. Prescindir de 100 l de hasta siete cepas adicionales de pozos B1-H1. </li><li> Utilice una pipeta multicanal para dispensar 90 l de dH 2 O estéril para pozos A2-H2 a través A6-H6. Finalmente, preparar una serie de diluciones 1:10 de cultivos de levadura normalizados. Iniciar pipeteando en serie 10 l de la columna 1 a la columna 2 con una pipeta multicanal, y continuar a través de la placa de 96 pocillos hasta que se hagan seis diluciones (es decir, 10 0 a 10 -5). </li><li> Transferencia de 2-5 l de cultivos de levadura diluidas en una cuadrícula utilizando un multicanalpipeta sobre YEPD medios sólidos que contienen sustancias químicas y el control del vehículo. Incubar las placas a la temperatura adecuada durante 24-48 h. </li><li> Inspeccionar placas y comparar la sensibilidad de seleccionar mutantes para química (en relación con BY4741 control). Las placas radiográficas a las 24 y 48 horas como en el paso 3.2. NOTA: Si bien la identificación de varias cepas hipersensibles con ontologías de genes relacionados o funciones presta confianza para la validez de la pantalla, se requiere la transformación de una cepa de deleción hipersensibles con un plásmido que contiene una copia de tipo salvaje del gen para establecer formalmente que un gen eliminación de interés (y las mutaciones no ocultos segundo sitio) provoca sensibilidad a los fármacos. </li></ol>

<ol>
	<li>Giaever, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+profiling+of+the+Saccharomyces+cerevisiae+genome.">Functional profiling of the Saccharomyces cerevisiae genome.</a> Nature. 418, 387-391 (2002).</li><li>Winzeler, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+the+S.+cerevisiae+genome+by+gene+deletion+and+parallel+analysis.">Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.</a> Science. 285, 901-906 (1999).</li><li>Breslow, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+strategy+enabling+high-resolution+functional+analysis+of+the+yeast+genome.">A comprehensive strategy enabling high-resolution functional analysis of the yeast genome.</a> Nat Methods. 5, 711-718 (2008).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+exploration+of+essential+yeast+gene+function+with+temperature-sensitive+mutants.">Systematic exploration of essential yeast gene function with temperature-sensitive mutants.</a> Nat Biotechnol. 29, 361-367 (2011).</li><li>Costanzo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+landscape+of+a+cell.">The genetic landscape of a cell.</a> Science. 327, 425-431 (2010).</li><li>Parsons, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+chemical-genetic+and+genetic+interaction+data+links+bioactive+compounds+to+cellular+target+pathways.">Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways.</a> Nat Biotechnol. 22, 62-69 (2004).</li><li>Parsons, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+mode-of-action+of+bioactive+compounds+by+chemical-genetic+profiling+in+yeast.">Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast.</a> Cell. 126, 611-625 (2006).</li><li>Hillenmeyer, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+genomic+portrait+of+yeast:+uncovering+a+phenotype+for+all+genes.">The chemical genomic portrait of yeast: uncovering a phenotype for all genes.</a> Science. 320, 362-365 (2008).</li><li>Hillenmeyer, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+analysis+of+genome-wide+fitness+data+in+yeast+reveals+novel+gene+function+and+drug+action.">Systematic analysis of genome-wide fitness data in yeast reveals novel gene function and drug action.</a> Genome Biol. 11, R30 (2010).</li><li>Smith, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competitive+genomic+screens+of+barcoded+yeast+libraries.">Competitive genomic screens of barcoded yeast libraries.</a> J Vis Exp. (54), (2011).</li><li>Bowie, D., Parvizi, P., Duncan, D., Nelson, C. J., Fyles, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical-genetic+identification+of+the+biochemical+targets+of+polyalkyl+guanidinium+biocides.">Chemical-genetic identification of the biochemical targets of polyalkyl guanidinium biocides.</a> Organic &amp; Biomolecular Chemistry. 11, 4359-4366 (2013).</li><li>Alamgir, M., Erukova, V., Jessulat, M., Azizi, A., Golshani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical-genetic+profile+analysis+of+five+inhibitory+compounds+in+yeast.">Chemical-genetic profile analysis of five inhibitory compounds in yeast.</a> BMC Chem Biol. 10 (6), (2010).</li><li>Amberg, D. C., Burke, D., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cold Spring Harbor Laboratory. Methods In Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. , (2005).</li><li>Baryshnikova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+genetic+array+(SGA)+analysis+in+Saccharomyces+cerevisiae+and+Schizosaccharomyces+pombe.">Synthetic genetic array (SGA) analysis in Saccharomyces cerevisiae and Schizosaccharomyces pombe.</a> Methods Enzymol. 470, 145-179 (2010).</li><li>Young, B. P., Loewen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balony:+a+software+package+for+analysis+of+data+generated+by+synthetic+genetic+array+experiments.">Balony: a software package for analysis of data generated by synthetic genetic array experiments.</a> BMC Bioinformatics. 14, 354 (2013).</li><li>Wagih, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SGAtools:+one-stop+analysis+and+visualization+of+array-based+genetic+interaction+screens.">SGAtools: one-stop analysis and visualization of array-based genetic interaction screens.</a> Nucleic Acids Res. 41, W591-W596 (2013).</li><li>Dittmar, J. C., Reid, R. J., Rothstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScreenMill:+a+freely+available+software+suite+for+growth+measurement,+analysis+and+visualization+of+high-throughput+screen+data.">ScreenMill: a freely available software suite for growth measurement, analysis and visualization of high-throughput screen data.</a> BMC Bioinformatics. 11, 353 (2010).</li><li>Longley, D. B., Harkin, D. P., Johnston, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-fluorouracil:+mechanisms+of+action+and+clinical+strategies.">5-fluorouracil: mechanisms of action and clinical strategies.</a> Nat Rev Cancer. 3, 330-338 (2003).</li><li>Lum, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+modes+of+action+for+therapeutic+compounds+using+a+genome-wide+screen+of+yeast+heterozygotes.">Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes.</a> Cell. 116, 121-137 (2004).</li><li>Toussaint, M., Conconi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+and+sensitive+assay+to+measure+yeast+cell+growth:+a+bench+protocol+for+testing+genotoxic+agents.">High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents.</a> Nat Protoc. 1, 1922-1928 (2006).</li><li>Robinson, M. D., Grigull, J., Mohammad, N., Hughes, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FunSpec:+a+web-based+cluster+interpreter+for+yeast.">FunSpec: a web-based cluster interpreter for yeast.</a> BMC Bioinformatics. 3, 35 (2002).</li><li>Huang da, W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+bioinformatics+resources.">Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.</a> Nat Protoc. 4, 44-57 (2009).</li><li>Huang da, W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatics+enrichment+tools:+paths+toward+the+comprehensive+functional+analysis+of+large+gene+lists.">Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.</a> Nucleic Acids Res. 37, 1-13 (2009).</li><li>Hong, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Ontology+annotations+at+SGD:+new+data+sources+and+annotation+methods.">Gene Ontology annotations at SGD: new data sources and annotation methods.</a> Nucleic Acids Res. 36, D577-D581 (2008).</li><li>Fang, F., Hoskins, J., Butler, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-fluorouracil+enhances+exosome-dependent+accumulation+of+polyadenylated+rRNAs.">5-fluorouracil enhances exosome-dependent accumulation of polyadenylated rRNAs.</a> Mol Cell Biol. 24, 10766-10776 (2004).</li><li>Baba, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+Escherichia+coli+K-12+in-frame,+single-gene+knockout+mutants:+the+Keio+collection.">Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.</a> Mol Syst Biol. 2, 0008 (2006).</li><li>Nichols, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+landscape+of+a+bacterial+cell.">Phenotypic landscape of a bacterial cell.</a> Cell. 144, 143-156 (2011).</li><li>Dai, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+nucleosome+function:+a+highly+versatile+library+of+synthetic+histone+H3+and+H4+mutants.">Probing nucleosome function: a highly versatile library of synthetic histone H3 and H4 mutants.</a> Cell. 134, 1066-1078 (2008).</li></ol>

The authors declare they have no competing financial interests.

Esbozado aquí es un enfoque para la generación de perfiles de interacción química y genética. El método es simple: mediante la comparación de tamaños de colonia de cada cepa en colecciones integrales de supresión de genes en presencia y ausencia de un producto químico, todos los genes necesarios para tolerar un insulto química se identifican. Análisis de enriquecimiento de anotación de la lista resultante de cepas sensibles a continuación, se puede utilizar para proporcionar información sobre un modo de p...

Las herramientas genéticas y los recursos disponibles en el organismo modelo Saccharomyces cerevisiae han permitido a gran escala estudios de genómica funcional que proporcionan colectivamente una nueva visión de cómo los genes funcionan como redes para cumplir con los requisitos de los sistemas biológicos. La piedra angular de estas herramientas fue la creación en colaboración de un conjunto completo de no esenciales supresiones de genes de todos los marcos de lectura abierta en la levadura 1,2. Una observación sorprendente fue que sólo ~ 20% de los genes de levadura se requiere para la viabilidad cuando se cultiva como haploides en condiciones estándar de laboratorio. Esto pone de relieve la capacidad de una célula para amortiguar contra perturbaciones genómico a través de la utilización de las vías biológicas alternativas. Mutantes genéticos que sean viables individualmente, pero letal en combinación, señalan caminos biológicos paralelos conectados o convergentes y forman redes de interacción genéticos que describen la función biológica. Con el desarrollo de te condicionalalelos mperatura sensibles y hypomorphic de genes esenciales de la tecnología no se ha limitado al estudio de los genes no esenciales 3,4. Este concepto ha sido aplicado en una escala genómica producir un imparcial mapa interacción genética que ilustra cómo los genes implicados en los procesos celulares similares se agrupan 5. Perturbaciones químicas de redes genéticas imitan supresiones de genes (Figura 1) 6. Consulta de compuestos inhibidores del crecimiento contra un array de alta densidad de cepas de deleción para la hipersensibilidad identifica un perfil de interacción química y genética, es decir, una lista de genes que se requiere para tolerar el estrés químico. Al igual que las interacciones genéticas, pantallas de gran escala de las bibliotecas químicas han demostrado que los compuestos con un modo similar de racimo acción juntos 7. Por lo tanto, mediante el establecimiento del perfil de interacción química y genética de un compuesto el modo de acción se puede deducir mediante la comparación con LARinteracción genética genética y química sintética ge-escala datasets 8,9. Pantallas de química y genética a gran escala, donde son interrogados decenas de compuestos, se han realizado ensayos de competición por código de barras. En este enfoque, la colección agrupada de las cepas de deleción se cultiva en masa durante varias generaciones en un pequeño volumen de medio que contiene una sustancia química. Dado que cada mutante de deleción alberga un código de barras genético único, la viabilidad / crecimiento de mutantes individuales dentro de la piscina de supresión cepas se realiza un seguimiento de microarrays o de alto rendimiento de secuenciación 10. Inferir la aptitud mediante el control de tamaño de las colonias de mutantes físicamente dispuestos cultivadas en agar sólido que contiene un compuesto bioactivo es también un método eficaz para identificar las interacciones química y genética 11,12. Este enfoque proporciona una alternativa rentable a la selección basada en la competencia y es muy adecuado para el ensayo de las pequeñas bibliotecas de productos químicos.Esbozado aquí es una metodología sencilla para producir una lista de interacciones químicas genética en S. cerevisiae que no se basa en la manipulación molecular o la infraestructura de biología. Se requiere sólo una colección deleción de levadura, un aparato de colocación de clavos robótico o manual, y el software de análisis de imagen disponible libremente.

<table border="0" cellpadding="0" cellspacing="0" width="987">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Name of Material/ Equipment&#160;</td>
			<td style="width:161px;">Company&#160;</td>
			<td style="width:156px;">Catalog Number&#160;</td>
			<td style="width:457px;">Comments/Description&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:213px;">Yeast Extract</td>
			<td style="width:161px;">BioBasic</td>
			<td>G0961</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 1% final concentration (w/v)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:213px;">Tyrptone Powder</td>
			<td style="width:161px;">BD Biosciences</td>
			<td>211820</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 2% final concentration (w/v)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Dextrose</td>
			<td style="width:161px;">Anachemia</td>
			<td style="width:156px;">31096-380</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 2% final concentration (w/v) - Do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Agar A</td>
			<td style="width:161px;">Bio Basic</td>
			<td>FB0010</td>
			<td style="width:457px;">For YEPD solid media add to 2% final concentration (w/v)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:213px;">G418</td>
			<td style="width:161px;">A.G. Scientific Inc.</td>
			<td style="width:156px;">G-1033</td>
			<td style="width:457px;">Prepare 1000x stock at 200mg/ml in dH2O and filter sterilize.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">12 well plate</td>
			<td>Greiner Bio One</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">5ml culture tubes</td>
			<td>Evergreen Scientific</td>
			<td>222-2376-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">10cm Petri Dish</td>
			<td style="width:161px;">VWR</td>
			<td>25384-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROTOR HDA</td>
			<td>Singer Instruments&#160;</td>
			<td>ROT-001</td>
			<td>high-throughput microbial array pinning robot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PLUSPLATE&#169; Petri Dish</td>
			<td>Singer Instruments&#160;</td>
			<td>PLU-001</td>
			<td>Box of 200 dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384 Short-Pin RePad</td>
			<td>Singer Instruments&#160;</td>
			<td>RP-MP-384</td>
			<td>Box of 1000 pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1536 Short-Pin RePad</td>
			<td>Singer Instruments&#160;</td>
			<td>RP-MP-1536</td>
			<td>Box of 1000 pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alternative Pinning Tools:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fully Automated Robtic Systems</td>
			<td>S&#38;P robotics</td>
			<td>http://www.sprobotics.com</td>
			<td>Several automated colony handling robitic and imagining systems available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manual Pinning Tools</td>
			<td>V&#38;P Scientific</td>
			<td>http://www.vp-scientific.com</td>
			<td>Handheld replication tools and accessories.&#160;</td>
		</tr>
	</tbody>
</table>

rapid identification of chemical genetic interactions in saccharomyces cerevisiae

Determinar el modo de acción de los productos químicos bioactivos es de interés para una amplia gama de académico, farmacéutico, y los científicos industriales. Saccharomyces cerevisiae, o la levadura en ciernes, es un modelo eucariota para la que una colección completa de ~ 6000 mutantes de deleción de gen y gen esencial hypomorphic mutantes están disponibles comercialmente. Estas colecciones de mutantes se pueden utilizar para detectar sistemáticamente la interacción química de genes, es decir, genes necesarios para tolerar una sustancia química. Esta información, a su vez, informa sobre el modo probable de acción del compuesto. Aquí se describe un protocolo para la identificación rápida de las interacciones químicas genética de la levadura en ciernes. Demostramos el método que utiliza el agente quimioterapéutico 5-fluorouracilo (5-FU), que tiene un mecanismo bien definido de acción. Nuestros resultados muestran que la TRAMP nuclear RNA se necesitan enzimas de exosoma y de reparación del ADN para la proliferación en presencia de 5-FU, que es coherente con m anterioricroarray enfoques basados ​​de códigos de barras químicos genéticos y de los conocimientos que el 5-FU afecta negativamente tanto el metabolismo de ARN y ADN. También se describen los protocolos de validación solicitada de estas pantallas de alto rendimiento.

Como una validación de este enfoque se realizó una pantalla genética química representante interacción del agente quimioterapéutico 5-fluorouracilo (5-FU) siguiendo el protocolo descrito anteriormente. 5-FU es conocido para interrumpir la timidilato sintasa, así como ADN y ARN metabolismo 18. Las interacciones genéticas químicas de 5-FU son bien estudiado y se han investigado mediante técnicas de microarrays de código de barras de la levadura utilizando ambas colecciones supresión heterocigotos y ...

Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

La rápida identificación de químicos interacciones genéticas en<em&gt; Saccharomyces cerevisiae</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

rapid-identification-chemical-genetic-interactions-saccharomyces

Research

JoVE Journal

Biology

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

10.3K vistas.  University of Victoria. Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Video: Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

La disección de Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Investigación

Biología

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identificación de los complejos de proteínas con la proteómica cuantitativa en S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Phosphoproteomics cuantitativo de ácidos grasos estimulada Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

El análisis de microarrays para Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Un ensayo de microscopía de fluorescencia para Mitophagy Vigilancia de la levadura Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Expresión y purificación de la proteína transmembrana de la fibrosis quística regulador de la conductancia en la Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

El análisis genético rápido de epitelio-mesenquimal de señalización durante la regeneración del cabello

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Los ensayos acoplados para Monitoreo replegamiento de proteínas en<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Determinación basada en el crecimiento y en la confirmación bioquímica de Requisitos Genéticos para la degradación de proteínas en<em&gt; Saccharomyces cerevisiae</em

La rápida identificación de químicos interacciones genéticas en

Resumen

Explorar más videos

Capítulos en este video

La disección de Saccharomyces cerevisiae Asci

Identificación de los complejos de proteínas con la proteómica cuantitativa en S. cerevisiae

Phosphoproteomics cuantitativo de ácidos grasos estimulada Saccharomyces cerevisiae

El análisis de microarrays para Saccharomyces cerevisiae

Un ensayo de microscopía de fluorescencia para Mitophagy Vigilancia de la levadura Saccharomyces cerevisiae

Expresión y purificación de la proteína transmembrana de la fibrosis quística regulador de la conductancia en la Saccharomyces cerevisiae

El análisis genético rápido de epitelio-mesenquimal de señalización durante la regeneración del cabello

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia

Los ensayos acoplados para Monitoreo replegamiento de proteínas en

Determinación basada en el crecimiento y en la confirmación bioquímica de Requisitos Genéticos para la degradación de proteínas en