El trabajo en el laboratorio del autor está financiado por los Institutos Canadienses de Investigación en Salud, la Fundación Canadiense para la Innovación, el de Ciencias Naturales e Ingeniería de Investigación y la Universidad de Concordia.

Construcción de cepas diploides Protocolo para la producción de diploides en el apareamiento <ol><li> Las cepas inicial debe ser ponchado en medio YPD, o selectiva, si es necesario, para producir colonias aisladas. Las dos cepas haploides debe ser de tipo de apareamiento opuesto (es decir, Mata y MATα). </li><li> El uso de un asa de siembra estéril recoger una pequeña parte de una colonia de una de las dos cepas haploides. En una racha de nueva placa YPD el circuito una vez en una línea recta a través de la placa. Marca la dirección de la veta con una flecha en la parte inferior de la placa. </li><li> Repita esta operación para la segunda cepa de la placa de YPD mismo. Esta vez la racha perpendicular a la primera colonia, cruzar la primera racha con el segundo. Marca la dirección de la segunda racha y tenga en cuenta la zona en la segunda racha cruza el primero. Esta área será donde se producirán los diploides. </li><li> Incubar la placa de YPD a 30 ° C durante la noche. </li><li> Replica la placa de la placa de YPD en medios selectivos que permiten selectivamente el crecimiento de los diploides recién formado. </li><li> Elija una sola colonia, si es posible, o una pequeña porción de la racha de diploides y raya en el medio selectivo misma para generar colonias aisladas. </li></ol> Esporulación y la digestión de los ascos Protocolo <ol><li> Elige colonias aisladas de la placa por encima y hacer pequeños (1 cm x 1 cm) en los parches de esporulación (SPO) placas e incubar a 30 ° C durante 3-7 días a esporas. </li><li> La esporulación se puede comprobar observando las células de cada parche en un microscopio de luz. Células de lugar en un portaobjetos de 5.10 l de agua, coloque un cubreobjetos en la parte superior de la gota, y buscar la presencia de ascos. Si ascos no son fáciles de encontrar a continuación, poner de nuevo en la incubadora. </li><li> Si hay esporulación efectiva en uno de los parches, preparar un tubo cónico de 15 ml estéril con 100μL de jugo de tétrada. </li><li> Recoger las células frente a este parche esporulación con un asa de siembra estéril de 3 mm (células que cubren ¼ del bucle) y el lugar en el jugo de la tétrada. Haga girar suavemente el bucle para permitir que las células se caiga el circuito y mezclar con la solución. </li><li> Agregar el jugo tétrada (que contiene zymolyase) de 4-10 minutos (dependiendo de la concentración y la actividad de zymolyase y el propio esfuerzo). Esto permite la digestión de la pared celular que rodea el ascosporas. </li><li> Marque la parte superior e inferior de la región de inóculo en una placa de YPD. </li><li> Opcional. La reacción puede ser detenida mediante la adición de 1 ml de hielo frío estéril dH 2 O. Permitir que las células que conformarse con 5 minutos. Retire 1 ml del sobrenadante y mezclar suavemente con la mano y mantener en hielo. </li><li> Tome 5μL de las células digieren desde el paso 5 y suavemente poner gotas en una línea en la región de inóculo de la placa de YPD pre-marcadas. </li><li> Use un asa estéril la inoculación de 3 mm para difundir con mucho cuidado los tiempos de 1-3 gotas en una línea, mientras que apenas hacer contacto con el agar. </li><li> Deje que el líquido se absorba en el agar (Si se omite el paso 7, esto es cuando la digestión de la pared celular externa se reducirá o parada). Continúe con el microscopio de disección. </li></ol> La disección de Asci Protocolo Tenga en cuenta que el siguiente protocolo se describe de un microscopio cantante MSM Sistema de micromanipulación 200. Este protocolo se puede seguir con ligeras modificaciones, si se utiliza un micromanipulador manual como un micromanipulador Zeiss MR. <ol><li> Centrarse en una región de la placa donde la mitad del campo contiene las células y la otra mitad está vacía. Poco a poco poner la aguja en el foco de tal manera que toca la región vacía del campo. Esto asegurará que la aguja no contiene células de un uso anterior. El contacto entre la aguja y el agar es visto como un círculo negro oscuro que poco a poco entra en el foco. </li><li> Ir a la matriz en la posición A1 y hacer una marca por la perforación del agar de nuevo. Esto es para ayudar a determinar la orientación de la placa si se saca el microscopio antes de su finalización. </li><li> Cambiar al modo de búsqueda. Localice una ascos. Preferiblemente elija ascos que se encuentran en zonas donde las células están bien hacia fuera. Evite las zonas con grupos de células. Los cuatro ascosporas de un ascos aún debe ser unidas unas a otras. Dos ascosporas pueden ser ligeramente más grande que los otros dos. Evitar la captación de ascos en un ascosporas está suelto y no está conectada con el resto de las ascosporas. </li><li> Recoger un asco con la aguja. Poco a poco poner la aguja en el plato. Cuando la sombra de las microagujas que se ve, esta línea con el asco para ser recogidos. Nuevo enfoque de la aguja hasta que el contorno negro de un círculo (la punta de la aguja) que se ve. Toque la tétrada con este círculo y rápidamente sacar la aguja. Asegúrese de que los cuatro ascosporas de un asca se han adherido a la aguja y que no hay células adicionales se reunieron en la microaguja que no forman parte de este asco. </li><li> Tire de la microaguja lejos de la placa e ir a la matriz (por el asco en primer lugar, vaya aposición A1). Tomar nota de cada posición en la que se traslada un asco. Coloque el asco hacia abajo tocando la placa con la microaguja y alejándose hasta que los cuatro ascosporas salir de la microagujas. Suaves golpecitos en el brazo o la aguja de la plataforma que sostiene la placa YPD a menudo es útil en este paso. </li><li> Repita los pasos 3-5 y lugar ascos abajo en posiciones consecutivas de la matriz hasta el número deseado de ascos han sido recogidos. </li><li> Volver a cada asca y romper las cuatro celdas separadas por las burlas con la aguja, tocando suavemente la plataforma o una combinación de ambos. Recoger las células dejando uno en una nueva posición en la matriz hasta que cada ascosporas en cada asca se separa en filas de cuatro. </li><li> Coloque la placa de YPD en una incubadora a 30 ° C durante varios días para observar el patrón de crecimiento. </li><li> Replica la placa de las células diseccionado a un medio selectivo si es necesario. </li></ol> Reubicación de los cigotos Protocolo <ol><li> Racha de dos cepas haploides con los tipos de apareamiento opuesto en el medio apropiado para obtener colonias aisladas. </li><li> Compañero de estas dos cepas como se describió anteriormente y permitir que crezca de 4 a 6 horas (o toda la noche si es necesario). </li><li> Compruebe que los cigotos con un microscopio óptico. Cigotos aparecerá como pesas en forma de células, ya sea con o sin raíz. </li><li> Recoger las células con un asa de siembra de 3 mm (células que cubren ¼ del bucle) y el lugar suavemente en 100μL de agua estéril dH 2 O. </li><li> Marque la parte superior e inferior de la región de inóculo en una placa de YPD. </li><li> Tome 5μL y suavemente poner gotas en una línea en la región de inóculo de la placa de YPD pre-marcadas. </li><li> Use un asa de siembra de 3 mm se esparzan las gotas en una línea muy suavemente, mientras que apenas hacer contacto con el agar. </li><li> Deje que el líquido se absorba en el agar. Continúe con el microscopio de disección. </li><li> Centrarse en una región de la placa donde la mitad del campo contiene las células y la otra mitad está vacía. Poco a poco poner la aguja en el foco de tal manera que toca la región vacía del campo. Esto asegurará que la aguja no contiene células de un uso anterior. El contacto entre la aguja y el agar es visto como un círculo negro oscuro que poco a poco entra en el foco. </li><li> Ir a la matriz en la posición A1 y hacer una marca por la perforación del agar de nuevo. Esto es para ayudar a determinar la orientación de la placa si se saca el microscopio antes de su finalización. </li><li> Cambiar al modo de búsqueda. Localizar a un cigoto. </li><li> Recoger un cigoto con la aguja. </li><li> Cigotos lugar individual en la matriz. </li><li> Permita que los cigotos a crecer durante varios días. </li><li> Placa de réplica de los cigotos en el medio selectivo apropiado. </li></ol> Resultados representante <img src="/files/ftp_upload/1146/1146fig1.jpg" alt="" /> Figura 1: El apareamiento de dos cepas haploides de los tipos de apareamiento opuesto genera una cepa diploide. <img src="/files/ftp_upload/1146/1146fig2.jpg" alt="" /> Figura 2: (A) Una placa de YPD con resultados thre de un cruzamiento entre una cepa sensible a la temperatura (con un auxotrofía leu2) y una cepa de tipo salvaje (LEU2). Las células digieren se distribuyen en la región de inóculo de la izquierda donde se ve el crecimiento de espesor entre las dos líneas de color negro. Ascos que han sido disecados crecen espaciados uniformemente en la matriz de la derecha. (B) La placa en (A) se reprodujo en una SD-leucina de abandono de placas. Tenga en cuenta el crecimiento de 2:02 para el marcador que indica un LEU2 ascosporas validado fue disecada. (C) La placa en (A) se reprodujo en una placa de YPD y se incubaron a 37 º C. Tenga en cuenta el crecimiento de 2:2 que valida aún más que un ascosporas fue disecada.

<ol>
	<li>Sherman, F., Hicks, J., Guthrie, C., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+and+Dissection+of+Asci.">Micromanipulation and Dissection of Asci.</a> Guide to Yeast Genetics and Molecular Biology. , 21-37 (1991).</li></ol>

Disección de la levadura es una herramienta útil para seleccionar nuevas variedades con marcadores deseado. Al determinar el patrón de crecimiento teórico de cuatro ascosporas es importante tener en cuenta el genotipo de la célula vegetativa. Si un plásmido está presente, hay que saber si es solo ejemplar, de baja o alta ya que esto afectará la que las ascosporas (s) recibe el plásmido, y afectará a las predicciones y conclusiones con respecto a las cepas siendo manipulados. Cierta...

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Singer MSM System 200</td>
<td>Microscope</td>
<td>Singer Instruments</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>D-sorbitol</td>
<td>Reagent</td>
<td>BioShop</td>
<td>SOR508</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>Reagent</td>
<td>BioShop</td>
<td>TRS001</td>
<td>&nbsp;</td>
<tr>
<td>Zymolyase 100T</td>
<td>Reagent</td>
<td>Seikagaku Corporation</td>
<td>120493</td>
<td>&nbsp;</td>
<tr>
<td>Yeast extract</td>
<td>Reagent</td>
<td>BioShop</td>
<td>YEX401</td>
<td>&nbsp;</td>
<tr>
<td>Peptone</td>
<td>Reagent</td>
<td>BioShop</td>
<td>PEP 403</td>
<td>&nbsp;</td>
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<td>D-glucose</td>
<td>Reagent</td>
<td>BioShop</td>
<td>GLU501</td>
<td>&nbsp;</td>
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<td>Yeast nitrogen base</td>
<td>Reagent</td>
<td>BioShop</td>
<td>YNB406</td>
<td>&nbsp;</td>
<tr>
<td>Bio-tryptone</td>
<td>Reagent</td>
<td>BioShop</td>
<td>TRP402</td>
<td>&nbsp;</td>
<tr>
<td>Sodium chloride</td>
<td>Reagent</td>
<td>BioShop</td>
<td>SOD002</td>
<td>&nbsp;</td>
<tr>
<td>Potassium acetate</td>
<td>Reagent</td>
<td>BioShop</td>
<td>POA 301</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>Reagent</td>
<td>BioShop</td>
<td>AGR003</td>
<td>&nbsp;</td>
<tr>
<td>Adenine sulfate</td>
<td>Reagent</td>
<td>BioShop</td>
<td>ADS201</td>
<td>&nbsp;</td>
<tr>
<td>L-uracil</td>
<td>Reagent</td>
<td>BioShop</td>
<td>URA 241</td>
<td>&nbsp;</td>
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<td>L-tryptophan</td>
<td>Reagent</td>
<td>BioShop</td>
<td>TRP 100</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>BioShop</td>
<td>HIS 200</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Amresco</td>
<td>0877-100G</td>
<td>&nbsp;</td>
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<td>L-methionine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>MET 222</td>
<td>&nbsp;</td>
<tr>
<td>L-tyrosine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>TYR 333</td>
<td>&nbsp;</td>
<tr>
<td>L-isoleucine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>ISO 910</td>
<td>&nbsp;</td>
<tr>
<td>L-valine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>VAL 201</td>
<td>&nbsp;</td>
<tr>
<td>L-lysine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>LYS 101</td>
<td>&nbsp;</td>
<tr>
<td>L-phenylalanine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>PHA 302</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamic acid</td>
<td>Reagent</td>
<td>BioShop</td>
<td>GLU 202</td>
<td>&nbsp;</td>
<tr>
<td>L-threonine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>THR002</td>
<td>&nbsp;</td>
<tr>
<td>L-leucine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>LEU 222</td>
<td>&nbsp;</td>		</tbody>
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dissection of saccharomyces cerevisiae asci

La levadura es un sistema modelo muy manejable que se utiliza para estudiar muchos procesos celulares diferentes. La cepa de laboratorio común<em&gt; Saccharomyces cerevisiae</em&gt; Existe, ya sea en un estado haploide o diploide. La capacidad de combinar los alelos de dos haploides y la posibilidad de introducir modificaciones en el genoma requiere la producción y la disección de los ascos. La producción de células haploides ascos comienza con el apareamiento de dos cepas de levaduras haploides con tipos de acoplamiento compatible para producir una cepa diploide. Esto se puede lograr de varias maneras, ya sea en un medio sólido o líquido. Es ventajoso para seleccionar a los diploides en un medio que promueve selectivamente su crecimiento en comparación con cualquiera de las cepas haploides. Los diploides se dejan de esporas en medio pobre en nutrientes para formar ascos, un paquete de cuatro células hijas haploides resultantes de reproducción meiótica de los diploides. Una mezcla de células vegetativas y ascos se trata con la enzima para digerir zymolyase de distancia de la membrana del saco que rodea el ascosporas de los ascos. El uso de micromanipulación con una microaguja bajo un microscopio de disección se puede recoger y separar cada ascos y reubicar a los cuatro ascopores. Ascos disecados se cultivan durante varios días y la prueba de los marcadores o alelos de interés por la chapa adecuada réplica en los medios selectivos.

Watch this Scientific Journal Video about Dissection of Saccharomyces Cerevisiae Asci at JoVE.com

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulación de células de levadura que se necesita para el análisis genético meiótica o para seleccionar cigotos diploides. Estos micromanipulaciones se llevan a cabo con la microaguja de un microscopio de disección. La microaguja se utiliza para trasladar las células y es controlado por un micromanipulador que están disponibles con diferentes grados de automatización.

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 ...

dissection-of-saccharomyces-cerevisiae-asci

Research

JoVE Journal

Biology

Dissection of Saccharomyces Cerevisiae Asci

18.5K vistas.  Concordia University. Micromanipulación de células de levadura que se necesita para el análisis genético meiótica o para seleccionar cigotos diploides. Estos micromanipulaciones se llevan a cabo con la microaguja de un microscopio de disección. La microaguja se utiliza para trasladar las células y es controlado por un micromanipulador que están disponibles con diferentes grados de automatización.

Video: Dissection of Saccharomyces Cerevisiae Asci

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 ...

Investigación

Biología

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  ...

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 ...

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

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

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

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

La purificación de la fibrosis quística regulador de conductancia transmembrana de proteínas expresadas en<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 &#181;M. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

EPR supervisado Redox La titulación de los cofactores de<em&gt; Saccharomyces cerevisiae</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

La medición de mRNA decadencia Tarifas en<em&gt; Saccharomyces cerevisiae</em&gt; Uso<em&gt; Rpb1-1</em&gt; Cepas

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

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. ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Dirigida Método Evolución en<em&gt; Saccharomyces cerevisiae</em&gt;: Mutant Biblioteca Creación y Proyección

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...