Este trabajo fue apoyado por una beca Joseph P. Healey de la Universidad de Massachusetts Boston (LSH) y R15 GM86805 de los NIH (LSH). SMP es apoyado en parte por una beca de Sanofi-Genzyme en la Universidad de Massachusetts, Boston.

1. Preparación para la esporulación Nota:. Los medios descritos en este protocolo se realizan utilizando las recetas y los métodos estándar 13,17 La tabla 1 muestra la formulación de 1 l de los diferentes medios utilizados en este protocolo. <table border="1" cellpadding="0" cellspacing="0" height="180" width="469"><colgroup><col /><col /><col span="3" /><col /><col span="2" /></colgroup><tbody><tr height="15"><td height="15" style="height:20px;"></td><td style="width: 55px;"> Bacto peptona </td><td style="width: 50px;"> Extracto de levadura </td><td style="width: 50px;"> Bacto Agar </td><td style="width: 60px;"> Dextrosa </td><td style="width: 70px;"> Acetato de potasio </td><td style="width: 60px;"> Glicerol </td><td style="width: 60px;"> ddH2O </td></tr><tr height="15"><td height="15" style="height:20px;"> placas YPG </td><td> 20 g </td><td> 10 g </td><td> 20 g </td><td> - </td><td> - </td><td> 30 ml </td><td> 970 ml </td></tr><tr height="15"><td height="15" style="height:20px;"> placas de YPD </td><td> 20 g </td><td> 10 g </td><td> 20 g </td><td> 20 g </td><td> - </td><td> - </td><td> 1.000 ml </td></tr><tr height="15"><td height="15" style="height:20px;"> líquido YPD </td><td> 20 g </td><td> 10 g </td><td> - </td><td> 20 g </td><td> - </td><td> - </td><td> 1.000 ml </td></tr><tr height="15"><td height="15" style="height:20px;"> líquido YPA </td><td> 20 g </td><td> 10 g </td><td> - </td><td> - </td><td> 20 g </td><td> - </td><td> 1.000 ml </td></tr><tr height="15"><td style="height: 20px; width: 80px;"> medios de esporulación </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 10 g </td><td> - </td><td> 1.000 ml </td></tr></tbody></table> Tabla 1:. Formulaciones de medios montos son dados con 1 l de medio. Específicamente acerca de los mediosingredientes se pueden encontrar en la Tabla de Materiales. <ol><li> Si todas las células en esporulación será el mismo genotipo, siga este procedimiento: <ol><li> Descongelar la cepa de levadura que se utiliza en una placa de YPG (YPGlycerol). Crecer la levadura de 12-16 horas a 30 ° C. Es importante destacar que también están preparados células SK1 esporular, por lo cepas SK1 no se deben dejar demasiado tiempo en placas YPG, o pueden esporular en el plato. Nota: Se prefiere una cepa recientemente descongelado crecido en YPG, como la utilización de glicerol como fuente de carbono requiere la fosforilación oxidativa, y por lo tanto selecciona para células que tienen ADN mitocondrial funcional. células SK1 tienen una tendencia a perder el ADN mitocondrial funcional y convertirse petites. se necesita ADN mitocondrial funcional para la esporulación. </li><li> levadura consecutivas de la placa YPG sobre una placa de YPD (YPDextrose) para colonias individuales. Crecer la levadura en la placa YPD 24-28 horas a 30 ° C. </li><li> Después de alrededor de 36-48 h de crecimiento en YPD medios sólidos, iniciar un cultivo de 25 ml de YPD utilizando una sola coloniade la placa de YPD. Crecer este cultivo hasta que las células son aproximadamente DO600 = 4,0 a 7,0. Realice este paso a 30 ° C durante la noche en una incubadora con agitación, agitando a 220 rpm. </li><li> A partir del cultivo YPD, utilice una cantidad adecuada de células para iniciar 1.000 ml de YPA cultivo (YPAcetate) que es OD 600 = 0,1 para cada necesidad placa de 96 pocillos en la Sección 2. crecer la levadura en una incubadora de agitación 30 ° C (con agitación a 220 rpm) hasta que el cultivo OD 600 es entre 1,3 a 1,5. Nota: inocular 100 ml de cultivo YPA para cada placa de múltiples pocillos 96 que se proyectarán en la Sección 2. Por ejemplo, si el examen de cuatro placas de múltiples pocillos 96, serán necesarios 400 ml de cultivo YPA. A partir de 10% volumen extra en la cultura YPA es necesaria para proteger contra la pérdida de volumen por evaporación. Por lo general, se tardará entre 12-16 horas para que las células alcanzan la densidad óptica apropiada 600; la cantidad de tiempo puede variar dependiendo del genotipo de la cepa de levadura. YPA es los medios de comunicación presporulation. Pregrowthen acetato de ayuda a inducir la expresión de genes (como IME1) necesarios para la esporulación eficiente y síncrono 6. </li><li> Centrifugado a 1.800 xg en una centrífuga de mesa durante 5 minutos para sedimentar las células cultivadas en YPA. Lavar las células 1 vez con 0,5 volúmenes de agua desionizada estéril tratada en autoclave. Girar de nuevo, y desechar lavar. Resuspender las células en medios volumen esporulación 1x. Continúe con la Sección 2. </li></ol></li><li> Si el uso de células de esporulación que son de diferentes genotipos, (es decir, transformadas con plásmidos diferentes o que contienen diferentes mutaciones, normalmente obtenidos y almacenados como reservas congeladas), células de agrupación de los diferentes genotipos en un formato de múltiples pocillos 96. A continuación, siga este procedimiento: <ol><li> Use un frogger 96 puntas estériles para transferir las células procedentes de las existencias congeladas descongeladas en placas de 96 pocillos (s) de múltiples pocillos en medios sólidos YPG. Crecer durante la noche de levadura a 30 ° C. </li><li> El uso de un frogger 96 prong estéril, la transferencia de células de YPG medios sólidos en medios sólidos YPD o selectivamedios sólidos (es decir, medios mínimos sintéticos carece de los aminoácidos, si las células contienen plásmidos que requieren la selección). Crecer levadura a 30 ° C hasta que las colonias se forman para cada cepa, típicamente 1 día en YPD y 2 días si los medios de comunicación selectiva es necesario. </li><li> Alícuota de 1,2 ml de medio líquido YPD o medios líquidos selectivos en cada pocillo de una placa de múltiples pocillos de 96 profundas 2 ml. La transferencia de células a partir de medios sólidos en medios líquidos utilizando un frogger, cubrir con una tapa de plástico en su lugar por cinta, y crecen durante la noche a 30 ° C agitador, agitación a 220 rpm. Nota: Para minimizar la contaminación, placas de cubierta utilizando las tapas de plástico de una placa de Petri rectangular. Mantenga las tapas en su lugar con un trozo de cinta, por lo que la circulación de aire está restringido mínimamente. </li><li> placas de centrifugar a 1800 xg durante 5 min, utilizando una cubeta oscilante centrífuga con adaptadores para acomodar placas de 96 pocillos. Retirar los medios de comunicación de las células; añadir 500 YPA l a cada pocillo y resuspender las células sedimentadas en el YPA. Cubrircon tapa de plástico en su lugar por cinta. </li><li> Se cultivan las células durante 15 horas a 30 ° C en una incubadora con agitación, agitando a 220 rpm. Continúe con la Sección 2. </li></ol></li></ol> 2. Las células esporuladas en un formato de 96 multipocillos <ol><li> Añadir o bien un talón sólida estéril 3 mm de vidrio o una estériles 5 mm x 2 mm barra de agitación magnética en cada pocillo de una placa de 96 pocillos con 1,3 ml pozos. (Ver resultados representativos de abajo para una discusión sobre la conveniencia de utilizar un cordón o una barra de agitación.) </li><li> Si todas las células que se proyectarán son del mismo genotipo, ir a 2.2.1. Si las células que se proyectarán son de diferentes genotipos, ir a 2.2.2. <ol><li> Tomar células de la etapa 1.1.5 y alícuota de 500 l de células más medios de comunicación en cada pocillo de la placa de múltiples pocillos 96. Cubra con una tapa de plástico en su lugar por cinta. Proceder a 2,3. </li><li> Centrifugar las células que se encuentran en YPA de 1.2.4 a 1800 xg durante 5 min, utilizando una cubeta oscilante centrífuga con adaptadores para acomodar placas de 96 pocillos. verter def medios de las células. Añadir 500 l de medios de esporulación a cada pocillo y resuspender las células sedimentadas en los medios de esporulación. Cubra con una tapa de plástico en su lugar por cinta. Proceder a 2,3. </li></ol></li><li> Si se utiliza una cuenta de vidrio, colocar las muestras en una incubadora de agitación a 30 ° C y agitar a 220 rpm durante la esporulación. Si se utiliza una barra de agitación magnética, colocar las muestras en una placa de agitación en el interior de una incubadora a 30 ° para la esporulación. Nota: La velocidad de agitación exacto para las barras de agitación magnéticas no es muy importante, siempre y cuando todas las barras se mueven con energía y la cultura se airea. </li><li> Para controlar la progresión a través de la esporulación, retirar muestras de la cultura de esporulación para la visualización, Añadir 6 l de células de los cultivos a las 24 esporulación l de medios de esporulación en una placa de 96 pocillos cubreobjetos (dilución 1: 5). Deja células que conformarse con 5 min antes de examinar las células usando un microscopio invertido. Nota: En las células de tipo salvaje SK1, esporas maduras compactos formarán un 36 horas después de la transferencia al medio de esporulación. La progresión a través de esporulación puede ser monitoreada por microscopía utilizando marcadores fluorescentes, tales como un marcador nuclear (es decir, HTB2-mCherry 18) para examinar la división meiótica o un marcador de membrana prospore (es decir, G20 19) para examinar la morfogénesis de esporas. Alternativamente, la formación de esporas refráctiles se puede ver usando microscopía de Nomarski DIC a partir de 12 h después de la transferencia al medio de esporulación. </li></ol>

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The authors have no competing financial interests.

Aquí se presenta un protocolo para la esporulación de levadura SK1 en un formato de múltiples pocillos 96. La aireación es clave para la esporulación eficiente, que requiere el uso de cualquiera de una barra de agitación o una perla de vidrio en cada pocillo. Cuando las células son esporulados en una placa de múltiples pocillos 96 en una incubadora de agitación sin ya sea un cordón o una barra de agitación, las células no esporulan eficiente. Sólo un pequeño aumento en la eficiencia de la esporulación se ve cuando se esporulados células sin ya sea un cordón o una barra de agitación en un incubador con agitación, en comparación con ser a 30 ° C sin agitación (Tabla 1; 17% en agitador vs 10% colocada en un revuelo plato). Del mismo modo, el uso de una barra de agitación 7 mm dio como resultado una pobre eficiencia esporulación (28%), probablemente debido a los 7 mm agitar barras no airean la cultura en forma adecuada debido a las interacciones con barras de agitación en los pozos adyacentes. Este protocolo describe las condiciones para la esporulación mediante el SK1 cepa de levadura esporulación de forma sincrónica y eficiente 7,8 comúnmente utilizado para estudiar sporulation; eficiencias de esporulación que se pueden obtener con otras cepas de levadura que utilizan este protocolo deben ser examinados antes de emprender una gran pantalla utilizando estas técnicas. Aunque unos 5 mm x 2 mm resultados barra de agitación en un poco mejor esporulación en comparación con el uso de una perla de vidrio (66% con 5 mm x 2 mm barra de agitación vs 56% con una perla de vidrio), el coste requerido para comprar suficiente revuelo barras para cada bien pueden ser prohibitivos. Las eficiencias de esporulación obtenidos usando una perla de vidrio proporciona una solución de bajo costo que crea suficiente aireación para cribado de alto rendimiento en un formato de múltiples pocillos 16 96. Desafortunadamente, la adición de una barra de agitación o una perla de vidrio no alcanzó las eficiencias de esporulación muy altas (típicamente ~ 80% o mayor) visto cuando esporulación en frascos, y fenotipos esporulación así sutiles puede ser difícil de detectar para el cribado en un 96 así formato. A diferencia de los métodos descritos anteriormente esporulación 9-13 </sup&gt; que utilizan medios sólidos o líquidos volúmenes de medios más grandes usando frascos o tubos de cultivo, este protocolo se describen las condiciones para la esporulación eficiente en un formato de múltiples pocillos 96. El uso de un formato de múltiples pocillos 96 es compatible con diferentes tipos de pantallas de alto rendimiento que se pueden llevar a cabo para examinar los fenotipos de esporulación. Por ejemplo, las pantallas de químicos que afectan los procesos de esporulación se pueden llevar a cabo usando esta técnica, donde las células en esporulación se tratan con diferentes compuestos que han sido dispuestos en un formato de 96 pocillos. En este caso, las células que se esporulados serían todos del mismo genotipo, y se añadieron los compuestos en el paso 2.2 de la esporulación. Este protocolo también es compatible con la detección de células de diferentes genotipos de fenotipos de esporulación, tales como células de levadura transformadas con una biblioteca de la copia o una colección cepa mutante de levadura. Para este caso, sería necesario que se vista de primera en un formato de 96 pocillos las cepas y el protocolo descrito a partir de unat paso 1.2 sería apropiado.

La esporulación en la levadura en ciernes ha sido estudiado para proporcionar información sobre muchos aspectos de la biología, incluyendo el control de la segregación de cromosomas durante la meiosis 1, los mecanismos de recombinación genética 2, el control del desarrollo de células de señalización 3, control nutricional de desarrollo 4, la transcripción regulación del desarrollo 5, y el examen de la formación de esporas 6. La formación de esporas incluye una novela caso la división celular que implica la formación de nuevos compartimentos de membrana dentro de la célula madre seguido de la deposición de una pared de la espora de protección 6. Estos estudios que examinan células esporuladas a menudo se aprovechan de la cepa de levadura rápida esporulación SK1, que puede someterse al proceso de esporulación en aproximadamente 24 hr de una manera relativamente eficiente 7,8. Aunque optimización de las condiciones de esporulación de levadura en ciernes se han descrito 9-13, estos experimentoss examinó la esporulación en medios sólidos o en cultivos líquidos de mayor escala, donde la esporulación se lleva a cabo utilizando tubos de cultivo o frascos. Aquí se describe un método para la esporulación de levadura en un formato de placa de múltiples pocillos 96. Encontramos que para este método, la aireación es crítica para la esporulación síncrono y eficiente, y hemos ideado técnicas para asegurar la suficiente esporulación un formato de múltiples pocillos de pequeño volumen. Esporulación en un formato de 96 placa de múltiples pocillos permite para las células para ser examinadas usando técnicas de alto rendimiento optimizados y reactivos para un formato de placa de múltiples pocillos, como la detección de altos supresores de copias deseado con una biblioteca de azulejos 14-16.

<table><tbody><tr><td>Nunc 1.3 ml DeepWell Plates</td><td>ThermoScientific</td><td>260251</td><td>Used for sporulation</td></tr><tr><td>Nunc 2.0 ml DeepWell plates</td><td>ThermoScientific</td><td>278743</td><td>Used for presporulation growth, step 1.2.3</td></tr><tr><td>3 mm glass bead</td><td>Fisher</td><td>11-312A</td><td>Used for sporulation</td></tr><tr><td>5 mm x 2 mm stir bar, pack of 12</td><td>Fisher</td><td>14-511-82</td><td>Used for sporulation</td></tr><tr><td>96 well frogger</td><td>V&amp;amp;P Scientific</td><td>VP407</td><td>needed for step 1.2</td></tr><tr><td>library copier</td><td>V&amp;amp;P Scientific</td><td>VP381</td><td>needed for step 1.2; to be used with the frogger</td></tr><tr><td>rectangular Petri dish</td><td>ThermoScientific</td><td>264728</td><td>needed for step 1.2</td></tr><tr><td>Bacto Peptone</td><td>BD</td><td>211677</td><td>needed for media</td></tr><tr><td>Yeast Extract</td><td>BD</td><td>212750</td><td>needed for media</td></tr><tr><td>Bacto Agar</td><td>BD</td><td>212750</td><td>needed for media</td></tr><tr><td>Dextrose</td><td>Fisher</td><td>D16-3</td><td>needed for media</td></tr><tr><td>Potassium Acetate</td><td>Fisher</td><td>P171-500</td><td>needed for media</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>G33-500</td><td>needed for media</td></tr><tr><td>Black 96 well glass bottom plate</td><td>MatTek</td><td>PBK96G-1.5.5-F</td><td>needed for step 2.4</td></tr></tbody></table>

efficient sporulation of saccharomyces cerevisiae in a 96 multiwell format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

<html> Para evaluar este protocolo, las eficiencias de esporulación obtenidos de esporulación las células en placas de pocillos múltiples (como se describió anteriormente) se compararon a las células esporulados utilizando volúmenes más grandes en matraces (Tabla 2). El uso de placas de múltiples pocillos no alcanzó la alta eficiencia se ve cuando esporulación en frascos, donde la eficiencia ~ 80% se puede ver de forma rutinaria. Esporulación en placas de pocillos múltiples con aireación adecuada (proporcionado por perlas de vidrio o barras de agitación) puede alcanzar la suficiente eficiencia esporulación mayor que 50%, con los mejores resultados (eficacia 66%) obtenida utilizando un 5 mm x 2 mm barra de agitación. Un representante de la esporulación dos cepas diferentes (tipo salvaje y smk1Δ) se muestra aquí (Figura 1). Estas células se examinan después de 36 horas en un medio de esporulación y se visualizaron utilizando una placa de fondo de cristal de 96 pocillos. 84 / 54584fig1.jpg &quot;/&gt; Figura 1:. Esporulantes células de un representante de la esporulación de tipo salvaje y las células smk1Δ 20 esporulados en unas placas de 96 pocillos múltiples. Las células se visualizaron utilizando un objetivo 63X en un microscopio invertido. Tétradas refráctiles (puntas de flecha) se pueden ver en la cultura de tipo salvaje, pero no en el cultivo que contiene células smk1Δ. Barra de escala = 10 micras. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54584/54584fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> La esporulación se ensayó usando o bien un 5 mm o mm bar 7 de agitación, cada uno de los cuales se inscribe en el pocillo de una 96 1,3 ml placa de múltiples pocillos (Tabla 2). El uso de una barra de agitación 7 mm dio como resultado una pobre eficiencia esporulación (28%) en comparación con el rendimiento más alto (66%) consigue utilizando la barra de agitación 5 mm. La barra de agitación 7 mm en un pozo tiende a interactuar con una barra de agitación en unan bien adyacente (Figura 1), la prevención de las barras de ser capaz de agitar correctamente y proporcionar una aireación adecuada del cultivo. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> células de esporulación (%) </td><td> SEM (%) </td></tr><tr><td> frasco en la coctelera </td><td> 82 </td><td> 1.8 </td></tr><tr><td> sin cordón en la coctelera </td><td> 17 </td><td> 2.4 </td></tr><tr><td> talón en la coctelera </td><td> 56 </td><td> 3 </td></tr><tr><td> sin barra de agitación en placa de agitación </td><td> 10 </td><td> 2.2 </td></tr><tr><td> 5 mm a revolver la barra en la placa de agitación </td><td> 66 </td><td> 3.9 </td></tr><tr><td> 7 mm revolver la barra en la placa de agitación </td><td> 28 </td><td> 6,7 </td></tr><tr><td> EEM = error estándar de la media </td><td></td><td></td></tr></tbody></table> Tabla 2:Eficiencias de esporulación utilizando diferentes condiciones. La esporulación se llevó a cabo en placas de 96 pocillos de múltiples pocillos como se describe anteriormente, o, si se ha señalado, en matraces de 500 ml que contienen 50 ml de medio de esporulación. Ocho pozos diferentes se promediaron para cada condición llevado a cabo en una placa de múltiples pocillos. Tres frascos diferentes fueron esporulados. Todas las culturas son repeticiones técnica, partieron de la misma colonia de HTB2-etiquetados mCherry pero por lo demás el tipo salvaje SK1 cepa de levadura diploide (LH902 18). Estas células se cultivaron en un solo matraz de YPD y un solo matraz de YPA, y luego se divide para inocular placas de 96 pocillos que contienen medio de esporulación, como se describe anteriormente. eficiencia esporulación se ensayó mediante el recuento de esporas que refractan la luz 36 horas después de la transferencia al medio de esporulación utilizando microscopía de campo claro DIC Nomarski. <img alt="Figura 2" src="/files/ftp_upload/54584/54584fig2.jpg" /> Figura 2: Stirbares en 96 1,3 ml placas de múltiples pocillos. cultura esporulación que contienen ya sea (A) de 5 mm x 2 mm barras de agitación (rojos) o (B) bares 7 mm x 2 mm stir (azul) en una placa de múltiples pocillos 96 1,3 ml en un agitador magnético plato. Barra de escala = 10 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54584/54584fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format at JoVE.com

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

La esporulación eficiente de<em&gt; Saccharomyces cerevisiae</em&gt; En un formato de 96 multipocillos

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

efficient-sporulation-saccharomyces-cerevisiae-96-multiwell

Nanyang Technological University

Research

JoVE Journal

Biology

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

10.0K vistas.  University of Massachusetts Boston. Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

Video: Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

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

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Un método de alto rendimiento a nivel mundial para estudiar la morfología orgánulo en S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

Los ensayos ACT1-CUP1 determinan las sensibilidades específicas del sustrato de los mutantes espliceosomales en levaduras en ciernes

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

Genética

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Metabolómica cuantitativa de Saccharomyces cerevisiae mediante cromatografía líquida combinada con espectrometría de masas en tándem

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Bioquímica

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Preparación y micromecanizado Crio-FIB de Saccharomyces cerevisiae para Crio-Tomografía Electrónica

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Pantallas competitiva Genómica de Bibliotecas de levadura con código de barras

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

Un flujo de trabajo de Análisis Cuantitativo Gimnasio

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

El Proceso de Monstruo Verde para la generación de cepas de levadura que llevan deleciones múltiples genes

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

Proyección de alto rendimiento para la herencia basados en proteína en S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Here we present a protocol to estimate the respiratory and fermentative metabolism by fitting the exponential growth of Saccharomyces cerevisiae to the exponential growth equation. Calculation of the kinetic parameters allows for the screening of influences of substances/compounds on fermentation or mitochondrial respiration.

Saccharomyces cerevisiae Cinética de crecimiento exponencial en la cultura de lote para analizar el metabolismo respiratorio y fermentativo

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The ...

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