This work was supported by NIH grant GM50936 to DKB.

NOTA: Algunos de los pasos del protocolo a continuación requieren trabajar en una campana de extracción limpia. Además, el método requiere un microscopio de disección tétrada levadura equipado con un objetivo 10X distancia larga de trabajo para supervisar formación de esferoplastos. El microscopio debe establecerse en la campana antes de tiempo. Retire el brazo micromanipulador y soporte de la placa del ámbito de aplicación y colocar estos elementos en un lugar seguro lejos de la zona de trabajo. 1. Preparación de esferoplastos <ol><li> Crecer levadura bajo las condiciones deseadas. Utilice aproximadamente 2 x 10 8 células para cada muestra, por ejemplo, 4 ml de un cultivo a DO600 = 1,4 5 x 10 7 células / ml. Aunque el procesamiento inmediato de las muestras puede ser preferible en algunos casos, para la mayoría de aplicaciones, alícuotas de células pueden ser almacenadas en hielo durante hasta 8 horas antes de ser procesados. </li><li> Transferir la suspensión celular a un tubo de centrífuga de 15 ml y centrifugado en una centrifugadora clínica en la configuración 5 (2345 rpm / 857,5 xg) durante 3 min para sedimentar las células. </li&gt; <li> Decantar el medio toma cuidado de no perder las células del sedimento. Resuspender las células en tampón de ZK 1 ml y luego añadir 40 l 1 M ditiotreitol (DTT). Incubar 2 minutos con agitación suave. Células de pellets como antes (ver el paso 1.2). </li><li> Gránulos Resuspender en tampón de ZK 1 ml. Añadir 5 l de una solución recién preparada de 100T zimoliasa que se ha mezclado a fondo. Incubar durante 20 a 30 minutos a 30 ° C para eliminar la pared celular y producir esferoplastos. </li><li> Ensayar una gotita de 10 l de suspensión de células en un portaobjetos de microscopio para determinar si es completa formación de esferoplastos. Las células vistos bajo el microscopio de disección sin cubreobjetos. Las células deben aparecer hinchada y redonda en lugar de un poco oblonga y deben lisar tras la adición de 20 l de agua. Si las células no pueden lisar, devolver la muestra a 30 ° C y re-ensayo cada 10 min hasta que se observa fácilmente inducida agua de lisis. Células de pellets como antes (ver el paso 1.2). </li><li> Resuspender suavemente y lavar las células de pellets in 2,5 ml MES / tampón sorbitol frío. Células de pellets como antes (ver el paso 1.2). Resuspender suavemente el sedimento celular en 300-400 l MES frío / tampón sorbitol. Las células se pueden mantener en el hielo en esta etapa durante varias horas. </li></ol> 2. Difundir Cromosoma NOTA: Las superficies de vidrio sobre el cual los cromosomas se diapositivas o bien repartidas-cubreobjetos-debe estar preparado antes de tiempo. Cada portaobjetos o cubreobjetos deben ser sumergidos en agua, a continuación, EtOH, después se dejó secar, y se pule con papel para lentes. Si los cromosomas se repartirán en cubreobjetos, el cubreobjetos debe ser colocada en una diapositiva con cinta adhesiva o pegamento de caucho. A menos que se indique lo contrario, el resto del protocolo describirá la difusión ya sea en un portaobjetos o cubreobjetos <ol><li> Usando una pipeta P20, pipeta de 20 l de suspensión celular sobre la superficie de un portaobjetos limpio. </li><li> Utilizando una pipeta P200, añadir 40 l de la PFA / solución de sacarosa al 3% y mezclar suavemente el solution por &quot;remolinos&quot; la diapositiva con la mano hasta que las líneas Schlieren desaparecen. Coloque diapositiva bajo el microscopio y confirmar que las células están en foco. La solución PFA debe ser recién preparado en la campana de humos en el día de uso. NOTA: Si la proteína de interés no ha sido examinada por el método anteriormente, es aconsejable preparar diapositivas adicionales utilizando soluciones que contienen 2 y 4% PFA para determinar si la retención de la proteína es sensible a las condiciones de fijación. Una solución / sacarosa 4% PFA se utiliza en los casos en los que es deseable para visualizar los microtúbulos de tubulina asociados. La desventaja de la concentración fijador superior es que los cromosomas serán &quot;underspread,&quot; 2% PFA también tiende a producir cromosomas &quot;underspread&quot;. </li><li> Usando una pipeta P200, añadir 80 l de 1% Lipsol, girar como antes para mezclar soluciones tan completamente como sea posible, se inicia un temporizador. Si Lipsol no está disponible, 80 l 1% NP40 o 80 l dH 2 O, se puedensustituido (ver resultados representativos, Figura 2). </li><li> Ver las células con cuidado y suavemente girar la diapositiva cada 15 seg. Cuando más o menos 80% de las células se han lisado (desaparecido) detener el temporizador, retire inmediatamente el portaobjetos del microscopio, añadir 80 l de PFA / solución de sacarosa, y agitar para mezclar. Lisis debe ocurrir de entre 30 y 90 seg después de comenzar el temporizador. NOTA: Si varias diapositivas muestran lisis en aproximadamente el mismo tiempo, uno eventualmente puede omitir la supervisión microscópica para el resto de las diapositivas y simplemente el tiempo cuidadosamente el periodo entre la adición de lipsol y la adición de la alícuota final del fijador. Sin embargo, este atajo sólo es fiable cuando se preparan varias diapositivas de la misma muestra. Lo mejor es controlar la lisis de cada muestra microscópicamente cuando diferentes diapositivas se preparan a partir de muestras tomadas en diferentes épocas o de diferentes culturas. </li><li> Colocar el portaobjetos en una superficie plana y limpia y difundir el líquido a través de todo el surfac unfrostede de la corredera usando el lado de A, pipeta desechable limpio Pasto mantenido por debajo del menisco de la &quot;charco&quot;, pero por encima de la superficie de la misma diapositiva, es decir. no &quot;rastrillo&quot; la superficie de la corredera con la pipeta. No vuelva a utilizar la pipeta en diferentes diapositivas para evitar la contaminación de las muestras. </li><li> Deja las diapositivas que se seque durante la noche en la campana de humos. Componentes nucleares insolubles se depositan sobre la superficie de deslizamiento y se unen a ella. Para grandes experimentos, la planificación del uso del espacio para la etapa de secado es importante. </li><li> Una vez secos, los resultados óptimos se obtienen por progresar a la etapa de inmunotinción en el mismo día. Sin embargo, la solución de sacarosa se secó incrusta los cromosomas de propagación en un &quot;miel&quot; que permite la congelación de las muestras: las diapositivas pueden ser transferidos a una caja de portaobjetos de plástico y se almacenaron a -20 ° C durante años. </li></ol> 3. La inmunotinción <ol><li> Diapositivas Dip en 0,2% Foto-Flo para 30 seg. para eliminar la miel. Diapositivas de Lean enun borde, con el borde que descansa sobre una toalla de papel, para eliminar residuos Photo-Flo. </li><li> Diapositivas Dip en 1x TBS durante 5 min para lavar. Retire el exceso de líquido apoyándose diapositivas en un borde, pero no deje que se sequen. </li><li> Coloque las diapositivas helado hacia arriba, pipeta 300 l TBS / BSA sobre el portaobjetos través de la porción no glaseado de la diapositiva. </li><li> Incubar en una cámara húmeda a temperatura ambiente durante 15 min. (Recipiente grande de plástico llena de agua saturada de toallas de papel debajo de una caja de diapositivas placa de metal o de plástico perforada con toallas de papel saturadas). </li><li> Escurrir diapositivas descansando un borde corto de la diapositiva en una toalla de papel y se inclina la diapositiva contra un soporte apropiado (como un bastidor de tubo de ensayo). No permita que la superficie se seque. </li><li> Inmediatamente aplique 80 l de tampón TBS / BSA que contiene la dilución apropiada de anticuerpo primario. Si el anticuerpo está limitando, utilice 40 ly un mm cubreobjetos de 22 x 22. Para suero de conejo crudo, esto es típicamente entre una dilución de 1/50 y 1/500. NOTA:Realizar las valoraciones para nuevos preparaciones de anticuerpos. Se elige la preparación más diluida que da la señal de alto por encima del fondo. Para controlar el nivel de la tinción de fondo, los núcleos deben prepararse a partir de la cepa mutante de deleción apropiado y / o diapositivas duplicados deben teñirse con suero pre-inmune. </li><li> Coloque una pulida 22 x 50 mm cubreobjetos sobre el portaobjetos evitando burbujas. Para ello, la celebración de la cubreobjetos entre el pulgar y el dedo índice a lo largo del borde largo en posiciones cerca de un borde corto. Sosteniendo el cubreobjetos en un ángulo de 30 ° con respecto al deslizamiento, la más alejada del borde corto de los dedos se baja hasta que descanse en la diapositiva justo dentro del borde de la &quot;charco&quot; más cercano a la región de esmerilado de la diapositiva. Entonces baje lentamente el cubreobjetos en un movimiento suave hasta que los dedos que sostienen el toque de diapositivas el banco, y luego suelte. No intente ajustar la posición del cubreobjetos vez aterriza. NOTA: Si extensiones de cromosomas se prepararon encubreobjetos adosadas (en lugar de directamente sobre la superficie de diapositivas), este cubreobjetos muestra permanecerá fijado a la corredera a través de los pasos de tinción y lavado. Un cubreobjetos adicional se colocará en la parte superior del cubreobjetos muestra para distribuir uniformemente la solución de anticuerpo durante la tinción y luego desechado. </li><li> Coloque los portaobjetos en una cámara húmeda sellado e incubar durante la noche a 4 ° C. </li><li> La celebración de la transferencia de borde esmerilado las diapositivas a un estante de tinción de portaobjetos de vidrio y sumergirse en un frasco de tinción que contiene TBS. </li><li> Sumergir suavemente la diapositiva arriba y abajo para quitar el cubreobjetos. Trate de no usar demasiada fuerza para hacer esto. Lave las diapositivas 2x 10 min sumergiendo en TBS. Eliminar diapositivas de cremallera y drenar el exceso de líquido al tocar el borde de la toalla de papel. No deje secar la superficie. </li><li> Trabajando bajo la luz tenue, añadir inmediatamente 80 l TBS / BSA que contiene una dilución veces 1/1000 del anticuerpo secundario conjugado con fluorocromo (o 40 l con un cubreobjetos de 22 x 22 mm). Una dd cubreobjetos como antes y se incuba a 4 ° C durante 2 horas en la cámara húmeda en la oscuridad. </li><li> Retire cubreobjetos como antes, drene diapositivas, y permitir que la superficie se seque al aire durante 1 a 2 horas en la oscuridad. Diapositivas de Lean sobre toallas de papel como antes. NOTA: Si la difusión se realizó en un cubreobjetos montados, separar el cubreobjetos de la corredera antes de secar. Los cubreobjetos se secan a continuación como se ha descrito para las diapositivas en el paso anterior. </li><li> Trabajando bajo la luz tenue, añadir unos 30 l de medio que contiene DAPI (10 gotas tres mu l) de montaje y luego baje cuidadosamente un cubreobjetos. Si los márgenes son en cubreobjetos, coloque las gotas de medio de montaje sobre un portaobjetos limpio y bajar el cubreobjetos, lado cromosoma abajo, sobre el portaobjetos. </li><li> Todavía bajo la luz tenue, espere 2 minutos para cubreobjetos para asentarse y sellar los bordes de cubreobjetos con esmalte de uñas transparente. Coloque las diapositivas en una caja de compuerta plana. NOTA: Para microscopía STED, monte con prolongar Gold. Utilice 30 l de prolongar Orosin DAPI y deje curar durante la noche. Sellado con esmalte de uñas es innecesario. </li><li> Ver diapositivas mediante microscopía de epifluorescencia de campo amplio con un 40 u objetivo 100X utilizando un conjunto de filtros apropiados para DAPI para enfocar. Opcionalmente almacenar el portaobjetos en la oscuridad a 4 ° C durante varias semanas. No lo congele. </li></ol>

<ol>
	<li>Loidl, J., Nairz, K., Klein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+chromosome+synapsis+in+a+haploid+yeast.">Meiotic chromosome synapsis in a haploid yeast.</a> Chromosoma. 100 (4), 221-228 (1991).</li><li>Loidl, J., Klein, F., Engebrecht, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+morphological+approaches+for+the+analysis+of+meiotic+chromosomes+in+yeast.">Genetic and morphological approaches for the analysis of meiotic chromosomes in yeast.</a> Methods in cell biology. 53, 257-285 (1998).</li><li>Lydall, D., Nikolsky, Y., Bishop, D. K., Weinert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+meiotic+recombination+checkpoint+controlled+by+mitotic+checkpoint+genes.">A meiotic recombination checkpoint controlled by mitotic checkpoint genes.</a> Nature. 383 (6603), 840-843 (1996).</li><li>Bishop, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecA+homologs+Dmc1+and+Rad51+interact+to+form+multiple+nuclear+complexes+prior+to+meiotic+chromosome+synapsis.">RecA homologs Dmc1 and Rad51 interact to form multiple nuclear complexes prior to meiotic chromosome synapsis.</a> Cell. 79, 1081-1092 (1994).</li><li>Rabitsch, K. P., Galova, M., Schleiffer, A., Buonomo, S. B., Nasmyth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+identifies+Monopolin:+a+kinetochore+protein+required+for+segregation+of+homologs+during+meiosis+I.">Functional genomics identifies Monopolin: a kinetochore protein required for segregation of homologs during meiosis I.</a> Cell. 103 (7), 1155-1168 (2000).</li><li>Gasior, S. L., Olivares, H., Ear, U., Hari, D. M., Weichselbaum, R., Bishop, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+RecA-like+recombinases:+Distinct+roles+for+mediator+proteins+in+mitosis+and+meiosis.">Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis.</a> PNAS. 98 (15), 8411-8418 (2001).</li><li>Biggins, S., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+budding+yeast+protein+kinase+Ipl1/Aurora+allows+the+absence+of+tension+to+activate+the+spindle+checkpoint.">The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint.</a> Genes &amp; Development. 15 (23), 3118-3129 (2001).</li><li>Dresser, M. E., Giroux, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+chromosome+behavior+in+spread+preparations+of+yeast.">Meiotic chromosome behavior in spread preparations of yeast.</a> The Journal of Cell Biology. , (1988).</li><li>Lao, J. P., Cloud, V., 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=Meiotic+crossover+control+by+concerted+action+of+Rad51-Dmc1+in+homolog+template+bias+and+robust+homeostatic+regulation.">Meiotic crossover control by concerted action of Rad51-Dmc1 in homolog template bias and robust homeostatic regulation.</a> PLOS Genetics. 9 (12), e1003978-e1003978 (2013).</li><li>Vonesch, C., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast+thresholded+landweber+algorithm+for+wavelet-regularized+multidimensional+deconvolution.+IEEE+transactions+on+image+processing+:+a+publication+of+the.">A fast thresholded landweber algorithm for wavelet-regularized multidimensional deconvolution. IEEE transactions on image processing : a publication of the.</a> IEEE Signal Processing Society. 17 (4), 539-549 (2008).</li></ol>

The authors declare no competing financial interests.

The appearance of spread nuclei critically depends upon the balance between fixation and lysis/detergent treatment. As discussed above, the preservation of varying cellular structures requires the use of different PFA concentrations. For most proteins, 3% PFA is optimal. However, preservation of spindles requires use of 4% PFA. Even with a single set of reagents, the timing of lysis relative to fixation can also affect the quality of spread nuclei. For the most consistent results, the time of lysis should be normalize...

La incipiente levadura Saccharomyces cerevisiae ofrece muchas ventajas para el estudio de los mecanismos moleculares de los procesos biológicos, incluyendo el estudio de las proteínas que controlan la función cromosoma. Aunque hay ventajas bien conocidas de la levadura en ciernes para estudios genéticos, moleculares, y bioquímicos, los estudios citológicos de la distribución de las proteínas en el núcleo de la célula se complica por su pequeño tamaño. El núcleo típico de levadura tiene un diámetro de menos de una micra, que está a sólo unos 5 veces el límite de resolución de la luz visible. Por lo tanto, la cantidad de información acerca de la distribución de las proteínas nucleares que se puede obtener a partir de la inmunotinción convencional o mediante el uso de etiquetas de proteínas fluorescentes, tales como la proteína fluorescente verde (GFP), es limitado. Un enfoque útil para caracterizar la distribución de las proteínas es subnuclear superficie cromosoma propagación. Este enfoque implica la eliminación de la pared celular, lo que altera las células y membranas nucleares, y unllowing los contenidos insolubles del núcleo se asienten sobre la superficie de un portaobjetos de microscopio. Estos componentes insolubles incluyen la matriz nuclear y los cromosomas. Además de permitir la eliminación de los contenidos nucleares solubles, lo que mejora la capacidad de detectar las proteínas unidas de la cromatina, el cromosoma propagación método da como resultado la descompresión sustancial de los cromosomas de tal manera que los núcleos de propagación tienen diámetros de alrededor de 3 a 5 micras (para núcleos diploides meióticas) y de 2 a 3 micras para núcleos mitóticos diploides. Esta descompresión permite la detección de la subestructura nuclear que es relativamente difícil o imposible de resolver en los núcleos intactos. Un inconveniente obvio para el cromosoma de ensanchamiento es la posibilidad de que el procedimiento de difusión puede interrumpir parcial o totalmente la estructura de interés. De particular preocupación es que una proteína unida cromosoma particular, podría perderse como consecuencia del procedimiento de difusión. Este potencial shou complicaciónld tenerse en cuenta al interpretar los datos. Un ejemplo de una proteína que es sensible a la propagación de procedimiento es beta-tubulina. Bajo algunas condiciones, el husillo, que se compone principalmente de la tubulina, se conserva durante la extensión 3. Visualización del husillo es a menudo útil para organizar los núcleos de interés. Sin embargo, la visualización de la tubulina requiere tratamiento con un fijador alta concentración; husillos se pierden en las condiciones estándar descritas a continuación. Este ejemplo ilustra que, al analizar la distribución de una proteína no caracterizado previamente, es importante para variar la concentración de fijador para determinar la sensibilidad de la proteína es a tal variación. A pesar de la preocupación por el impacto de las condiciones de propagación en la estructura cromosómica, la utilidad y el poder del método de propagación se ha demostrado en muchos contextos y tiene amplia utilidad en la caracterización de mitótico y, especialmente células meióticas 4-7. Two métodos de propagación se han utilizado ampliamente. El primero de estos métodos, desarrollados por Dresser and Giroux 8, evita el uso de detergente y puede producir preparaciones separadas que parecen tener relativamente bien conservado morfología cromosómica cuando se tiñen para el ADN específico de tinte DAPI. Sin embargo, este método es relativamente difícil de perfeccionar y la calidad de los núcleos de propagación varía dramáticamente, cuando una región de una diapositiva se compara con otras regiones. Este problema se puede complicar enfoques cuantitativos que implican la formación de imágenes muchos núcleos no seleccionadas de una diapositiva para evitar el sesgo de datos de adquisición. El segundo cromosoma método de propagación, desarrollado por Loidl y Klein 1, consiste en equilibrar la fijación de paraformaldehído, con la lisis y descompresión cromatina promovido por una solución de detergente. Cuando se realiza correctamente, este método da resultados muy reproducibles con menos variación de región a región en comparación con el método de Dresser y Giroux. Esta presentación se centra en un modified versión del método de Loidl y Klein, debido a su fiabilidad y la sencillez. Cromosoma propagación no es complicado ni requiere mucho tiempo; hasta 100 diapositivas se pueden preparar para inmunotinción en un solo día. Además, las preparaciones de cálculo se pueden almacenar en el congelador durante años antes de la inmunotinción, y por lo tanto los laboratorios pueden desarrollar un repositorio de extensiones de cromosomas congelados que se puede utilizar cuando surgen nuevas cuestiones biológicas o nuevos reactivos de tinción estén disponibles. El método de propagación de cromosoma se usa más comúnmente en combinación con inmunotinción y microscopía de fluorescencia de campo amplio, pero también es posible preparar diapositivas para super-resolución métodos microscópicos de luz tales como el agotamiento de la emisión estimulada (STED) microscopía.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Zymolyase</td>
			<td>US Biological&#160;</td>
			<td>Z1004</td>
			<td>Prepare 20 mg/mL solution in 50 mM Tris pH 7.5 supplemented with 2% glucose. Prepare fresh each experiment and store at 4&#176;C until ready for use.</td>
		</tr>
		<tr>
			<td>Lipsol&#160;</td>
			<td>L.I.P. Ltd&#160;</td>
			<td>no longer commercially available</td>
			<td>Prepare 1% (v/v) solution in water. Store on ice.</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>USB</td>
			<td>19628</td>
			<td>Prepare 1% (v/v) solution in water. Store on ice.</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Corning</td>
			<td>2948-75x25</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard coverslip</td>
			<td>Fisher</td>
			<td>12-544-E or 12-540-B</td>
			<td></td>
		</tr>
		<tr>
			<td>High resolution coverslips</td>
			<td>Fisher</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo-Flo 200 solution</td>
			<td>Kodak</td>
			<td>P-7417</td>
			<td>Prepare 0.2% (v/v) solution in water.&#160;</td>
		</tr>
		<tr>
			<td>TBS</td>
			<td></td>
			<td></td>
			<td>137 mM NaCl, 2.7 mM KCl, 24.7 mM Tris, pH 8</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td>Prepare a 1% (w/v) solution in TBS. Store at 4&#176;C for up to a month.</td>
		</tr>
		<tr>
			<td>Primary antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Rabbit</td>
			<td>Invitrogen</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG (H+L) Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting media with DAPI</td>
			<td>Vector 
			Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic slide box</td>
			<td>Fisher</td>
			<td>03-448-1</td>
			<td>Store slides containing dried spreads in slots at -20&#176;C. Also, use as a wet chamber.</td>
		</tr>
		<tr>
			<td>Cardboard slide box</td>
			<td>Fisher</td>
			<td>12-587-10</td>
			<td>Use to conveniently transport stained/sealed slides or store at 4&#176;C.</td>
		</tr>
		<tr>
			<td>Coplin jar</td>
			<td>Fisher</td>
			<td>08-816</td>
			<td>Use as a wash basin for slides.&#160;</td>
		</tr>
	</tbody>
</table>

surface spreading and immunostaining of yeast chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

La aparición de núcleos de propagación depende críticamente sobre el equilibrio entre el cromosoma fijación y de-compactación. Incluso cuando los reactivos se equilibran correctamente, la variación en el grado de cromosoma de-compactación puede ocurrir en diferentes regiones de la misma diapositiva y / o entre diferentes diapositivas. Por lo tanto, la calidad de los diferenciales en una región determinada de una diapositiva debe evaluarse antes se interpretan las imágenes. Los efec...

Watch this Scientific Journal Video about Surface Spreading and Immunostaining of Yeast Chromosomes at JoVE.com

Surface Spreading and Immunostaining of Yeast Chromosomes

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

Difusión de superficies y inmunotinción de cromosomas de la levadura

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

surface-spreading-and-immunostaining-of-yeast-chromosomes

Research

JoVE Journal

Biology

9.9K vistas.  University of Chicago. A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

Video: Surface Spreading and Immunostaining of Yeast Chromosomes

PDMS Device Fabrication and Surface Modification

PDMS fabricación de dispositivos y modificación de la superficie

Investigación

Biología

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Purificación de las mitocondrias de las células de levadura

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Colonia de levaduras Método de anidamiento

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

La levadura de alto rendimiento de la pantalla sobreexpresión plásmido

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

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 ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificación por cromatografía de ribosomas de levadura de gran actividad

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Inmunotinción de Disección del corazón embrionario de pez cebra

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Avidez detección basada en la interacción extracelular (AVEXIS) para la detección escalable de extracelular de baja afinidad interacciones ligando-receptor

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

De alto rendimiento mapeo físico de cromosomas utilizando automatizado<em&gt; In situ</em&gt; La hibridación

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

La disección y la inmunotinción de imaginal discos de<em&gt; Drosophila melanogaster</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...