Fondos para este proyecto fue proporcionada por el programa de investigación de ciencia básica a través de la nacional investigación Fundación de Corea (NRF) financiado por el Ministerio de ciencia y TIC (2016R1A2B2006354).

1. preparación de medios de
<ol><li>Preparar Extracto de levadura con suplementos (sí), sí sin adenina (Ade baja de sí), media de Pombe Glutamate (PMG12) y PMG sin adenina (Ade-PMG) mezclando los componentes como se describe en la tabla 1. SÍ-Ade (bajo Ade) y placas PMG-Ade tienen todos los mismos componentes, pero la adenina se omite de este último.<ol>
<li>Utilice el siguiente stock de sal (50 x): 52,5 g/L MgCl2·6H2O 2 g/L CaCl2·2H2O 50 g/L de KCl, 2 g/L Na2para4 en agua destilada. Filtro esterilice usando un filtro de tamaño de poro de 0,22 μm y almacenar a 4 ° C.</li>
		<li>Utilizar la vitamina siguiente (1, 000 x) stock: 10 g/L de ácido nicotínico, pantotenato de 1 g/L, biotina 10 mg/L en agua destilada, 10 g/L inositol. Filtro esterilice usando un filtro de tamaño de poro de 0,22 μm y almacenar a 4 ° C.</li>
		<li>Utilice el siguiente mineral (10, 000 x) stock: 5 g/L de ácido bórico, 4 g/L MnSO4, 4 g/L ZnSO4·7H2O, 2 g/L FECLAS2·4H2O 0.4 g/L MoO3, KI, 0.4 g/L CuSO4·5H2O de 1 g/L , 10 g/L de ácido cítrico en agua destilada. Filtro esterilice usando un filtro de tamaño de poro de 0,22 μm y almacenar a 4 ° C. Nota: Medio líquido sí puede realizarse omitiendo el agar.</li>
	</ol>
</li>
	<li>Medio de autoclave y enfriar a continuación 60 ° C mientras revuelve con una barra de agitación, a una velocidad de 200 rpm.</li>
	<li>Para las placas sí + G418 (geneticin), agregue solución 1 mL/L G418 al medio sí.<ol>
<li>Utilice el siguiente G418 (1, 000 x) stock: G418 100 mg/mL en agua destilada. Filtro de esterilización usando un filtro de tamaño de poro de 0.22 μm, alícuota a tubos de centrífuga de 1.5 mL y almacenar a-20 ° C.</li>
	</ol>
</li>
	<li>Revuelva con otro 5-10 min y alícuota los medios platos de Petri de 90 mm (a 1 L de medio alícuotas a aproximadamente 40 placas de Petri. Almacenar las placas a 4 ° C.</li>
</ol>2. clonación de rpt4 + y su 5' / 3' UTRs
<ol><li>Realizar la PCR con iniciadores p1 y p2 (figura 1A) usando la fisión levadura DNA genómico (gDNA) como la plantilla en un volumen total de 50 μl (~ 1 μg ADN, enzima de U 20, 1 x buffer de reacción), y aplicando los ciclos de reacción se describe en la tabla 2 para ampliar una base de 3.315 (bp) fragmento formado por rpt4 + y su 5' / 3' UTRs. Purificar el producto PCR utilizando un kit de purificación13 como lo indique el fabricante (figura 1A).</li>
	<li>Digerir la de vector pBlueScript KS(-)14 y el fragmento de ADN amplificado con rpt4 + con sus 5' / 3' UTRs con BamH1 y Xho1 en un volumen total de 20 μl (~ 1 μg de ADN, 20 de U de enzima, tampón de digestión x 1) a 37 ° C en un baño de agua durante 16-18 h (figura 1B).</li>
	<li>Gel purifica el vector digerido (gel de agarosa al 1,2%) e inserte el ADN por realizar la electroforesis en gel de agarosa TAE-basado (100 V, 1 h), suprimir las bandas de ADN de los tamaños deseados y aislar el ADN con una de kit de extracción de gel de15.</li>
	<li>Ligar el vector y el inserto de ADN con ligase de la DNA de T4 mediante la realización de una reacción de la ligadura de 20 μl que contiene 50-100 ng del vector de la DNA, una ~ 3-fold exceso molar del ADN inserto y 400 U T4 ADN ligasa 1 x buffer de la ligadura. Incubar la mezcla a 18 ° C durante 16-18 h.</li>
	<li>Transformar el ADN ligado en 100 μl de e. coli DH5α mediante la aplicación de choque térmico de 70 s a 42 ° C. Añadir 1 mL de LB e incubar 1 h a 37 ° C para la recuperación. Realizar la centrifugación (13.800 x g), descartar el sobrenadante, placa de la transformada de e. coli células en placas de LB-ampicilina (LA) e incubar a 37 ° C por 16 h.</li>
	<li>Tomar 4-8 colonias con palillos de dientes y crecer durante la noche a 37 ° C en medio de 3 mL LA.</li>
	<li>Aislar la plásmidos con un plásmido purificación kit16 utilizado según protocolo del fabricante y realizar análisis enzimático de la restricción digestiones con 5 μl del plásmido aislada, 5 U de cada enzima de restricción (BamH1 y Xho1) y 1 x de buffer de restricción. Incubar la mezcla de reacción a 37 ° C en un baño de agua durante 3 h. confirmar la clonación mediante la secuenciación de los plásmidos de candidato.</li>
</ol>3. Introducción de la mutación silenciosa (sitio de la restricción deXho1 )
<ol><li>Diseño de un par de cartillas 33-bp (p3 y p4) que contienen la mutación deseada en la secuencia complementaria de lo contrario.</li>
	<li>Realizar la polimerización en cadena con polimerasa de alta fidelidad17 (figura 1) en un volumen total de 25 μl (10 ng de plásmido clonado, 2 μl de 10 pmol cartilla mezcla, enzima U 2, 1 x buffer de reacción) utilizando los parámetros ciclismo enumerados en la tabla 3.</li>
	<li>Digerir el plásmido de plantilla con DpnI en un volumen total de 20 μl (20 U enzima, tampón de digestión 1 x) a 37 ° C en un agua de baño para 1 h.</li>
	<li>Transformar, propagar, aislar y secuenciar el plásmido que contiene la mutación silenciosa, como se describe en pasos de 2.5-2.7. Nota: La mutación silenciosa también puede introducirse mediante un método de clonación que permite la Unión de varios fragmentos de la DNA en una sola reacción18.</li>
</ol>4. al azar mutagénesis de rpt4 + por Error-prone PCR
<ol><li>Realizar PCR propensa a errores, utilizando el plásmido con la mutación silenciosa (Xho1 sitio) como la plantilla, cartillas p5 y p6, un volumen total de 50 μl (la cantidad de plantilla definida en el paso 4.1.1, 2 μl de mezcla de cartilla de 10 pmol, enzima U 2.5, tampón de reacción x 1) , y una polimerasa especial que está diseñada para generar un error de alta velocidad19 (figura 1). Realizar PCR como se describe en la tabla 2.<ol>
<li>Utilizar 4-5 μg de plásmido de plantilla para controlar la frecuencia de mutación a 1 bp/kb (kilobases). Nota: Una alta concentración (> 500 ng/μL) del plásmido, que se puede obtener utilizando un kit de preparación mini-para el20, se requiere.</li>
	</ol>
</li>
	<li>Utilice gel de electroforesis para confirmar un producto PCR de 2670 bp (gel de agarosa al 1,2%). Gel purifica este producto PCR como se describe en el paso 2.5.</li>
</ol>5. preparación de los fragmentos PCR de fusión (KAN, 3' UTR)
<ol><li>Realizar PCR utilizando pFA6a-KANMX621 como plantilla, cartillas p7 y p8, y las condiciones enumeran en la tabla 4 para obtener el fragmento de marcador (KAN). Gel purifica el fragmento resultante de 1.480-bp como se describe en el paso 2.5 (Figura 1E).<ol>
<li>Compruebe si el codón del gen para la mutación y la región de la codificación de su gen adyacente están separados por más de 500 bp. El terminador endógeno no puede utilizarse cuando la región es inferior a 500 bp; algo, el terminador de ADH puede usarse en lugar del terminator endógena. Los cambios de protocolo necesarios para utilizar el terminador de ADH se presentan en la tabla 5. Nota: Estos cambios sólo se aplican cuando el terminador de la ADH , que está disponible en el plásmido pFA6a-3HA-KANMX6, se utiliza en lugar del terminator endógena.</li>
	</ol>
</li>
	<li>Realizar la PCR con la clonada rpt4 +-que contiene el vector como la plantilla y las cartillas p9 y p10 en un volumen total de 50 μl (20 ng de plásmido clonado, 2 μl de 10 pmol cartilla mezcla, enzima U 2, 1 x buffer de reacción), utilizando las condiciones presentadas en el cuadro 6. Gel purifican obtenidos 506 bp 3' UTR fragmento (Figura 1E).</li>
</ol>6. generación de la construcción de la transformación por fusión PCR (Figura 1E)
<ol><li>Preparar los 3 fragmentos (mutagenized rpt4 +, KAN y el 3' UTR) a 50 ng/μl.</li>
	<li>Realizar fusión que polimerización en cadena de los tres fragmentos utilizando primers p11 y p12, tal como se describe en la tabla 7. (El protocolo de fusión PCR es una modificación del protocolo original22). Purificar el producto PCR de 4.398-bp principales.<ol>
<li>Utilice rpt4 +: KAN:3'UTR fragmentos en una proporción de 1:3:1 para obtener resultados óptimos. Nota: La cantidad total de ADN inserto no debe exceder 500 ng. La cantidad recomendada de rpt4 +: KAN:3'UTR es de 50 ng de ng:50 de ng:150. La región mutagenized es aproximadamente 1,6 kb, por lo que el número mínimo de colonias de levaduras que se representan todas las combinaciones posibles de cada base se mutó a los otros tres es 1.600 x 3 = 4.800 colonias. Debido a una reacción de transformación produce típicamente 400-500 colonias, por lo menos 10 reacciones digno de fusión PCR construir es necesario. Esta necesidad puede satisfacerse simplemente aumentando la cantidad de reacción (es decir, el número de tubos PCR) por un factor de diez.</li>
	</ol>
</li>
</ol>7. transformación de la levadura de fisión por electroporación (Figura 1F)
<ol><li>Inocular la levadura a 10 mL de medio sí saturación por incubar durante más de 16 h.</li>
	<li>Diluir las células a una densidad óptica a 600 nm (OD600) de 0.2 en 200 mL de sí medio e incubar a 30 ° C con agitación durante 5-6 h, para OD600 = 0.6 - 0.8.</li>
	<li>Concentrar las células OD600 = 30, dispensar a cuatro tubos cónicos de 50 mL y enfriar en hielo durante 10 minutos.</li>
	<li>La cosecha de las células mediante la realización de centrifugación a 1.050 x g durante 3 min a 4 ° C. Coloque los tubos y cubetas de electro 10 en hielo. Realice los pasos 7.3-7.8 en hielo.</li>
	<li>Deseche el sobrenadante y añadir 15 mL de sorbitol de 1,2 M. Agitar suavemente los tubos para resuspender las células.</li>
	<li>Centrifugar las células a 1050 x g durante 3 min a 4 ° C.</li>
	<li>Repita los pasos 7.4 y 7.5. Deseche el sobrenadante. Añadir 500 μl de sorbitol de 1.2 M a cada tubo, resuspender las células y recoger todas las células en un tubo cónico de 15 ml.</li>
	<li>Añadir sorbitol M 1,2 a 2,4 mL (OD600 = 10 por 0,2 mL). Mantenga el tubo en hielo.</li>
	<li>Añadir 200 μL de células suspendidas de sorbitol (Asegúrese de que se suspenden completamente) a un tubo de EP que contiene la fusión PCR construir, mezclar bien y transferir la muestra a una cubeta de electro.</li>
	<li>Electroporate las células con las siguientes opciones: hongos, ShS (2.00kV, 1 pulso).</li>
	<li>Añadir 600 μl de 1,2 M sorbitol a cada cubeta de electro para un volumen total de 800 μl y luego las células en sí cuatro platos (200 μl/placa). Diez cubetas de electro dispensará a 40 sí las placas.</li>
	<li>Incubar las placas a 30 ° C durante 24 h.</li>
	<li>Realizar placas réplica a sí + G418 e Incube las placas a 30 ° C durante 3 días.</li>
</ol>8. selección de mutantes desestabilizadoras de heterocromatina y comprobación de falsos positivos
<ol><li>Para cada placa sí + G418, realizar chapado en réplica a sí-Ade (bajo Ade) y placas PMG-Ade (No Ade). Incubar las placas de réplica durante 1-2 días a 30 ° C, hasta que algunas de las colonias en las placas Ade sí muestran una coloración roja (figura 1).</li>
	<li>Comparar placas sí-Ade y PMG-Ade y seleccionar las células que muestran color rosa o blanco en la placa sí-Ade y también crecen en la placa de PMG-Ade. No seleccione colonias sin crecimiento en PMG-Ade, ya que son falsos positivos (por ejemplo, lo que refleja que el cassette de KAN se ha integrado en un sitio de destino no en otros lugares en el genoma).</li>
	<li>Recoger aproximadamente 1 x 105 células de cada colonia e incuban en 10 μl de solución SPZ que contiene 2,5 mg/mL zymolyase 100 T a 37 ° C durante al menos 30 min uso 1 μl de esta solución como la plantilla inicial para Colonia PCR (figura 1 H).<ol>
<li>Hacer los SPZ (50 mL) mezclando 30 mL de sorbitol de 2 M, 4,05 mL de 1 M de Na2HPO4, 0,95 mL NaH2PO4 y 15 mL de agua destilada (pH final, 7.5). Muestras (5 mL) pueden ser complementadas con zymolyase y almacenar en alícuotas de 500 μl a-20 ° C hasta su uso.</li>
	</ol>
</li>
	<li>Realizar la electroforesis en gel (gel de agarosa al 1.2%, 180V, 20 min) para visualizar los resultados de + la Colonia PCR. Seleccione las reacciones que exhiben bandas PCR del tamaño adecuado y digestión 5 μl de cada producto de la reacción con XhoI (4 U enzima, tampón de digestión x 1, baño de agua de 37 ° C, 1 h) para descartar falsos positivos (figura 1 H).</li>
	<li>Realizar la electroforesis en gel para visualizar los resúmenes XhoI (gel de agarosa 1.2%, 180 V, 20 min). Marque las colonias cuyos productos PCR se cortan por XhoIy parche a las placas sí + G418 (figura 1I).</li>
	<li>Aislar gDNA de las células de la levadura de fisión y realizar la secuencia de los productos PCR23. Comparar la secuencia obtenida con la secuencia wild type para identificar mutaciones (figura 1I).</li>
	<li>Directamente volver a introducir la mutación a las células de tipo salvaje transformándolos con fusión construcciones amplificadas por PCR de las células mutantes23. Utilice manchado para confirmar el fenotipo de las células mutantes recién hechas (Figura 1J).<ol>
<li>Fusión transformación construcción puede ser fácilmente amplificada por PCR con iniciadores p1 y p10 usando la DNA genomic de mutantes como la plantilla en un volumen total de 50 μl (~ 1 μg ADN, enzima de U 20, 1 x buffer de reacción) y la aplicación de los ciclos de reacción se describe en tabla 2 . Transformación de la construcción se puede hacer siguiendo los pasos 7.1-7.13.</li>
	</ol>
</li>
</ol>

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Mutagénesis al azar mediante error-prone PCR es una poderosa herramienta para la generación de un diverso grupo de mutantes en una región determinada. Esta técnica es especialmente útil para estudios que intentan evaluar la función de una proteína en una circunstancia específica. Por ejemplo, en el presente documento utilizamos error-prone PCR para evaluar la función de la subunidad del proteasoma 19S, Rpt4, en mantenimiento de heterocromatina. Variando la región dirigida por el error-prone PCR y ajustar las co...

Genética directa es un método clásico en el que los investigadores buscan a mutantes naturales que muestran un fenotipo particular y realizan análisis genéticos. En la genética reversa, se introducen mutaciones en un gen de interés y se examina el fenotipo. En este último caso, mutagénesis al azar de un gene de la blanco se utiliza a menudo para generar un grupo de mutantes que son posteriormente seleccionados para fenotipos deseados, tales como sensibilidad de temperatura o altera la actividad enzimática. Diversos métodos pueden utilizarse para mutagénesis al azar, incluyendo error-prone PCR1; De irradiación UV2; mutágenos químicos, tales como Metanosulfonato de etilo (EMS) o ácido nitroso3; el uso de cepas mutator temporal, tales como sobre-expresando mutD5 4; y barajado de ADN5.
Aquí, describimos una estrategia genética inversa en la cual utilizamos error-prone PCR para generar piscinas mutantes para un gen blanco en la levadura de fisión. Como uno puede imaginar por su nombre, este método genera mutaciones introduciendo deliberadamente errores durante el PCR. A diferencia de otros métodos de mutagénesis, error-prone PCR permite al usuario definir la región a ser mutagenized. Esto es particularmente útil en esfuerzos para estudiar la función de dominio y una proteína de interés.
Para demostrar este procedimiento mutagénesis al azar, en este documento utilizamos rpt4 +, que codifica la subunidad de proteasoma 19S, como un ejemplo. Rpt4 se ha demostrado que tienen funciones independientes de la proteólisis en organismos distintos de fisión levaduras6,7,8,9, y un defecto en la proteólisis puede causar efectos indirectos mediante la alteración de las proteínas niveles. Nosotros, por lo tanto, para los mutantes que desencadenó cambios independientes de la proteólisis, con el objetivo de investigar la función del proteasoma en la heterocromatina.
Error-prone PCR puede aplicarse a cualquier región del gene de templar la situación en la que se unen los cebadores. Mutantes que exhiben el cambio fenotípico deseado pueden identificarse con los reporteros apropiados. Aquí, utilizamos un ade6 + reportero insertado en el centrómero 1 exterior repita (otr) región10. Heterocromatina constitutiva es formada en esta región11, por lo que el reportero ade6 + está silenciado en la condición de tipo salvaje; Esto es indicado por las colonias rojo10. Una mutación que desestabiliza la heterocromatina constitutiva en el centrómero dará lugar a la expresión de la ade6 + reporter, que se visualiza como colonias blancas.

<table><tbody><tr><td>&lt;strong&gt;1. Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270-10KG</td><td></td></tr><tr><td>Yeast extract</td><td>BD Biosciences</td><td>212720</td><td></td></tr><tr><td>L-Leucine</td><td>JUNSEI</td><td>87070-0310</td><td></td></tr><tr><td>Adenine sulfate</td><td>ACR</td><td>16363-0250</td><td></td></tr><tr><td>Uracil&amp;nbsp;</td><td>Sigma-Aldrich</td><td>U0750</td><td></td></tr><tr><td>L-Histidine</td><td>Sigma-Aldrich</td><td>H8125</td><td></td></tr><tr><td>KH2PO4</td><td>Sigma-Aldrich</td><td>1.05108.0050</td><td></td></tr><tr><td>NaCl</td><td>JUNSEI</td><td>19015-0350</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>63140</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>12095</td><td></td></tr><tr><td>Potassium phthalate</td><td>Sigma-Aldrich</td><td>P6758-500g</td><td></td></tr><tr><td>Inositol</td><td>Sigma-Aldrich</td><td>I7508-50G</td><td></td></tr><tr><td>Biotin</td><td>Sigma-Aldrich</td><td>B4501-100MG</td><td></td></tr><tr><td>Boric Acid</td><td>Sigma-Aldrich</td><td>B6768-1KG</td><td></td></tr><tr><td>MnSO4</td><td>Sigma-Aldrich</td><td>M7634-500G</td><td></td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>JUNSEI</td><td>83060-031</td><td></td></tr><tr><td>FeCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>KANTO</td><td>CB8943686</td><td></td></tr><tr><td>Sodium molybdate dihydrate</td><td>YAKURI</td><td>31621</td><td></td></tr><tr><td>KI</td><td>JUNSEI</td><td>80090-0301</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>YAKURI</td><td>09605</td><td></td></tr><tr><td>D-myo-inositol</td><td>MP biomedicals</td><td>102052</td><td></td></tr><tr><td>Pantothenate</td><td>YAKURI</td><td>26003</td><td></td></tr><tr><td>Nicotinic Acid</td><td>Sigma-Aldrich</td><td>N4126-500G</td><td></td></tr><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A4418-100G</td><td></td></tr><tr><td>Agarose</td><td>Biobasic</td><td>D0012</td><td></td></tr><tr><td>G418, geneticin</td><td>LPS</td><td>G41805</td><td></td></tr><tr><td>&lt;strong&gt;2. Enzyme reactions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PfuUltra II Fusion HS DNA Polymerase</td><td>Aglient</td><td>600380</td><td>For site-directed mutagenesis</td></tr><tr><td>GeneMorphII Random mutagenesis Kit</td><td>Aglient</td><td>200500</td><td>For error-prone PCR</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>Thermo Fisher</td><td>F-530L</td><td>For fusion PCR</td></tr><tr><td>Ex Taq&amp;nbsp;DNA Polymerase</td><td>Takara</td><td>RR001b</td><td>For general PCR</td></tr><tr><td>BamH1</td><td>New England BioLabs</td><td>R0136S</td><td></td></tr><tr><td>Xho1</td><td>New England BioLabs</td><td>R0146S</td><td></td></tr><tr><td>Dpn1</td><td>New England BioLabs</td><td>R0176S</td><td></td></tr><tr><td>&lt;strong&gt;3. Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Velveteen square, black</td><td>VWR</td><td>89033-116</td><td>For replica</td></tr><tr><td>Replica-plating tool</td><td>VWR</td><td>25395-380</td><td>For replica</td></tr><tr><td>MicroPulser Electroporator</td><td>Biorad</td><td>1652100&amp;nbsp;</td><td>For fission yeast transformation</td></tr><tr><td>Electroporation Cuvettes, 0.2 cm gap</td><td>Biorad</td><td>1652086&amp;nbsp;</td><td>For fission yeast transformation</td></tr><tr><td>Thermal Cycler</td><td>Bioer</td><td>BYQ6078</td><td>For fusion PCR ramp reaction&amp;nbsp;</td></tr></tbody></table>

gene-targeted random mutagenesis to select heterochromatin-destabilizing proteasome mutants in fission yeast

Mutagénesis al azar de un gene de la blanco se utiliza comúnmente para identificar las mutaciones que producen el fenotipo deseado. De los métodos que pueden utilizarse para lograr la mutagénesis al azar, error-prone PCR es una estrategia conveniente y eficiente para la generación de un diverso grupo de mutantes (es decir., una biblioteca mutante). Error-prone PCR es el método de elección cuando un investigador intenta mutar una región previamente definida, como la región codificante de un gen dejando otras regiones genómicas inafectado. Después de que la biblioteca mutante se amplifica por PCR propensa a errores, debe ser clonado en un plásmido adecuado. El tamaño de la biblioteca generado por error-prone PCR está limitado por la eficiencia de la clonación del paso. Sin embargo, en la levadura de fisión Schizosaccharomyces pombe, el paso de la clonación puede reemplazarse por el uso de una fusión de un solo paso muy eficiente PCR para generar construcciones para la transformación. Mutantes de fenotipos deseados pueden seleccionarse entonces usando reporteros apropiados. Aquí, describimos esta estrategia en detalle, tomando como ejemplo, un reportero insertado en centromérica de la heterocromatina.

Los mutantes Rpt4 adquiridos siguiendo los procedimientos descritos en la figura 1 se pueden analizar mediante la evaluación de los colores de las colonias. Los colores de las colonias se han manchado en las placas correspondientes en la disminución de número de células en la figura 2. El reportero ade6 + insertado en la región de heterocromatina es silenciado en el tipo salvaje y colonias rojo muestra en la placa s...

Watch this Scientific Journal Video about Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast at JoVE.com

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Gene-dirigido mutagénesis al azar para seleccionar a mutantes de proteasoma desestabilizadoras de la heterocromatina en la levadura de fisión

Este artículo describe una metodología detallada para la mutagénesis al azar de un gene de la blanco en la levadura de fisión. Por ejemplo, apuntamos rpt4 +, que codifica una subunidad del proteasoma 19S y defender para las mutaciones que desestabilizan la heterocromatina.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

gene-targeted-random-mutagenesis-to-select-heterochromatin

Research

JoVE Journal

Genetics

10.6K vistas.  Korea Advanced Institute of Science and Technology. Este artículo describe una metodología detallada para la mutagénesis al azar de un gene de la blanco en la levadura de fisión. Por ejemplo, apuntamos rpt4 +, que codifica una subunidad del proteasoma 19S y defender para las mutaciones que desestabilizan la heterocromatina.

Video: Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Optogenética aleatoria de mutagénesis, utilizando la histona-miniSOG en<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Investigación

Genética

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

Targeted<em&gt; In situ</em&gt; mutagénesis de histona genes en la levadura en ciernes

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Proyección de biblioteca sobreexpresión basado en apareamiento en levaduras

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

Una pantalla de supresor asistida por secuenciación profunda, espontánea en la levadura de fisión Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Los estudios genéticos de proteínas humanas de reparación del ADN utilizando la levadura como un sistema modelo

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Biología

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Mutagénesis y funcional protocolos de selección de la evolución dirigida de proteínas en E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

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. ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

Aislar Potenciada Hsp104 Variantes Usando levadura Modelos proteinopatía

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

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

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

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

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

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

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

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

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

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

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

Gene-dirigido mutagénesis al azar para seleccionar a mutantes de proteasoma desestabilizadoras de la heterocromatina en la levadura de fisión

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Optogenética aleatoria de mutagénesis, utilizando la histona-miniSOG en

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Proyección de biblioteca sobreexpresión basado en apareamiento en levaduras

Una pantalla de supresor asistida por secuenciación profunda, espontánea en la levadura de fisión Schizosaccharomyces pombe

Los estudios genéticos de proteínas humanas de reparación del ADN utilizando la levadura como un sistema modelo

Mutagénesis y funcional protocolos de selección de la evolución dirigida de proteínas en E. coli

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

Aislar Potenciada Hsp104 Variantes Usando levadura Modelos proteinopatía

La rápida identificación de químicos interacciones genéticas en

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