Name: gene-targeted random mutagenesis to select heterochromatin-destabilizing proteasome mutants in fission yeast
Uploaded: 2018-05-15T14:00:00
Description: Watch this Scientific Journal Video about Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast at JoVE.com

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 CaCl2·2H...

<ol>
	<li>Pritchard, L., Corne, D., Kell, D., Rowland, J., Winson, 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+general+model+of+error-prone+PCR.">A general model of error-prone PCR.</a> J Theor Biol. 234 (4), 497-509 (2005).</li><li>Pfeifer, G. P., You, Y. H., Besaratinia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+induced+by+ultraviolet+light.">Mutations induced by ultraviolet light.</a> Mutat Res. 571 (1-2), 19-31 (2005).</li><li>Moreno, S., Klar, A., Nurse, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetic+analysis+of+fission+yeast+Schizosaccharomyces+pombe.">Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.</a> Methods Enzymol. 194, 795-823 (1991).</li><li>Selifonova, O., Valle, F., Schellenberger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+novel+traits+in+microorganisms.">Rapid evolution of novel traits in microorganisms.</a> Appl Environ Microbiol. 67 (8), 3645-3649 (2001).</li><li>Cohen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+DNA+shuffling+works.">How DNA shuffling works.</a> Science. 293 (5528), 237 (2001).</li><li>Sahu, P. P., Sharma, N., Puranik, S., Chakraborty, S., Prasad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+26S+Proteasome+subunit+RPT4a+regulates+ToLCNDV+transcription+and+activates+hypersensitive+response+in+tomato.">Tomato 26S Proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato.</a> Sci Rep. 6, 27078 (2016).</li><li>Xu, Q., Singer, R. A., Johnston, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sug1+modulates+yeast+transcription+activation+by+Cdc68.">Sug1 modulates yeast transcription activation by Cdc68.</a> Mol Cell Biol. 15 (11), 6025-6035 (1995).</li><li>Bhat, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+19S+proteasome+ATPase+Sug1+plays+a+critical+role+in+regulating+MHC+class+II+transcription.">The 19S proteasome ATPase Sug1 plays a critical role in regulating MHC class II transcription.</a> Mol Immunol. 45 (8), 2214-2224 (2008).</li><li>Chang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Gal4+activation+domain+binds+Sug2+protein,+a+proteasome+component,+in+vivo+and+in+vitro.">The Gal4 activation domain binds Sug2 protein, a proteasome component, in vivo and in vitro.</a> J Biol Chem. 276 (33), 30956-30963 (2001).</li><li>Ekwall, K., Cranston, G., Allshire, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fission+yeast+mutants+that+alleviate+transcriptional+silencing+in+centromeric+flanking+repeats+and+disrupt+chromosome+segregation.">Fission yeast mutants that alleviate transcriptional silencing in centromeric flanking repeats and disrupt chromosome segregation.</a> Genetics. 153 (3), 1153-1169 (1999).</li><li>Grewal, S. I., Jia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin+revisited.">Heterochromatin revisited.</a> Nat Rev Genet. 8 (1), 35-46 (2007).</li><li>Forsburg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Forsburg Lab Recipes: Fission Yeast Media. , (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> QIAquick PCR Purification Kit. , (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> pBlueScript II Vector. , (2005).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> QIAquick Gel Extraction Kit. , (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> QuickGene SP Kit Plasmid Kit II (SP-PL2). , (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PfuUltra High-fidelity DNA Polymerase. , (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gibson Assembly Master Mix. , (2017).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GeneMorph II Random Mutagenesis Kits. , (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plasmid Plus Midi Kit. , (1999).</li><li>Bahler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+modules+for+efficient+and+versatile+PCR-based+gene+targeting+in+Schizosaccharomyces+pombe.">Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.</a> Yeast. 14 (10), 943-951 (1998).</li><li>Szewczyk, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+PCR+and+gene+targeting+in+Aspergillus+nidulans.">Fusion PCR and gene targeting in Aspergillus nidulans.</a> Nat Protoc. 1 (6), 3111-3120 (2006).</li><li>Nurse, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fission Yeast Handbook. , (2000).</li><li>Seo, H. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+19S+proteasome+is+directly+involved+in+the+regulation+of+heterochromatin+spreading+in+fission+yeast.">The 19S proteasome is directly involved in the regulation of heterochromatin spreading in fission yeast.</a> J Biol Chem. , (2017).</li><li>Tang, N. H., 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=Generation+and+characterisation+of+temperature+sensitive+mutants+of+genes+encoding+the+fission+yeast+spindle+pole+body.">Generation and characterisation of temperature sensitive mutants of genes encoding the fission yeast spindle pole body.</a> PeerJ Preprints. 5, e3377v3371 (2017).</li><li>Rasooly, A., Weisz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+antibacterial+activities+of+phloxine+B+and+other+halogenated+fluoresceins+against+methicillin-resistant+Staphylococcus+aureus.">In vitro antibacterial activities of phloxine B and other halogenated fluoresceins against methicillin-resistant Staphylococcus aureus.</a> Antimicrob Agents Chemother. 46 (11), 3650-3653 (2002).</li><li>Wilson, D. S., Keefe, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+mutagenesis+by+PCR.">Random mutagenesis by PCR.</a> Curr Protoc Mol Biol. , (2001).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="825">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">1. Media</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Glucose</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-10KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Yeast extract</td>
			<td style="width:155px;">BD Biosciences</td>
			<td style="width:119px;">212720</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">L-Leucine</td>
			<td style="width:155px;">JUNSEI</td>
			<td style="width:119px;">87070-0310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Adenine sulfate</td>
			<td style="width:155px;">ACR</td>
			<td style="width:119px;">16363-0250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Uracil&#160;</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">U0750</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">L-Histidine</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">H8125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">KH2PO4</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">1.05108.0050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">NaCl</td>
			<td style="width:155px;">JUNSEI</td>
			<td style="width:119px;">19015-0350</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">MgSO4&#8226;7H2O</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">63140</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">CaCl2</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">12095</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Potassium phthalate</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6758-500g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Inositol</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">I7508-50G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Biotin</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">B4501-100MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Boric Acid</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">B6768-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">MnSO4</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7634-500G</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">ZnSO4&#8226;7H2O&#160;</td>
			<td style="width:155px;">JUNSEI</td>
			<td style="width:119px;">83060-031</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">FeCl2&#8226;4H2O</td>
			<td style="width:155px;">KANTO</td>
			<td style="width:119px;">CB8943686</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Sodium molybdate dihydrate</td>
			<td style="width:155px;">YAKURI</td>
			<td style="width:119px;">31621</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">KI</td>
			<td style="width:155px;">JUNSEI</td>
			<td style="width:119px;">80090-0301</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">CuSO4&#8226;5H2O</td>
			<td style="width:155px;">YAKURI</td>
			<td style="width:119px;">09605</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">D-myo-inositol</td>
			<td style="width:155px;">MP biomedicals</td>
			<td style="width:119px;">102052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Pantothenate</td>
			<td style="width:155px;">YAKURI</td>
			<td style="width:119px;">26003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Nicotinic Acid</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">N4126-500G</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">(NH4)2SO4&#160;</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4418-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Agarose</td>
			<td style="width:155px;">Biobasic</td>
			<td>D0012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">G418, geneticin</td>
			<td style="width:155px;">LPS</td>
			<td style="width:119px;">G41805</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">2. Enzyme reactions</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">PfuUltra II Fusion HS DNA Polymerase</td>
			<td style="width:155px;">Aglient</td>
			<td style="width:119px;">600380</td>
			<td>For site-directed mutagenesis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">GeneMorphII Random mutagenesis Kit</td>
			<td style="width:155px;">Aglient</td>
			<td style="width:119px;">200500</td>
			<td>For error-prone PCR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Phusion High-Fidelity DNA Polymerase</td>
			<td style="width:155px;">Thermo Fisher</td>
			<td>F-530L</td>
			<td>For fusion PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Ex Taq&#160;DNA Polymerase</td>
			<td style="width:155px;">Takara</td>
			<td style="width:119px;">RR001b</td>
			<td>For general PCR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">BamH1</td>
			<td style="width:155px;">New England BioLabs</td>
			<td style="width:119px;">R0136S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Xho1</td>
			<td style="width:155px;">New England BioLabs</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Dpn1</td>
			<td style="width:155px;">New England BioLabs</td>
			<td style="width:119px;">R0176S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">3. Equipment</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Velveteen square, black</td>
			<td style="width:155px;">VWR</td>
			<td style="width:119px;">89033-116</td>
			<td>For replica</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Replica-plating tool</td>
			<td style="width:155px;">VWR</td>
			<td style="width:119px;">25395-380</td>
			<td>For replica</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">MicroPulser Electroporator</td>
			<td style="width:155px;">Biorad</td>
			<td style="width:119px;">1652100&#160;</td>
			<td>For fission yeast transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Electroporation Cuvettes, 0.2 cm gap</td>
			<td style="width:155px;">Biorad</td>
			<td style="width:119px;">1652086&#160;</td>
			<td>For fission yeast transformation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Thermal Cycler</td>
			<td style="width:155px;">Bioer</td>
			<td style="width:119px;">BYQ6078</td>
			<td>For fusion PCR ramp reaction&#160;</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 ...

The overall goal of this random mutagenesis is to screen for mutations that destabilize heterochromatin. This method can help answer key questions in the heterochromatin field, such as factors that regulate the formation and maintenance of heterochromatin. The main advantage of this technique is that it can specifically target a desired gene for mutagenesis, even if the gene is essential.To begin, prepare yeast extract with supplements, or YES, without adenine, Pombe Glutamate Medium, or PMG, and PMG without adenine, by mixing the components as shown in this table. After autoclaving and adding G418 if necessary, stir the medium for another five to ten minutes. Then, aliquot the media into 90 milliliter Petri dishes and store the plates at four degrees Celsius.Using the cloned rpt4 positive, with its five prime and three prime UTRs and silent mutation, as well as primers p5 and p6 and a special polymerase, designed to generate a high error rate, perform error-prone PCR in a total volume of 50 microliters. After generating a transformation-fusion construct, according to the text protocol, inoculate ten milliliters of YES medium with yeast, and incubate it for more than 16 hours to saturation. Use 200 milliliters of YES medium to dilute the cells to an OD600 of 0.2, and incubate the culture at 30 degrees Celsius with shaking for five to six hours to an OD600 of 0.6 to 0.8.Calculate the volume of cells that add up to OD600 of 30. Then, dispense the cells into four 50 milliliter conical tubes and chill them on ice for 10 minutes. Harvest the cells by centrifugation at 1050 G and four degrees Celsius for three minutes.Then, place the microfuge tubes with cassette DNA and 10 electro cuvettes on ice. While still on ice, discard the supernatant and add 15 milliliters of 1.2-molar sorbitol, gently shake the tubes to re-suspend the cells, then centrifuge the cells at 1050 G and four degrees Celsius for three minutes. Discard the supernatant and perform a second sorbitol wash.After centrifugation, discard the supernatant. Then, re-suspend the cells and collect them all in one 15-milliliter conical tube. Next, add 1.2 molar sorbitol up to 2.4 milliliters.Keep the tube of cells on ice. Then, add 200 microliters of sorbitol-suspended cells to a tube containing the fusion PCR construct, mix well, and transfer the sample to an electro cuvette. It is important not to use an excess amount of PCR cassette, as it will give discharge errors.Electroporate the cells using the following options:add 600 microliters of 1.2-molar sorbitol to each electro cuvette for a total volume of 800 microliters. Then, spread the cells onto four YES plates. Incubate the plates at 30 degrees Celsius for 24 hours.Then, perform replica plating on YES plus G418 before incubating the plates for an additional three days. For each YES plus G418 plate, perform replica plating to YES without adenine and PMG without adenine plates. Incubate the replica plates at 30 degrees Celsius for one to two days until some of the colonies on the YES without adenine plates show a white coloration.Compare YES without adenine and PMG without adenine plates and select cells that show pink or white on the YES without adenine plate, and also grow on the PMG without adenine plate. Do not select colonies without growth on PMG without adenine, as they are false positives. Pick each colony and add it to 10 microliters of SPZ solution containing 2.5 milligrams per milliliter of zymolyase 100T.Then, incubate the colonies at 37 degrees Celsius for at least 30 minutes. Following the incubation, use one microliter of this solution as the starting template for colony PCR. After digesting selected colonies and carrying out gel electrophoresis according to the text protocol, mark the colonies whose PCR products were cut by XHO1 and patch them to YES plus G418 plates.Following the isolation of gDNA from fission yeast cells, and sequencing of the PCR products according to the text protocol, compare the obtained sequence with the wild-type sequence to identify mutations. Once the mutations are re-introduced into wild-type cells, use spotting to confirm the phenotype in the newly-made mutant cells. The rpt4 mutants generated using the protocol demonstrated in this video can be analyzed by assessing the colors of the colonies.As shown in this figure, the colonies were spotted onto the relevant plates in decreasing cell number. The adenine reporter inserted in the heterochromatin region is silenced in wild-type cells and produces red colonies on YES without adenine plates. Once the heterochromatin is destabilized and the adenine reporter is expressed, white colonies can be observed on the YES without adenine plates as seen with the clr4-delta mutant.The screened rpt4 mutants are as shown with the rpt4-1 mutant exhibiting the most severe heterochromatin destabilization. Once mastered, this technique can be done in two weeks to get the desired mutants. While attempting this procedure, it's important to eliminate the false positives as they are more frequent than expected.Following this procedure, other methods like protein purification and enzyme activity tests can be performed in order to answer additional questions, like the nature of the proteins. After watching this video, you should have a good understanding of how to generate mutants in a target gene with the desired phenotype.

Research

JoVE Journal

Genetics

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

Investigación

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High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

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De alto rendimiento de aislamiento robótica asistida sensible a la temperatura del letal mutantes en<em&gt; Chlamydomonas reinhardtii</em

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

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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 ...

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Mating-based Overexpression Library Screening in Yeast

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

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