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

Finansiering støtte til dette projekt blev leveret af den grundlæggende videnskab forskningsprogram gennem National Research Foundation af Korea (NRF) finansieret af Ministeriet for videnskab og IKT (2016R1A2B2006354).

1. forberedelse af medier
<ol><li>Forberede gær Extract med kosttilskud (ja), ja uden adenin (ja lav Ade), Pombe Glutamate medium (PMG12) og PMG uden adenin (PMG-Ade) ved at blande komponenterne som beskrevet i tabel 1. Ja-Ade (lav Ade) og PMG-Ade plader har alle de samme komponenter, men adenin udelades fra sidstnævnte.<ol>
<li>Brug de følgende salt (50 x) bestand: 52,5 g/L MgCl2·6H2O, 2 g/L CaCl2·2H2O, 50 g/L KCl, 2 g/L Na2...

<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>

Tilfældig mutagenese via fejlbehæftet PCR er et kraftfuldt værktøj til at generere en forskelligartet gruppe af mutanter i en given region. Denne teknik er især nyttig for undersøgelser, der forsøger at vurdere funktionen af en protein under en bestemt omstændighed. For eksempel, brugte vi heri fejlbehæftet PCR for at vurdere funktionen af 19S proteasom subunit, Rpt4, i heterochromatin vedligeholdelse. Ved at variere regionen målrettet af fejlbehæftede PCR og justere screening betingelser, vi var i stand til a...

Fremad genetik er et klassisk metode hvor forskere søger naturligt forekommende mutanter, der viser en særlig fænotype, og udføre genetiske analyser. I reverse genetik, mutationer er indført i et gen af interesse og fænotype er undersøgt. I sidstnævnte tilfælde bruges tilfældig mutagenese af et target gen ofte til at generere en pulje af mutanter, som efterfølgende vælges til ønskede fænotyper, såsom temperatur følsomhed eller ændret enzymatisk aktivitet. Forskellige metoder kan bruges til at opnå tilfældig mutagenese, herunder fejlbehæftet PCR1; UV bestråling2; kemiske mutagener, såsom ethyl methanesulfonate (EMS) eller salpetersyrling3; brugen af midlertidige mutator stammer, som dem over at udtrykke mutD5 4; og DNA blander5.
Her, beskriver vi en reverse-genetik strategi, hvor vi udnytter fejlbehæftet PCR for at generere mutant pools for et mål gen i fission gær. Som man kunne gætte fra navnet, genererer denne metode mutationer ved bevidst at indføre fejl under PCR. I modsætning til andre metoder til mutagenese tillader fejlbehæftet PCR bruger hen til definere regionen til at være mutagenized. Dette gør det særligt nyttig i bestræbelserne på at studere funktionen af en protein/domæne af interesse.
For at demonstrere denne tilfældig mutagenese procedure, bruger vi heri rpt4 +, som koder 19S proteasom subunit, som et eksempel. Rpt4 har vist sig at have proteolyse-uafhængig funktioner i organismer end fission gær6,7,8,9, og en defekt i proteolyse kunne forårsage indirekte effekter ved at ændre proteiner niveauer. Vi er derfor, screenes for mutanter, der udløste proteolyse-uafhængig ændringer, med mål at undersøge funktionen af proteasom på heterochromatin.
Fejlbehæftede PCR kan anvendes til nogen gene region ved tuning den placering, hvor primere binde. Mutanter, som udviser den ønskede fænotypiske forandringer kan identificeres med passende journalister. Her, vi udnyttet en ade6 + reporter indsat på centromer 1 ydre Gentag (otr) region10. Konstituerende heterochromatin er dannet på denne region11, så ade6 + reporter er tavshed i wild-type betingelse; Det angives ved røde kolonier10. En mutation, der destabiliserer det konstituerende heterochromatin på centromer vil føre til ekspression af ade6 + -reporter, der er visualiseret som hvide kolonier.

<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

Tilfældig mutagenese af et gen, målet er almindeligt anvendt til at identificere mutationer, der giver den ønskede fænotype. Af de metoder, der kan bruges til at opnå tilfældig mutagenese, fejlbehæftet PCR er en praktisk og effektiv strategi til at generere en forskelligartet gruppe af mutanter (dvs., en mutant bibliotek). Fejlbehæftede PCR er den foretrukne metode, når en forsker søger at mutere en pre-definerede region, som regionen kodning af et gen, mens upåvirket andre genomisk regioner. Når biblioteket mutant forstærkes af fejlbehæftede PCR, skal det blive klonet i en egnet plasmid. Størrelsen af biblioteket genereret af fejlbehæftede PCR er begrænset af effektiviteten af trinnet kloning. I fission gær, Schizosaccharomyces pombe, kan trinnet kloning udskiftes ved hjælp af en yderst effektiv one-step fusion PCR til at generere konstruktioner til transformation. Mutanter af ønskede fænotyper kan derefter vælges ved hjælp af passende journalister. Her beskriver vi denne strategi i detaljer, tager som et eksempel, en reporter indsat på centromeric heterochromatin.

De erhvervede Rpt4 mutanter ved at følge de procedurer, der er beskrevet i figur 1 kan analyseres ved at vurdere farver i kolonierne. Farverne på kolonierne er spottet på de relevante plader i faldende celle nummer i figur 2. Ade6 + reporter indsat på heterochromatin området er tavshed i wild-type og viser rød kolonier i ja-Ade plade. Når heterochromatin er destabiliseret og ade6 + reporter udtrykkes, kan...

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

Gen-målrettet tilfældig mutagenese at vælge Heterochromatin-destabiliserende proteasom mutanter i Fission gær

I denne artikel beskrives en detaljeret metode for tilfældig mutagenese af et target gen i fission gær. Som et eksempel målrette vi rpt4 +, som koder en underenhed af 19S proteasom og screene for mutationer, at destabilisere heterochromatin.

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

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Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Målrettet<em&gt; I Situ</em&gt; Mutagenese af histon-gener i knopskydende gær

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

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

Under anvendelse af et fluorescerende PCR-kapillær gelelektroforese Teknik til genotype CRISPR / Cas9-medieret Knockout Mutanter i et format med høj kapacitet

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

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CRISPR/Cas9 gen redigering at gøre betinget mutanter af menneskelige Malaria parasitten, P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Mating-based Overexpression Library Screening in Yeast

Parring-baserede overekspression bibliotek Screening i gær

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

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Kendetegner Histon posttranslationel modifikation ændringer i gær Neurodegenerative Proteinopathy modeller

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

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

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En dyb-sekventering-assisteret, spontane Suppressor skærm i Fission gær 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 ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Forbedret gær One-hybrid skærme til at identificere transskription faktor Binding til menneskelige DNA-sekvenser

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

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