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

Finanziamento per questo progetto è stato fornito dal programma di ricerca di scienza base attraverso la National Research Foundation di Corea (NRF) finanziato dal Ministero delle scienze e TIC (2016R1A2B2006354).

1. preparazione dei Media
<ol><li>Preparare Estratto di lievito con integratori (Sì), sì senza adenina (Sì basso Ade), Pombe Glutamate media (PMG12) e PMG senza adenina (PMG-Ade) mescolando i componenti come descritto nella tabella 1. Sì-Ade (bassa Ade) e piastre PMG-Ade hanno tutti gli stessi componenti, ma l'adenina è omesso da quest'ultimo.<ol>
<li>Utilizzare il seguente stock di sale (50x): 52,5 g/L MgCl2·6H2O, 2 g/L CaCl2·2H2</su...

<ol>
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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>

Mutagenesi casuale tramite PCR errori è un potente strumento per la generazione di un pool di diverso mutanti in una determinata regione. Questa tecnica è particolarmente utile per gli studi che cercano di valutare la funzione di una proteina in una circostanza specifica. Ad esempio, abbiamo utilizzato nel presente documento errori PCR per valutare la funzione della 19S proteasome subunità, Rpt4, nella manutenzione di eterocromatina. Variando la regione mirato dalla PCR errori e regolare le condizioni di selezione, si...

Genetica in avanti è un metodo classico, in cui i ricercatori cercano mutanti naturali che visualizzano un fenotipo particolare ed eseguire analisi genetiche. In genetica inversa, mutazioni vengono introdotti in un gene di interesse ed il fenotipo è esaminato. In quest'ultimo caso, mutagenesi casuale di un gene target viene spesso utilizzata per generare un pool di mutanti che sono successivamente selezionato per desiderata fenotipi, come sensibilità di temperatura o alterati di attività enzimatica. Possono essere utilizzati vari metodi per raggiungere mutagenesi casuale, tra cui errori PCR1; Di irradiazione UV2; mutageni chimici, ad esempio methanesulfonate etilico (EMS) o acido nitroso3; l'uso di ceppi di mutator temporanei, come quelli che sovraesprimono mutD5 4; e DNA shuffling5.
Qui, descriviamo una strategia inversa-genetica in cui utilizziamo errori PCR per generare mutanti piscine per un gene dell'obiettivo del lievito di fissione. Come si potrebbe intuire dal suo nome, questo metodo genera mutazioni introducendo deliberatamente errori durante la PCR. A differenza di altri metodi di mutagenesi, errori PCR permette all'utente di definire la regione a essere mutagenizzato. Questo rende particolarmente utile negli sforzi per studiare la funzione di una proteina/dominio di interesse.
Per illustrare questa procedura di mutagenesi casuale, qui usiamo rpt4 +, che codifica per la subunità del proteasoma 19S, come un esempio. Rpt4 ha dimostrato di avere funzioni di proteolisi-indipendente negli organismi diversi da fissione lievito6,7,8,9, e un difetto nella proteolisi potrebbe causare effetti indiretti alterando proteine livelli. Abbiamo, quindi, sottoposti a screening per mutanti che ha attivato la proteolisi-indipendente modifiche, con l'obiettivo di indagare la funzione del proteasoma su eterocromatina.
Errori PCR può essere applicato a qualsiasi regione del gene regolando la posizione a cui associare il primer. Mutanti che presentano la variazione fenotipica desiderata possono essere identificati con i giornalisti appropriati. Qui, abbiamo utilizzato un reporter ade6 + inserito al centromero 1 esterno ripetere (otr) regione10. Eterocromatina costitutiva è formata a questa regione11, così il ade6 + reporter viene messo a tacere nella condizione di selvaggio-tipo; Questo è indicato da colonie rosse10. Una mutazione che destabilizza l'eterocromatina costitutiva al centromero porterà all'espressione del ade6 + reporter, che è visualizzato come colonie bianchi.

<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

Mutagenesi casuale di geni bersaglio è comunemente usata per identificare le mutazioni che producono il fenotipo desiderato. Dei metodi che possono essere utilizzati per raggiungere mutagenesi casuale, errori PCR è una strategia conveniente ed efficiente per la generazione di un pool di diverso mutanti (i. e., una libreria mutante). Errori PCR è il metodo di scelta quando un ricercatore cerca di mutare una regione pre-definita, come la regione di codificazione di un gene lasciando inalterato altre regioni genomiche. Dopo la libreria mutante è amplificata dalla PCR errori, deve essere clonato in un plasmide adatto. La dimensione della biblioteca generata da errori PCR è limitata dall'efficienza del passaggio clonazione. Tuttavia, il lievito di fissione, Schizosaccharomyces pombe, il clonazione passo possa essere sostituito dall'uso di una fusione altamente efficiente One-Step PCR per generare costrutti per la trasformazione. Mutanti di fenotipi desiderati quindi sono selezionabili utilizzando appropriati ai giornalisti. Qui, descriviamo questa strategia in dettaglio, prendendo come esempio, un reporter inserito all'eterocromatina centromerica.

I mutanti di Rpt4 acquisiti seguendo le procedure descritte nella Figura 1 possono essere analizzati valutando i colori delle colonie. I colori delle colonie sono macchiati sulle piastre pertinenti nel fare diminuire il numero delle cellule nella Figura 2. Il reporter ade6 + inserito presso la regione di eterocromatina è messo a tacere nel selvaggio-tipo e Mostra colonie rosse in piastra sì-Ade. Una volta che l'eterocr...

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 targeting mutagenesi casuale per selezionare eterocromatina-destabilizzare Proteasome mutanti in lievito di fissione

Questo articolo descrive una metodologia dettagliata per mutagenesi casuale di un determinato gene in lievito di fissione. Ad esempio, noi target rpt4 +, che codifica per una subunità del proteasoma 19S e schermo per le mutazioni che destabilizzare eterocromatina.

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.

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