La ricerca in laboratorio CJN è sostenuto da sovvenzioni di funzionamento dal NSERC, la Canadian Cancer Society Research Institute (CCSRI), e la Canadian Breast Cancer Foundation (BC-Yukon Branch).

NOTA: Il flusso generale di questa procedura è illustrata nella figura 2. 1. Determinazione della Dose Growth-inibitorio <ol><li> Preparazione del lievito cultura durante la notte NOTA: I media la crescita del lievito estratto-peptone-destrosio (YEPD) utilizzato in questo protocollo è una ricetta standard 13. <ol><li> Streak out BY4741 (Mata his3 Δ1 leu2Δ0 met15Δ0 ura3Δ0) le cellule su un piatto agar YEPD e incubare per 48 ore a 30 ° C o fino a formare colonie visibili. </li><li> Preparare culture durante la notte inoculando 5 ml di YEPD mezzi liquidi in un recipiente di coltura sterile con una singola colonia. Incubare una notte a 30 ° C con rotazione continua o agitazione. La cultura tipicamente raggiungere la saturazione del mattino (~ 2 × 10 8 cellule / ml). </li></ol></li><li> Preparazione agar solido contenente vari dosaggi chimici <ol><li> Preparare alcuni stock diil prodotto chimico da testare a 100x la concentrazione finale desiderata in un solvente appropriato. NOTA: Le concentrazioni efficaci dipendono dalla chimica, e devono essere determinati empiricamente. Si consiglia pertanto di verificare inizialmente un&#39;ampia gamma concentrazione finale, vale a dire, dal basso all&#39;alto mM mM. </li><li> Per ciascuna concentrazione della sostanza chimica da testare, un&#39;aliquota 3 ml di agar fuso YEPD ad un vari provetta sterile. Luogo in 55 ° C bagnomaria per evitare di solidificazione. </li><li> Per ogni concentrazione di sostanza chimica da testare aggiungere 30 ml di composto 100x ai media fusi, vortice 2-3 secondi per mescolare, e pipetta 1 ml in ogni coppia di pozzetti duplicati in un 12-pozzetti. Assicurati di includere un solo veicolo di controllo. Lasciare piastre di impostare notte a temperatura ambiente. Eliminare qualsiasi supporto aggiuntivo. </li></ol></li><li> Cellule spread placcatura <ol><li> Seminare 10 ml di YEPD mezzi liquidi con 2 ml di una cultura lievito saturo, coltivate durante la notte, come nella fase 1.1.2. </li><li> Piatto 25 ml di cultura lievito diluito per pozzetto, distribuiti in modo uniforme con perline di vetro sterili o asta, e permettono la piastra si asciughi sotto una fiamma. Incubare la piastra a 30 ° C per 48 ore. </li><li> Valutare la crescita del lievito (sia numero totale e la dimensione delle colonie) sulle piastre. Selezionare una concentrazione sub-letale di composto che non inibisce la crescita di oltre l&#39;10% -15% per eseguire lo schermo. </li></ol></li></ol> 2. sistematica schermo chimica genetica <ol><li> Manutenzione di delezione-mutante-array (DMA) e preparazione della Sorgente Piastra <ol><li> Per una descrizione dettagliata sulla costruzione di matrici mutanti cancellazione dalle scorte glicerolo consultare Baryshnikova et al. 14. Una volta mutanti di delezione sono schierati con una densità di 384 colonie per piastra, il DMA può essere conservato per parecchi mesi a 4 ° C. Replica su agar YEPD contenente 200 mg / ml di G418, se necessario, per evitare le colonie di crescere in ognialtro. NOTA: Multiple repliche di serie dovrebbero essere evitati per prevenire la perdita dei ceppi a crescita lenta e ridurre al minimo la comparsa di mutanti soppressori. Ciò è particolarmente importante se il mantenimento delle collezioni come aploidi. </li><li> Eseguire il replica-placcatura usando un microbica arraying sistema robotico. In alternativa, manipolare le colonie usando strumenti pinning manuali schierati. NOTA: La collezione cancellazione lievito è disponibile come aploidi e diploidi da diverse fonti commerciali e in genere viene fornito come scorte glicerolo in piastre da 96 pozzetti. </li></ol></li><li> Condensazione la matrice delezione mutante <ol><li> Preparare 250 ml di agar YEPD contenente 200 mg / ml di G418. La concentrazione effettiva dei G418 può variare e ciascuna partita deve essere testato empiricamente. </li><li> Preparare cinque piastre di agar YEPD versando 50 ml di agar YEPD contenente 200 mg / ml di G418 per piastra. Fare questo un giorno prima condensazione matrice e consentono le piastre raffreddare a temperatura ambienteerature durante la notte su una superficie piatta. NOTA: Quattro piastre saranno necessari per la raccolta a densità 1.536-deformazione, il quinto piatto viene versato come un extra. </li><li> Rimuovere le 16 piastre Petri contenenti raccolta delezione mutante con una densità di 384 colonie per piastra e lasciare riposare a temperatura ambiente (~ 1 ora). </li><li> Asciugare la condensa che si è formata sul coperchio di ogni piatto con un compito delicato pulire. In caso contrario si può causare gocce d&#39;acqua depositato sulla matrice e la contaminazione incrociata dei mutanti schierati. </li><li> Utilizzando una matrice pinning robot microbica, condensare il DMA da una densità di 384 colonie per piastra (16 piatti Petri) a 1.536 colonie per piastra (4 piatti Petri). Eseguire questo in modo che il 1 ° colonia dalla piastra 1 perni alla riga 1 colonna 1 della matrice condensata, il 1 ° colonia di piastra 2 alla riga 1, colonna 2, 1 ° colonia di piastra 3 alla riga 2 e colonna 1, e la colonia 1 ° piatto 4 a Row 2colonna 2. </li><li> Incubare la matrice consolidato a 30 ° C per una notte. </li><li> Esaminare la matrice di garantire il trasferimento uniforme delle colonie da lastre di origine. Ci saranno diverse posizioni vuote, appositamente incorporate nella matrice, che fungono da guida per garantire la DMA è stato condensato correttamente (Figura 4A). NOTA: Questi saranno i piatti di origine per la placcatura replica sul supporto contenente il composto in esame. Una piastra singola fonte può essere utilizzata per più pinnings. Anche molti robot avranno una funzione di compensazione, che garantirà un numero simile di lievito vengono trasferiti ogni volta. </li></ol></li><li> Piastra Replica matrice eliminazione mutante <ol><li> Preparare 700 ml di controllo (solo veicolo) e 700 ml di mezzi sperimentali (-chimici contenenti). Questo è sufficiente per l&#39;esecuzione del test in triplice copia (12 piastre per condizione, più due extra). </li><li> Versare 50 ml di YEPD agar contenenti test chimico o di controllo per piastra. Lasciare le piastre to raffreddare a temperatura ambiente per una notte su una superficie piatta. </li><li> Utilizzando il robot piatto replica, inoculare il DMA su tre serie di piastre contenenti sostanze chimiche a concentrazione determinato sperimentalmente, così come tre set di piastre contenenti il ​​controllo del veicolo. Incubare le piastre a temperatura ambiente per 24-48 ore. </li></ol></li></ol> 3. piastrine e analisi dei dati <ol><li> Rimuovere le piastre da incubatore e permettere loro di venire a temperatura ambiente (~ 1 ora) prima di imaging. Questo consentirà di evitare la formazione di condensa, mentre l&#39;imaging le piastre. </li><li> Fosfori a 24 e 48 ore rimuovendo il coperchio e mettendo la faccia in giù su un letto piano dello scanner. Catturare immagini con una risoluzione di almeno 300 dpi. In alternativa, utilizzare una fotocamera digitale per catturare le immagini. Se così facendo, piatti segnaposto faccia in su uno sfondo scuro e rimuovere i coperchi di immagine. NOTA: Il formato del file e posizionamento delle piastre dipenderanno dal programma di quantificazione utilizzato. </li&gt; <li> Eseguire la quantificazione e il confronto delle dimensioni della colonia di matrice utilizzando uno dei numerosi programmi open source, tra cui Balony 15, SGAtools 16, e ScreenMill 17. </li></ol> 4. Validazione di schermo NOTA: In questa fase diverse eliminazioni di lievito mutanti segnerà come ipersensibili alla chimica di interesse. Queste interazioni chimiche-genetici devono essere convalidati prossimo in due modi. Innanzitutto l&#39;identità di ceppi sensibili dovrebbe essere confermata mediante PCR. In secondo luogo, la sensibilità chimica deve essere segnato in modo indipendente. Per eseguire un breve protocollo per l&#39;esecuzione di test avvistamento per convalidare ceppo ipersensibilità, una tecnica comune in lievito biologia. <ol><li> Streak out desiderato (ipersensibilità) mutanti della collezione eliminazione lievito su agar YEPD contenente 200 mg / ml di G418. Incubare a 30 ° C fino formano colonie. Verificare il genotipo del ceppo mediante PCR diagnostico con primer secondoprotocollo del produttore. </li><li> Seminare 5 ml del liquido YEPD per ogni ceppo e incubare una notte a 30 ° C con rotazione o agitazione. </li><li> Misurare OD 600 e diluire ogni ceppo di OD 600 = 1 in dH sterile 2 O. Queste sono oggi le colture di lieviti normalizzati. </li><li> Dispensare 100 l di normalizzato wild-type di lievito (BY4741) cultura e A1 di una piastra a 96 pozzetti. Dispensare 100 l di fino a sette ceppi supplementari per pozzi B1-H1. </li><li> Utilizzare una pipetta multicanale per erogare 90 ml di dH 2 O sterile per pozzi A2-H2 attraverso A6-H6. Infine, preparare una serie di diluizioni 1:10 di colture di lieviti normalizzati. Inizia in serie pipettando 10 ml di colonna 1, colonna 2, con una pipetta multicanale, e continuare attraverso la piastra a 96 pozzetti prima di aver effettuato sei diluizioni (ie, 10 0-10 -5). </li><li> Trasferire 2-5 ml di colture di lievito diluito in una griglia con un multicanalepipetta su terreni solidi YEPD contenenti prodotti chimici e dei veicoli di controllo. Incubare le piastre a temperatura appropriata per 24-48 ore. </li><li> Ispezionare le piastre e confrontare la sensibilità di selezionare mutanti ad agenti chimici (rispetto al BY4741 controllo). Fosfori a 24 e 48 ore come al punto 3.2. NOTA: Mentre l&#39;identificazione di diversi ceppi ipersensibili con ontologie geniche correlate o funzioni presta fiducia alla validità dello schermo, la trasformazione di un ceppo delezione ipersensibile con un plasmide contenente una copia del gene wild-type è necessaria per stabilire formalmente che un gene cancellazione di interesse (e le mutazioni non nascosti secondo sito) provoca la sensibilità ai farmaci. </li></ol>

<ol>
	<li>Giaever, G., 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=Functional+profiling+of+the+Saccharomyces+cerevisiae+genome.">Functional profiling of the Saccharomyces cerevisiae genome.</a> Nature. 418, 387-391 (2002).</li><li>Winzeler, E. A., 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=Functional+characterization+of+the+S.+cerevisiae+genome+by+gene+deletion+and+parallel+analysis.">Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.</a> Science. 285, 901-906 (1999).</li><li>Breslow, D. K., 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=A+comprehensive+strategy+enabling+high-resolution+functional+analysis+of+the+yeast+genome.">A comprehensive strategy enabling high-resolution functional analysis of the yeast genome.</a> Nat Methods. 5, 711-718 (2008).</li><li>Li, Z., 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=Systematic+exploration+of+essential+yeast+gene+function+with+temperature-sensitive+mutants.">Systematic exploration of essential yeast gene function with temperature-sensitive mutants.</a> Nat Biotechnol. 29, 361-367 (2011).</li><li>Costanzo, M., 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+genetic+landscape+of+a+cell.">The genetic landscape of a cell.</a> Science. 327, 425-431 (2010).</li><li>Parsons, A. B., 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=Integration+of+chemical-genetic+and+genetic+interaction+data+links+bioactive+compounds+to+cellular+target+pathways.">Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways.</a> Nat Biotechnol. 22, 62-69 (2004).</li><li>Parsons, A. B., 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=Exploring+the+mode-of-action+of+bioactive+compounds+by+chemical-genetic+profiling+in+yeast.">Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast.</a> Cell. 126, 611-625 (2006).</li><li>Hillenmeyer, M. 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=The+chemical+genomic+portrait+of+yeast:+uncovering+a+phenotype+for+all+genes.">The chemical genomic portrait of yeast: uncovering a phenotype for all genes.</a> Science. 320, 362-365 (2008).</li><li>Hillenmeyer, M. 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=Systematic+analysis+of+genome-wide+fitness+data+in+yeast+reveals+novel+gene+function+and+drug+action.">Systematic analysis of genome-wide fitness data in yeast reveals novel gene function and drug action.</a> Genome Biol. 11, R30 (2010).</li><li>Smith, A. M., 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=Competitive+genomic+screens+of+barcoded+yeast+libraries.">Competitive genomic screens of barcoded yeast libraries.</a> J Vis Exp. (54), (2011).</li><li>Bowie, D., Parvizi, P., Duncan, D., Nelson, C. J., Fyles, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical-genetic+identification+of+the+biochemical+targets+of+polyalkyl+guanidinium+biocides.">Chemical-genetic identification of the biochemical targets of polyalkyl guanidinium biocides.</a> Organic &amp; Biomolecular Chemistry. 11, 4359-4366 (2013).</li><li>Alamgir, M., Erukova, V., Jessulat, M., Azizi, A., Golshani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical-genetic+profile+analysis+of+five+inhibitory+compounds+in+yeast.">Chemical-genetic profile analysis of five inhibitory compounds in yeast.</a> BMC Chem Biol. 10 (6), (2010).</li><li>Amberg, D. C., Burke, D., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cold Spring Harbor Laboratory. Methods In Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. , (2005).</li><li>Baryshnikova, A., 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=Synthetic+genetic+array+(SGA)+analysis+in+Saccharomyces+cerevisiae+and+Schizosaccharomyces+pombe.">Synthetic genetic array (SGA) analysis in Saccharomyces cerevisiae and Schizosaccharomyces pombe.</a> Methods Enzymol. 470, 145-179 (2010).</li><li>Young, B. P., Loewen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balony:+a+software+package+for+analysis+of+data+generated+by+synthetic+genetic+array+experiments.">Balony: a software package for analysis of data generated by synthetic genetic array experiments.</a> BMC Bioinformatics. 14, 354 (2013).</li><li>Wagih, O., 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=SGAtools:+one-stop+analysis+and+visualization+of+array-based+genetic+interaction+screens.">SGAtools: one-stop analysis and visualization of array-based genetic interaction screens.</a> Nucleic Acids Res. 41, W591-W596 (2013).</li><li>Dittmar, J. C., Reid, R. J., Rothstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScreenMill:+a+freely+available+software+suite+for+growth+measurement,+analysis+and+visualization+of+high-throughput+screen+data.">ScreenMill: a freely available software suite for growth measurement, analysis and visualization of high-throughput screen data.</a> BMC Bioinformatics. 11, 353 (2010).</li><li>Longley, D. B., Harkin, D. P., Johnston, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-fluorouracil:+mechanisms+of+action+and+clinical+strategies.">5-fluorouracil: mechanisms of action and clinical strategies.</a> Nat Rev Cancer. 3, 330-338 (2003).</li><li>Lum, P. Y., 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=Discovering+modes+of+action+for+therapeutic+compounds+using+a+genome-wide+screen+of+yeast+heterozygotes.">Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes.</a> Cell. 116, 121-137 (2004).</li><li>Toussaint, M., Conconi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+and+sensitive+assay+to+measure+yeast+cell+growth:+a+bench+protocol+for+testing+genotoxic+agents.">High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents.</a> Nat Protoc. 1, 1922-1928 (2006).</li><li>Robinson, M. D., Grigull, J., Mohammad, N., Hughes, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FunSpec:+a+web-based+cluster+interpreter+for+yeast.">FunSpec: a web-based cluster interpreter for yeast.</a> BMC Bioinformatics. 3, 35 (2002).</li><li>Huang da, W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+bioinformatics+resources.">Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.</a> Nat Protoc. 4, 44-57 (2009).</li><li>Huang da, W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatics+enrichment+tools:+paths+toward+the+comprehensive+functional+analysis+of+large+gene+lists.">Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.</a> Nucleic Acids Res. 37, 1-13 (2009).</li><li>Hong, E. L., 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=Gene+Ontology+annotations+at+SGD:+new+data+sources+and+annotation+methods.">Gene Ontology annotations at SGD: new data sources and annotation methods.</a> Nucleic Acids Res. 36, D577-D581 (2008).</li><li>Fang, F., Hoskins, J., Butler, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-fluorouracil+enhances+exosome-dependent+accumulation+of+polyadenylated+rRNAs.">5-fluorouracil enhances exosome-dependent accumulation of polyadenylated rRNAs.</a> Mol Cell Biol. 24, 10766-10776 (2004).</li><li>Baba, T., 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=Construction+of+Escherichia+coli+K-12+in-frame,+single-gene+knockout+mutants:+the+Keio+collection.">Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.</a> Mol Syst Biol. 2, 0008 (2006).</li><li>Nichols, R. 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=Phenotypic+landscape+of+a+bacterial+cell.">Phenotypic landscape of a bacterial cell.</a> Cell. 144, 143-156 (2011).</li><li>Dai, 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=Probing+nucleosome+function:+a+highly+versatile+library+of+synthetic+histone+H3+and+H4+mutants.">Probing nucleosome function: a highly versatile library of synthetic histone H3 and H4 mutants.</a> Cell. 134, 1066-1078 (2008).</li></ol>

The authors declare they have no competing financial interests.

Delineato qui è un approccio per la generazione di profili di interazione chimico-genetico. Il metodo è semplice: confrontando le dimensioni delle colonie di ciascun ceppo in complete collezioni delezione genica in presenza e in assenza di una sostanza chimica, tutti i geni necessari per tollerare un insulto chimica sono identificati. Analisi annotazione arricchimento della lista risultante dei ceppi sensibili può quindi essere utilizzato per fornire una visione in una modalità di azione chimica. Mentre questo proto...

Gli strumenti e le risorse disponibili nel organismo modello Saccharomyces cerevisiae genetici hanno permesso grandi studi di genomica funzionale che forniscono insieme nuovi indizi su come i geni funzionano come reti per soddisfare i requisiti dei sistemi biologici. La pietra angolare di questi strumenti è stata la creazione collaborativa di un set completo di delezioni geniche non essenziali di tutte le fasi di lettura aperte nel lievito 1,2. Un&#39;osservazione sorprendente è che solo il ~ 20% dei geni di lievito sono necessari per la vitalità quando cresciuto come aploidi in condizioni standard di laboratorio. Ciò evidenzia la capacità di una cellula per tamponare contro perturbazioni genomiche attraverso l&#39;utilizzo di percorsi biologici alternativi. Mutanti genetici che sono vitali singolarmente, ma letali in combinazione, segnalano percorsi biologici paralleli collegati o convergenti e formano reti di interazione genetica che descrivono la funzione biologica. Con lo sviluppo di te condizionalealleli mperature-sensibili e hypomorphic di geni essenziali della tecnologia non è stato limitato allo studio di geni non essenziali 3,4. Questo concetto è stato applicato su scala genomica produce un imparziale mappa interazione genetica che illustra come i geni coinvolti in processi cellulari simili raggruppano 5. Perturbazioni chimiche di reti genetiche delezioni geniche mimici (Figura 1) 6. Interrogazione composti inibitori della crescita contro una matrice ad alta densità di ceppi di eliminazione per ipersensibilità identifica un profilo di interazione chimico-genetico, cioè una lista di geni che è richiesto di tollerare lo stress chimico. Come interazioni genetiche, schermi di grandi dimensioni di librerie chimiche hanno dimostrato che i composti con una modalità d&#39;azione simile grappolo insieme 7. Pertanto, stabilendo il profilo interazione chimica-genetica di un composto della modalità di azione può dedurre confrontandola con larinterazione genetica e chimica sintetica ge scala genetica Dataset 8,9. Schermi chimico-genetico su larga scala, in cui vengono interrogati decine di composti, sono stati eseguiti da saggi concorrenza barcode. In questo approccio, la raccolta aggregata dei ceppi di delezione è coltivato in massa per diverse generazioni in un piccolo volume di supporti contenenti una sostanza chimica. Dal momento che ogni mutante eliminazione nasconde un codice a barre genetico unico, la redditività / crescita dei singoli mutanti nel pool di ceppi cancellazione è monitorata da microarray o high-throughput sequencing 10. La presunzione di idoneità monitorando dimensioni colonia di mutanti fisicamente Arrayed coltivate su agar solido contenente un composto bioattivo è anche un metodo efficace per identificare interazioni chimiche-genetica 11,12. Questo approccio offre un&#39;alternativa conveniente per lo screening basato sulla concorrenza ed è adatto per saggiare le piccole librerie di sostanze chimiche.Delineato qui è una semplice metodologia per la produzione di un elenco di interazioni chimico-genetico in S. cerevisiae che non si basa su manipolazioni biologia molecolare o infrastrutture. Si richiede solo una collezione di eliminazione di lievito, un apparato pinning robotizzato o manuale, e liberamente disponibili software di analisi dell&#39;immagine.

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		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Name of Material/ Equipment&#160;</td>
			<td style="width:161px;">Company&#160;</td>
			<td style="width:156px;">Catalog Number&#160;</td>
			<td style="width:457px;">Comments/Description&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:213px;">Yeast Extract</td>
			<td style="width:161px;">BioBasic</td>
			<td>G0961</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 1% final concentration (w/v)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:213px;">Tyrptone Powder</td>
			<td style="width:161px;">BD Biosciences</td>
			<td>211820</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 2% final concentration (w/v)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Dextrose</td>
			<td style="width:161px;">Anachemia</td>
			<td style="width:156px;">31096-380</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 2% final concentration (w/v) - Do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Agar A</td>
			<td style="width:161px;">Bio Basic</td>
			<td>FB0010</td>
			<td style="width:457px;">For YEPD solid media add to 2% final concentration (w/v)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:213px;">G418</td>
			<td style="width:161px;">A.G. Scientific Inc.</td>
			<td style="width:156px;">G-1033</td>
			<td style="width:457px;">Prepare 1000x stock at 200mg/ml in dH2O and filter sterilize.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">12 well plate</td>
			<td>Greiner Bio One</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">5ml culture tubes</td>
			<td>Evergreen Scientific</td>
			<td>222-2376-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">10cm Petri Dish</td>
			<td style="width:161px;">VWR</td>
			<td>25384-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROTOR HDA</td>
			<td>Singer Instruments&#160;</td>
			<td>ROT-001</td>
			<td>high-throughput microbial array pinning robot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PLUSPLATE&#169; Petri Dish</td>
			<td>Singer Instruments&#160;</td>
			<td>PLU-001</td>
			<td>Box of 200 dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384 Short-Pin RePad</td>
			<td>Singer Instruments&#160;</td>
			<td>RP-MP-384</td>
			<td>Box of 1000 pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1536 Short-Pin RePad</td>
			<td>Singer Instruments&#160;</td>
			<td>RP-MP-1536</td>
			<td>Box of 1000 pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alternative Pinning Tools:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fully Automated Robtic Systems</td>
			<td>S&#38;P robotics</td>
			<td>http://www.sprobotics.com</td>
			<td>Several automated colony handling robitic and imagining systems available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manual Pinning Tools</td>
			<td>V&#38;P Scientific</td>
			<td>http://www.vp-scientific.com</td>
			<td>Handheld replication tools and accessories.&#160;</td>
		</tr>
	</tbody>
</table>

rapid identification of chemical genetic interactions in saccharomyces cerevisiae

Determinare la modalità di azione delle sostanze chimiche bioattive è di interesse per una vasta gamma di accademici, farmaceutico, e gli scienziati industriali. Saccharomyces cerevisiae, o lievito in erba, è un modello eucariote che una collezione completa di ~ 6.000 mutanti di delezione genica e hypomorphic gene essenziale mutanti sono disponibili in commercio. Queste collezioni di mutanti possono essere utilizzati per rilevare sistematicamente interazioni chimico-gene, cioè geni necessari a tollerare una sostanza chimica. Queste informazioni, a sua volta, riporta sulla modalità probabile di azione del composto. Qui si descrive un protocollo per la rapida identificazione di interazioni chimiche-genetico in erba lievito. Noi dimostriamo il metodo utilizzando l&#39;agente chemioterapico 5-fluorouracile (5-FU), che ha un meccanismo ben definito di azione. I nostri risultati mostrano che il vagabondo nucleare rna exosome e riparazione del DNA enzimi sono necessari per la proliferazione in presenza di 5-FU, che è coerente con le precedenti microarray approcci basati su codice a barre chimico genetiche e le conoscenze che il 5-FU influisce negativamente sia il metabolismo RNA e DNA. Sono descritti anche i protocolli di validazione richieste di questi schermi ad alto rendimento.

Come una convalida di questo approccio abbiamo effettuato un rappresentante chimica schermo interazione genetica dell&#39;agente chemioterapico 5-fluorouracile (5-FU) seguendo il protocollo descritto sopra. 5-FU si caratterizza per interrompere timidilato sintasi così come DNA e RNA metabolismo 18. Le interazioni genetiche chimiche su 5-FU sono ben studiati e sono stati studiati con tecniche di lievito barcode microarray utilizzando collezioni cancellazione eterozigoti e omozigoti 8,19. Qui mostri...

Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

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.

Rapida identificazione dei chimici genetici Interactions in<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. ...

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Research

JoVE Journal

Biology

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

10.3K Views.  University of Victoria. 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.

Video: Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Dissezione di Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Ricerca

Biologia

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

L&#39;identificazione di complessi proteici con la proteomica quantitativa in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Fosfoproteomica quantitativa in acidi grassi Stimolati Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Espressione e purificazione della proteina transmembrana cistica regolatore fibrosi conduttanza in Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Rapida analisi genetica di epitelio-mesenchimale segnalazione durante la rigenerazione dei capelli

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Saggi accoppiati per il monitoraggio delle proteine ​​in ripiegamento<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

Misura di mRNA Decay Prezzi in<em&gt; Saccharomyces cerevisiae</em&gt; Utilizzo<em&gt; Rpb1-1</em&gt; Ceppi

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

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.

Determinazione base alla crescita e conferma biochimica dei requisiti genetici per Protein Degradazione in<em&gt; Saccharomyces cerevisiae</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Diretto Evolution Metodo<em&gt; Saccharomyces cerevisiae</em&gt;: Mutant Biblioteca Creazione e screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...