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>

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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.

<table><tbody><tr><td>Yeast Extract</td><td>BioBasic</td><td>G0961</td><td>For YEPD liquid/solid media add to 1% final concentration (w/v)</td></tr><tr><td>Tyrptone Powder</td><td>BD Biosciences</td><td>211820</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v)</td></tr><tr><td>Dextrose</td><td>Anachemia</td><td>31096-380</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v) &amp;#8212; do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.</td></tr><tr><td>Agar A</td><td>Bio Basic</td><td>FB0010</td><td>For YEPD solid media add to 2% final concentration (w/v)</td></tr><tr><td>G418</td><td>A.G. Scientific Inc.</td><td>G-1033</td><td>Prepare 1,000x stock at 200&amp;nbsp;mg/ml in dH&lt;sub&gt;2&lt;/sub&gt;O and filter sterilize.&amp;nbsp;</td></tr><tr><td>12-well plate</td><td>Greiner Bio One</td><td>655180</td><td></td></tr><tr><td>5 ml culture tubes</td><td>Evergreen Scientific</td><td>222-2376-080</td><td></td></tr><tr><td>10 cm Petri Dish</td><td>VWR</td><td>25384-302</td><td></td></tr><tr><td>ROTOR HDA</td><td>Singer Instruments&amp;nbsp;</td><td>ROT-001</td><td>high-throughput microbial array pinning robot</td></tr><tr><td>PLUSPLATE&amp;copy; Petri Dish</td><td>Singer Instruments&amp;nbsp;</td><td>PLU-001</td><td>Box of 200 dishes</td></tr><tr><td>384 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-384</td><td>Box of 1,000 pads</td></tr><tr><td>1536 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-1536</td><td>Box of 1,000 pads</td></tr><tr><td colspan="4">Alternative Pinning Tools:</td></tr><tr><td>Fully Automated Robtic Systems</td><td>S&amp;amp;P robotics</td><td>http://www.sprobotics.com</td><td>Several automated colony handling robitic and imagining systems available.</td></tr><tr><td>Manual Pinning Tools</td><td>V&amp;amp;P Scientific</td><td>http://www.vp-scientific.com</td><td>Handheld replication tools and accessories.&amp;nbsp;</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. ...

rapid-identification-chemical-genetic-interactions-saccharomyces

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

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Mappatura di interazioni proteine-DNA con ChEC-seq nel gene-wide<em&gt; Saccharomyces cerevisiae</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Ricerca

Genetica

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

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

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

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

Una schermata di soppressore Deep-sequenziamento-assistita, spontanea nel lievito a fissione Schizosaccharomyces pombe

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

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

Mappatura genetica delle differenze di termotolleranza tra le specie di Saccamici Yeast tramite l'analisi dell'emizigosità reciproca genomica

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

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 ...

Biologia

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

Individuazione di intermedi di ricombinazione omologhi tramite legatura di prossimità e PCR quantitativa in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Utilizzo di Grafix per la rilevazione di interattori transitori di sottocomplessi di saccharomyces cerevisiae spliceosomi

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Biochimica

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Microarray analisi per Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Schermi competitivo genomica delle Biblioteche di lievito con codice a barre

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

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 ...

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Screening genetico per l'identificazione di soppressori multicopia in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

Rapida identificazione dei chimici genetici Interactions in

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Capitoli in questo video

Mappatura di interazioni proteine-DNA con ChEC-seq nel gene-wide

Una schermata di soppressore Deep-sequenziamento-assistita, spontanea nel lievito a fissione Schizosaccharomyces pombe

Mappatura genetica delle differenze di termotolleranza tra le specie di Saccamici Yeast tramite l'analisi dell'emizigosità reciproca genomica

L'identificazione di complessi proteici con la proteomica quantitativa in S. cerevisiae

Individuazione di intermedi di ricombinazione omologhi tramite legatura di prossimità e PCR quantitativa in Saccharomyces cerevisiae

Utilizzo di Grafix per la rilevazione di interattori transitori di sottocomplessi di saccharomyces cerevisiae spliceosomi

Microarray analisi per Saccharomyces cerevisiae

Schermi competitivo genomica delle Biblioteche di lievito con codice a barre

Determinazione base alla crescita e conferma biochimica dei requisiti genetici per Protein Degradazione in

Screening genetico per l'identificazione di soppressori multicopia in Schizosaccharomyces pombe