Recherche dans le laboratoire CJN est soutenu par des subventions de fonctionnement du CRSNG, l&#39;Institut de recherche Société canadienne du cancer (IRSCC), et la Fondation du cancer (Direction BC-Yukon) Canadian Breast.

NOTE: Le flux de travail général de cette procédure est décrite dans la figure 2. 1. Détermination de la croissance inhibitrice Dose <ol><li> Préparation de levure culture d&#39;une nuit NOTE: Les médias de croissance extrait de levure-peptone-dextrose (YEPD) utilisé dans ce protocole est une recette standard 13. <ol><li> Streak sur BY4741 (Δ1 leu2Δ0 met15Δ0 ura3Δ0 de Mata) cellules sur une plaque de gélose YEPD et incuber pendant 48 heures à 30 ° C ou jusqu&#39;à ce que des colonies visibles forment. </li><li> Préparer cultures d&#39;une nuit en inoculant 5 ml de YEPD milieux liquides dans un récipient de culture stérile avec une seule colonie. Incuber une nuit à 30 ° C avec rotation continue ou en secouant. La culture sera typiquement atteindre la saturation par le matin (~ 2 × 10 8 cellules / ml). </li></ol></li><li> Solide préparation des milieux agar contenant diverses doses chimiques <ol><li> Préparer plusieurs stocks dele produit chimique à tester à 100x la concentration finale souhaitée dans un solvant approprié. REMARQUE: Les concentrations efficaces dépendront de la chimie, et doivent être déterminées empiriquement. Il est donc conseillé de tester d&#39;abord une gamme de concentration finale de large; ce est à dire, d&#39;une faible uM à haute mM. </li><li> Pour chaque concentration de produit chimique à tester, aliquote de 3 ml de gélose YEPD fondu dans un tube de plusieurs culture stérile. Place à 55 ° C bain d&#39;eau pour garder de se solidifier. </li><li> Pour chaque concentration de produit chimique à tester ajouter 30 ul du composé à 100x milieu en fusion, vortex pour mélanger 2-3 secondes, et la pipette 1 ml dans chaque paire de puits en double dans une plaque à 12 puits. Veillez à inclure un véhicule seul contrôle. Permettre aux plaques de fixer pendant une nuit à température ambiante. Jeter ne importe quel média supplémentaire. </li></ol></li><li> cellules Spread placage <ol><li> Inoculer 10 ml de milieu liquide YEPD avec 2 ul d&#39;une culture de levure saturée, cultivées pendant une nuit comme dans l&#39;étape 1.1.2. </li><li> Plate 25 ul de la culture de levure diluée par puits, répartis uniformément en utilisant des billes de verre stériles ou tige, et permettent la plaque sécher sous une flamme. Incuber la plaque à 30 ° C pendant 48 heures. </li><li> Évaluer la croissance des levures (à la fois le nombre total et la taille des colonies) sur les plaques. Sélectionner une concentration sublétale d&#39;un composé qui ne inhibe pas la croissance de plus de 10% à 15% pour effectuer la totalité. </li></ol></li></ol> 2. Systematic crible génétique chimique <ol><li> Maintien de suppression-mutant-array (DMA) et la préparation de plaque source <ol><li> Pour une description détaillée sur la construction de réseaux de mutants suppression des stocks de glycérol se il vous plaît se référer à Baryshnikova et al. 14. Une fois que les mutants de deletion ont été revêtu à une densité de 384 colonies par plaque, la DMA peut être stocké pendant plusieurs mois à 4 ° C. Répliquer sur gélose YEPD contenant 200 ug / ml de G418 que nécessaire pour empêcher les colonies de se développer dans chaqueautre. Note: plusieurs répétitions de série devraient être évités pour prévenir la perte de souches à croissance lente et de minimiser l&#39;apparition de mutants suppresseurs. Ceci est particulièrement pertinent si le maintien des collections que haploïdes. </li><li> Effectuez toutes les étapes réplique placage utilisant un microbienne rangeant système robotique. Alternativement, manipuler colonies en utilisant des outils manuels d &#39;ancrage vêtu. NOTE: La collection de levure de suppression est disponible en haploïdes et diploïdes provenant de plusieurs sources commerciales et est généralement expédié en stocks de glycérol dans des plaques à 96 puits. </li></ol></li><li> Condenser le tableau suppression de mutant <ol><li> Préparer 250 ml de milieu de gélose YEPD contenant 200 ug / ml de G418. La concentration efficace de G418 peut varier et chaque lot doit être testé empiriquement. </li><li> Préparer cinq plaques de gélose YEPD en versant 50 ml d&#39;agar YEPD contenant 200 ug / ml de G418 par plaque. Pour ce faire, un jour avant la condensation du tableau et laisser les plaques refroidir à la température ambianterature nuit sur une surface plane. NOTE: Quatre plaques seront requis pour la collecte à une densité 1536-souche, la cinquième plaque est versé comme un supplément. </li><li> Retirer les 16 boîtes de Pétri contenant le mutant de deletion de collecte à une densité de 384 colonies par plaque et laisser revenir à la température ambiante (environ 1 heure). </li><li> Essuyez toute condensation qui se est formée sur le couvercle de chaque plaque en utilisant une tâche délicate essuyer. Ne pas le faire peut entraîner des gouttelettes d&#39;eau se dépose sur le tableau et la contamination croisée de mutants disposés. </li><li> L&#39;utilisation d&#39;un tableau épinglant robots microbienne, condenser la DMA d&#39;une densité de 384 colonies par plaque (16 boîtes de Pétri) à 1536 colonies par plaque (4 boîtes de Pétri). Effectuez cette sorte que le 1 er colonie de la plaque 1 pins à la ligne 1 colonne 1 du tableau condensé, le 1 er colonie de la plaque 2 à la ligne 1 colonne 2, le 1 er colonie de la plaque 3 à la ligne 2 et la colonne 1, et la colonie 1 er de la plaque 4 à la ligne 2colonne 2. </li><li> Incuber le tableau consolidé à 30 ° C pendant la nuit. </li><li> Examinez le tableau pour assurer un transfert uniforme de colonies à partir de plaques de source. Il y aura plusieurs positions vierges, à dessein incorporés dans la matrice, qui servent de guide pour assurer le DMA a été condensé correctement (figure 4A). NOTE: Il se agira des plaques de base pour des répliques sur des milieux contenant le composé d&#39;essai. Une plaque de source unique peut être utilisé pour plusieurs pinnings. Aussi de nombreux robots auront une fonction de compensation, ce qui assurera un nombre similaire de la levure sont transférés à chaque fois. </li></ol></li><li> plaque de gamme Replica suppression de mutant <ol><li> Préparez 700 ml de contrôle (véhicule seulement) et 700 ml de milieu expérimental (contenant de produits chimiques). Ce est suffisant pour la réalisation du test en trois exemplaires (12 plaques par condition, plus deux extras). </li><li> Verser 50 ml de gélose YEPD contenant lutte chimique ou par plaque test. Autoriser les plaques to refroidir à la température ambiante pendant une nuit sur une surface plane. </li><li> En utilisant le robot à la plaque de réplique, inoculer le DMA sur trois jeux de plaques contenant des produits chimiques à la concentration déterminée expérimentalement, ainsi que trois jeux de plaques contenant le contrôle du véhicule. Incuber les plaques à température ambiante pendant 24-48 h. </li></ol></li></ol> 3. Les plaques d&#39;imagerie et d&#39;analyse de données <ol><li> Retirer les plaques de l&#39;incubateur et leur permettre de revenir à température ambiante (~ 1 h) avant l&#39;imagerie. Cela permettra d&#39;éviter la formation de condensation en imagerie les plaques. </li><li> plaques d&#39;image au 24 et 48 h en enlevant le couvercle et placer face vers le bas sur un scanner à plat. Capturez des images à une résolution d&#39;au moins 300 dpi. Sinon, utilisez un appareil photo numérique pour capturer des images. Si faisant, place des plaques face vers le haut sur un fond sombre et retirez les couvercles à l&#39;image. NOTE: Le format de fichier et le positionnement des plaques dépendront sur le programme de quantification utilisée. </li&gt; <li> Effectuer la quantification et la comparaison de la taille des colonies de tableau en utilisant l&#39;un des nombreux programmes open source, y compris Balony 15, SGAtools 16 et 17 ScreenMill. </li></ol> 4. Validation de l&#39;écran NOTE: A ce stade, plusieurs mutants de suppressions de levure seront marquer une hypersensibilité à la substance chimique d&#39;intérêt. Ces interactions chimiques-génétiques doivent être validés prochaine de deux façons. D&#39;abord l&#39;identité des souches sensibles devrait être confirmée par PCR. En second lieu, la sensibilité chimique doit être reçu de façon indépendante. Décrit ci-dessous est un bref protocole pour effectuer des dosages de repérage pour valider hypersensibilité de la souche, une technique courante dans la levure biologie. <ol><li> Tués sur le souhaite (hypersensible) de la collection de mutants de deletion de la levure sur de la gélose YEPD contenant 200 ug / ml de G418. Incuber à 30 ° C jusqu&#39;à ce que des colonies se forment. Vérifier le génotype de la souche de diagnostic par PCR avec des amorces selon lale protocole du fabricant. </li><li> Inoculer 5 ml du liquide YEPD pour chaque souche et incuber une nuit à 30 ° C avec agitation ou rotation. </li><li> Mesurer 600 OD et diluer chaque souche à DO 600 = 1 en DH stérile 2 O. Ce sont maintenant les cultures de levure normalisées. </li><li> Distribuer 100 ul de normalisée levure de type sauvage (BY4741) la culture de bien A1 d&#39;une plaque de 96 puits. Distribuer 100 ul de jusqu&#39;à sept souches supplémentaires aux puits B1-H1. </li><li> Utilisez une pipette multicanaux pour distribuer 90 ul de dH 2 O stérile aux puits A2-H2 à A6-H6. Enfin, préparer une série de dilutions de 1:10 cultures de levures normalisées. Commencez par pipetage en série 10 ul de la colonne 1 à la colonne 2 avec une pipette multicanaux, et continuer à travers la plaque 96 puits jusqu&#39;à six dilutions sont faites (ce est à dire, 10 0 à 10 -5). </li><li> 5.2 Transfert ul de cultures de levure dilués dans une grille à l&#39;aide d&#39;un multicanalpipette sur YEPD milieux solides contenant chimique et la maîtrise du véhicule. Incuber les plaques à la température appropriée pour 24 à 48 h. </li><li> Inspectez plaques et de comparer la sensibilité de certains mutants aux produits chimiques (par rapport à BY4741 commande). plaques d&#39;image à 24 et 48 heures comme dans l&#39;étape 3.2. REMARQUE: Bien que l&#39;identification de plusieurs souches hypersensibles ontologies de gène apparenté ou fonctions prête confiance à la validité de l&#39;écran, la transformation d&#39;une souche de suppression hypersensible avec un plasmide contenant une copie de type sauvage du gène est nécessaire pour établir formellement qu&#39;un gène suppression d&#39;intérêt (et seconde mutations site pas cachés) provoque la sensibilité aux médicaments. </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.

Décrit ici est une approche pour générer des profils d&#39;interaction chimiques génétique. La méthode est simple: en comparant les tailles des colonies de chacune des souches dans des collections complètes de deletion de gène en présence et en absence d&#39;un produit chimique, tous les gènes nécessaires pour tolérer une insulte chimique sont identifiés. analyse de l&#39;enrichissement d&#39;annotation de la liste résultant de souches sensibles peut alors être utilisé pour fournir un aperçu d&#39;un mo...

Les outils et les ressources disponibles dans l&#39;organisme modèle Saccharomyces cerevisiae génétiques ont permis à grande échelle de génomique fonctionnelle études qui fournissent collectivement un nouvel éclairage sur la façon dont les gènes fonctionnent comme des réseaux pour répondre aux exigences des systèmes biologiques. La pierre angulaire de ces outils a été la création en collaboration d&#39;un ensemble complet de délétions de gènes non-essentiels de tous les cadres de lecture ouverts dans la levure 1,2. Une observation frappante était que seulement environ 20% des gènes de levure sont nécessaires pour la viabilité lorsqu&#39;il est cultivé comme haploïdes dans des conditions de laboratoire standard. Cela met en évidence la capacité d&#39;une cellule à tampon contre les perturbations génomiques grâce à l&#39;utilisation des voies biologiques alternatives. Mutants génétiques qui sont viables individuellement, mais en combinaison mortelle, signalent les voies biologiques parallèles ou convergentes connectés et forment des réseaux d&#39;interactions génétiques qui décrivent la fonction biologique. Avec le développement de te conditionnelleallèles mpérature-sensibles et hypomorphic de gènes essentiels de la technologie n&#39;a pas été limité à l&#39;étude des gènes non essentiels 3,4. Ce concept a été appliqué à l&#39;échelle génomique produire une carte d&#39;interaction génétique impartiale illustrant comment les gènes impliqués dans les processus cellulaires similaires se regroupent 5. Perturbations chimiques des réseaux génétiques suppressions de gènes imiter (Figure 1) 6. Interrogation composés inhibiteurs de croissance contre une matrice haute densité de souches de suppression de l&#39;hypersensibilité identifie un profil d&#39;interaction chimique génétique, ce est à dire une liste de gènes qui est nécessaire pour tolérer le stress chimique. Comme les interactions génétiques, grands écrans de bibliothèques chimiques ont montré que les composés avec un mode similaire de se regroupent sept d&#39;action. Par conséquent, en établissant le profil d&#39;interaction chimique génétique d&#39;un composé du mode d&#39;action peut être déduite par comparaison avec large échelle interaction génétique génétique et chimique de synthèse datasets 8,9. Écrans chimiques génétique à grande échelle, où des dizaines de composés sont interrogés, ont été réalisées par des analyses de la concurrence de codes à barres. Dans cette approche, la collecte groupée des souches de suppression est cultivé en masse depuis plusieurs générations dans un petit volume de milieu contenant un produit chimique. Depuis chaque mutant de suppression recèle un code-barres génétique unique, la viabilité / croissance des mutants individuels au sein du pool de souches de suppression est suivi par microarray ou séquençage à haut débit 10. Inférer remise en forme en surveillant la taille des colonies de mutants physiquement disposés en réseau cultivés sur gélose solide contenant un composé bioactif est aussi une méthode efficace pour identifier les interactions chimiques-génétiques 11,12. Cette approche offre une alternative rentable au dépistage fondé sur la concurrence et est bien adapté pour le dosage de petites bibliothèques de produits chimiques.Décrite ici est une méthodologie simple pour produire une liste des interactions chimiques-génétiques dans S. cerevisiae qui ne repose pas sur les manipulations moléculaire ou d&#39;infrastructure biologie. Elle exige seulement une collection de levure de suppression, un appareil de brochage robotisé ou manuel, et librement disponibles des logiciels d&#39;analyse d&#39;images.

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			<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

Déterminer le mode d&#39;action des produits chimiques bioactifs est d&#39;intérêt pour un large éventail d&#39;universitaires, pharmaceutiques, et les scientifiques industriels. Saccharomyces cerevisiae, ou levure bourgeonnante, est un eucaryote modèle pour lequel une collection complète de ~ 6000 mutants de délétion du gène et gène essentiel hypomorphic mutants sont disponibles dans le commerce. Ces collections de mutants peuvent être utilisés pour détecter systématiquement des interactions chimiques de gènes, à savoir les gènes nécessaires pour tolérer un produit chimique. Cette information, à son tour, des rapports sur le mode d&#39;action probable du composé. Ici, nous décrivons un protocole pour l&#39;identification rapide des interactions chimiques-génétiques dans la levure bourgeonnante. Nous démontrons le procédé utilisant l&#39;agent chimiothérapeutique 5-fluorouracile (5-FU), qui possède un mécanisme bien défini de l&#39;action. Nos résultats montrent que l&#39;ARN nucléaire TRAMP exosomes réparation de l&#39;ADN et les enzymes sont nécessaires pour la prolifération en présence de 5-FU, qui est compatible avec m précédenteicroarray approches génétiques basés codes barres chimiques et la connaissance que le 5-FU néfaste à la fois l&#39;ARN et l&#39;ADN métabolisme. Les protocoles de validation requis de ces écrans à haut débit sont également décrits.

En tant que validation de cette approche nous avons effectué un écran d&#39;interaction chimique génétique représentant de l&#39;agent chimiothérapeutique 5-fluorouracile (5-FU) en suivant le protocole décrit ci-dessus. 5-FU est connu de perturber la thymidylate synthase, ainsi que le métabolisme de l&#39;ADN et de l&#39;ARN 18. Les interactions génétiques chimiques de 5-FU sont bien étudiés et ont été étudiés par des techniques de levure code à barres de puces à ADN en utilisant les deux co...

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.

Identification rapide des interactions chimiques génétiques dans<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

10.2K vues.  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.

Vidéo: 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.

Dissection de Saccharomyces cerevisiae Asques

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

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Biologie

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.

Identification des complexes protéiques avec la protéomique quantitative chez 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.

Phosphoprotéomique quantitatives en acide gras Stimulé 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 ...

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.

Analyse de Microarray pour 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 ...

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.

Analyse rapide de la génétique des épithélio-mésenchymateuse signalisation lors de la régénération des cheveux

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.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<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.

Les analyses couplées pour la surveillance protéines repliement<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 ...

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

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

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

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Purification de la fibrose kystique régulateur de la conductance transmembranaire protéine exprimée dans<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 &#181;M. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

EPR surveillé Redox titrage des cofacteurs de<em&gt; Saccharomyces cerevisiae</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

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.

Mesure de la dégradation de l&#39;ARNm Tarifs en<em&gt; Saccharomyces cerevisiae</em&gt; Utilisation<em&gt; Rpb1-1</em&gt; Les souches

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