Nous tenons à remercier le Dr Rich Rogers pour l&#39;assistance technique avec des analyses par spectrométrie de masse et des discussions utiles, et les Drs. Rob-Moritz, Jeff Ranish, et Hamid Mirzai des discussions utiles. Ce travail a été financé par le NIH Centres d&#39;excellence.

La croissance cellulaire et les médias <ol><li> Une seule colonie de cellules BY4742Δarg4Δlys1 nuit dans 100 ml rich media à une DO 600 de 1,0, puis de graines en deux cultures de 1 litre d&#39;un milieu de levure minime (0,17% de base azotée de levure sans sulfate d&#39;ammonium ou des acides aminés, le sulfate d&#39;ammonium 0,5%) contenant un gamme complète d&#39;acides aminés, complétées par des 20mg / L de l&#39;arginine isotopiquement normale ou lourde (13 C 6 15 N 4; Isotec) et la lysine (13 C 6 15 N 2; Isotec). </li><li> Les cellules ont été cultivées pendant 18 heures, à une DO 600 de 1,8. Il est essentiel que les cellules passent par au moins 9 générations pour atteindre la pleine intégration des isotopes marqués. L&#39;échantillon a été la lumière granulés, lavé à l&#39;eau stérile, réensemencées dans un milieu contenant oléate (isotopiquement normale arginine et la lysine, oléate de 0,2% (Sigma Chemicals) et 0,5% de Tween 40 (Sigma Chemicals)) et stimulés pour un autre 85 minutes. Cela donne une isotopiquement léger, à l&#39;égard de l&#39;arginine et la lysine, oléate-stimulé l&#39;échantillon et un échantillon de référence isotopiquement lourds glucose-cultivés. </li></ol> Lyse cellulaire, l&#39;isolement et le fractionnement des peptides <ol start="3"><li> Peser bouteille de centrifugeuse. Récolte des échantillons par centrifugation pendant 3 minutes. Retirez le support et aspirer le liquide en excès. Peser granulés et bouteille (typiquement de rendement entre 1-2 grammes) de geler éclair, puis dans l&#39;azote liquide. Ajouter un volume équivalent au poids de granules de &quot;rectification&quot; tampon pour les boulettes congelées dans l&#39;azote liquide (tampon phosphate salin (PBS, Gibco), 10% de glycérol, inhibiteurs de protéase (comprimés SigmaFAST inhibiteur de protéase, Sigma) et d&#39;inhibiteurs de phosphatase HALT (Thermo Scientific )). </li><li> Gel du récipient de broyage dans l&#39;azote liquide. Attendez que l&#39;azote liquide cesse d&#39;ébullition. Transfert du culot à la cuve de broyage, avec roulements à billes gelées. </li><li> Grind à 600 rpm avec un cycle de 1 min 20 sec avec une inversion de sens pendant 3 minutes. Recongeler le récipient de broyage dans l&#39;azote liquide. Répétez 4 fois plus de 15 minutes au total du temps de broyage. </li><li> Recueillir les grindate congelé dans une glace 50ml Falcon placer le tube sec. Conserver à -80 ° C jusqu&#39;au moment de servir. </li><li> Faire une solution de 10ml de l&#39;urée 8M, bicarbonate d&#39;ammonium 0,1 M, Tris 0,1 M pH 8,6. Ajouter 3 volumes du tampon urée pour 1 volume de grindate congelés (pour 3mls exemple du tampon urée ajoutée à une boulette de 1 gramme par 1 ml de tampon PBS). Immédiatement soniquer le mélange avec un sonicateur pointe de la sonde (2 - 10 impulsions seconde). Garder la pointe près du fond du tube pour empêcher le moussage de la solution. Le grindate doit immédiatement passer en solution. </li><li> Effacer le lysat par centrifugation pendant 5 minutes à 4 ° C. </li><li> Transférer le surnageant dans des tubes frais. </li><li> Faites une partie aliquote de 0,5 M tris [2-carboxyéthyl] phosphine (PTCE). Ajouter à l&#39;échantillon à 1:100 pour une concentration 5 mM finale. Incuber à 37 ° C pendant 1 h. </li><li> Alkylation des cystéines réduites. Laisser refroidir à température ambiante. Faites une partie aliquote de iodoacétamide 1M. Gardez à l&#39;obscurité (nous envelopper le tube dans une feuille). Ajouter à une concentration finale de 20 mM. Incuber dans l&#39;obscurité pendant 1 h à température ambiante. </li><li> Étancher la iodoacétamide 20 mM avec la TNT à partir d&#39;un stock de 1M à température ambiante pendant 1 h. </li><li> Quantifier les protéines avec un standard ou Bradford dosage BCA (non décrit). Prélevez un échantillon pour analyse par SDS-PAGE ci-dessous. </li><li> Pour valider l&#39;incorporation des isotopes lourds, utilisez de 100 ul de lysat réduite et alkylée. Diluer 1:4 avec dH 2 O, soniquer et ajouter la trypsine à 50:1 protéines à la trypsine. Digest pour un minimum de 4 heures, l&#39;échantillon sec dans une speed vac puis dessaler avec une colonne C18 Ultramicrospin (Le groupe Nest). <ol><li> Hydratez colonne avec 100 ul d&#39;acétonitrile </li><li> S&#39;équilibrer avec 200 ul 0,1% de TFA. </li><li> Resuspendre échantillon sec dans 200 ul 0,1% de TFA. Charge sur la colonne. Centrifuger à 200 xg ~ ou la vitesse minimale requise pour circuler à travers la colonne. </li><li> Laver deux fois avec 200 ul 0,1% de TFA. </li><li> Éluer avec deux fois avec 50 ul acétonitrile 60%, 0,1% de TFA. </li><li> L&#39;échantillon sec dans un speed vac. </li><li> Remettre en suspension dans l&#39;échantillon 20 ul d&#39;acide formique 0,1%. Run 2 pl sur un spectromètre de masse. Vérifiez l&#39;incorporation de l&#39;arginine et la lysine isotopiquement lourds par un décalage de 10 et 8 Dalton dans le respectifs m / z spectres. L&#39;incorporation de chaque acide aminé doit être entre 96 - 98%. </li></ol></li><li> Mélanger des quantités équivalentes (mg de protéines) des échantillons isotopiquement lourds et légers. </li><li> Diluer l&#39;échantillon avec du méthanol 01:04 20%. </li><li> Ajouter la trypsine à 1:200. Incuber une nuit à 37 ° C. </li><li> Vérifiez que la digestion est allé à son terme par SDS-PAGE. Run 2 et 4 pl de l&#39;échantillon prédigestion et 8 uneD 16 ul du produit de digestion après la trypsine. Visualisez par coloration au bleu de Coomassie que la digestion est terminée. Si elle n&#39;est pas complète, soniquer l&#39;échantillon avec une pointe de sonde brièvement sonicateur et digérer à nouveau à la trypsine. </li><li> Sécher l&#39;échantillon dans une vitesse ACC. Lorsque ajouter à sec 200 pl de filtrer dH 2 O et le vortex jusqu&#39;à la pastille passe en solution. L&#39;échantillon à sec dans une vitesse ACC. Resuspendre nouveau avec dH 2 O. Sec de l&#39;échantillon à nouveau. </li><li> Resuspendre le culot dans l&#39;acide trifluoroacétique à 0,1% (TFA; Sigma). Vérifier que le pH se situe autour de 3 utilisant des bandes de pH. Si nécessaire acidifier avec TFA 10%. </li><li> Pellet hors tout précipité. </li><li> Dessaler les échantillons sur un Waters Sep-Pak Vac 500 mg colonne C18. Ne surchargez pas ces colonnes. Le montant maximum est de 5 mg, mais il est préférable de charger un peu moins. Divisez l&#39;échantillon entre plusieurs colonnes si nécessaire. <ol><li> Hydrater la colonne avec 5 ml d&#39;acétonitrile 100%. Centrifuger à 200 xg ~ ou la vitesse minimale requise pour circuler à travers la colonne. </li><li> Equilibrer la colonne avec du TFA 0,1% 5ml. Charge de l&#39;échantillon. Recueillir des flux et charger à nouveau. </li><li> Laver avec 5 ml de TFA 0,1%. Répéter. </li><li> Éluer avec 2ml 60% d&#39;acétonitrile, 0,1% de TFA. </li><li> L&#39;échantillon à sec dans une vitesse ACC. </li></ol></li></ol> Fractionnement de peptides et d&#39;isolement de phosphopeptides <ol><li> Les peptides sont fractionnés sur une colonne HILIC. Nous utilisons un TOSOH TSK-Gel Amide-80 4,6 mm x 25 cm colonne analytique avec une colonne de garde. Prenez votre temps avec cette étape, exécutez la colonne pendant 2 heures à 90% A deux puis exécutez gradients vierge avant le chargement de votre échantillon. La charge maximale sur une colonne de cette taille est estimée à environ XX, nous typiquement ne chargez pas plus de 5 mg par courir. </li><li> Solvant A - 98% ACN/0.1% de TFA Solvant B - 2% ACN/0.1% de TFA Charge à 90% A plus de 20 &#39;, 90% A 85% A plus de 5&#39;, 85% A 60% A plus de 40 &#39;, 60% de A à 0% A plus de 5&#39;, 0% A% à 90 A sur 5 &#39;. </li><li> Le débit est 1mL/min et les fractions ont été collectées à intervalles minutes 2 à partir de 85% A% à 60 Un gradient. </li><li> Combinez les fractions de réduire le nombre de fractions à 10. Fractions accrue donnera probablement une plus grande couverture de l&#39;phosphoprotéome. </li></ol> Enrichissement de phosphopeptides <ol><li> Phosphopeptides sont enrichis en utilisant PHOS-Select gel d&#39;affinité de fer. Nous aliquote de notre résine IMAC pour éviter le gel répétées décongélation. Faire 50 ml d&#39;acide acétique 250 mM, solution à 30% la charge d&#39;ACN. Amener le pH à 2,7 avec HCl. Resuspendre granulés dans 500 pl de solution de charge. Vérifiez le pH, ajuster avec HCl ou NaOH nécessaire. </li><li> Prélavage 200 pi de perles IMAC dans une colonne appropriée. Nous utilisons des colonnes de centrifugation Molbiotec. Ajouter 15 ul d&#39;une suspension de 50% (gel de charger la solution) à chaque fraction, et incuber les échantillons pendant 30 &#39;avec la fin de rotation plus fin. Gardez le flux pour l&#39;analyse. </li><li> Lavez échantillons 3 fois avec une solution de chargement et une fois avec 500 pl filtrée dH 2 O. </li><li> Peptides Eluer avec 2 - 3 min avec 400 ul de 50 mM Na 2 HPO 4 (pH 8,4) de mémoire tampon. Transférer dans un flacon en verre. </li><li> Acidifier à pH 3 avec 5 ul d&#39;acide formique à 100%, gel rapide dans l&#39;azote liquide, et l&#39;échantillon séché dans une vitesse ACC. </li><li> Phosphopeptides Remettre en suspension dans 200 pl de TFA à 0,1% et C18 nettoyage comme décrit ci-dessus. Sécher les échantillons élues dans un speed vac. </li><li> Resuspendre peptides dans 20 ul d&#39;acide formique 0,1%. Utilisez 2 ul par l&#39;analyse du spectromètre de masse ou au besoin. </li></ol> Remarques Pour une grande partie de ce protocole vous allez travailler «en aveugle». La lyse cellulaire peut être surveillé par Western Blot et des dosages de Bradford ou BCA, et la digestion par la trypsine peut également être facilement évaluée. La colonne HILIC sera donné une trace semblable à celui-ci, ou comme on le voit par d&#39;autres [1, 2] en fonction de la machine de chromatographie liquide qui est utilisé. Les échantillons sont contrôlés par spectrométrie de masse; tester vos échantillons sur des machines peu coûteuses telles que LCQs ou MALDIs avant d&#39;aller à l&#39;analyse coût élevé sur une haute précision de la masse, une machine à haute résolution. Enfin, si vous assèchement d&#39;un échantillon avec de l&#39;acide qu&#39;il contient, utiliser du verre.

<ol>
	<li>Albuquerque, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multidimensional+chromatography+technology+for+in-depth+phosphoproteome+analysis.">A multidimensional chromatography technology for in-depth phosphoproteome analysis.</a> Mol Cell Proteomics. 7 (7), 1389-1396 (2008).</li><li>McNulty, D. E., Annan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+interaction+chromatography+reduces+the+complexity+of+the+phosphoproteome+and+improves+global+phosphopeptide+isolation+and+detection.">Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.</a> Mol Cell Proteomics. 7 (5), 971-980 (2008).</li><li>Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+complex+protein+mixtures+using+isotope-coded+affinity+tags.">Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.</a> Nat Biotechnol. 17 (10), 994-999 (1999).</li><li>Ross, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Mol Cell Proteomics. 3 (12), 1154-1169 (2004).</li><li>Gruhler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteomics+applied+to+the+yeast+pheromone+signaling+pathway.">Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.</a> Mol Cell Proteomics. 4 (3), 310-327 (2005).</li><li>Wilson-Grady, J. T., Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+analysis+of+fission+yeast.">Phosphoproteome analysis of fission yeast.</a> J Proteome Res. 7 (3), 1088-1097 (2008).</li><li>Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SCX/IMAC+enrichment+approach+for+global+phosphorylation+analysis+by+mass+spectrometry.">The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.</a> Nat Protoc. 3 (10), 1630-1638 (2008).</li><li>Jensen, S. S., Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+impact+of+some+experimental+procedures+on+different+phosphopeptide+enrichment+techniques.">Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.</a> Rapid Commun Mass Spectrom. 21 (22), 3635-3645 (2007).</li><li>Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+selective+enrichment+of+phosphorylated+peptides+from+peptide+mixtures+using+titanium+dioxide+microcolumns.">Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.</a> Mol Cell Proteomics. 4 (7), 873-886 (2005).</li><li>Pinkse, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+isolation+at+the+femtomole+level+of+phosphopeptides+from+proteolytic+digests+using+2D-NanoLC-ESI-MS/MS+and+titanium+oxide+precolumns.">Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.</a> Anal Chem. 76 (14), 3935-3943 (2004).</li><li>Tao, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteome+analysis+using+a+dendrimer+conjugation+chemistry+and+tandem+mass+spectrometry.">Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.</a> Nat Methods. 2 (8), 591-598 (2005).</li><li>Zhou, H., Watts, J. D., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+approach+to+the+analysis+of+protein+phosphorylation.">A systematic approach to the analysis of protein phosphorylation.</a> Nat Biotechnol. 19 (4), 375-378 (2001).</li><li>Bodenmiller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+isolation+of+distinct,+overlapping+segments+of+the+phosphoproteome.">Reproducible isolation of distinct, overlapping segments of the phosphoproteome.</a> Nat Methods. 4 (3), 231-237 (2007).</li></ol>

Cette méthode donnera un enrichissement de phosphopeptides qui peuvent être analysées quantitativement. Un certain nombre de paramètres peuvent être modifiés dans ce protocole, mais l&#39;aspect le plus important à retenir est de préserver votre phosphorylation. Préparation des cellules pour la quantification peut être réalisé dans un certain nombre de manières différentes, par exemple, si vous ne pouvez pas l&#39;étiquette de vos cellules in vivo, des étiquettes telles que l&#39;ICAT [3] ou ITR...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Isotec&trade; L-Arginine-13C6,15N4</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>608033</td>
<td>&nbsp;</td>
<tr>
<td>Isotec&trade; L-Lysine-15N2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>609021</td>
<td>&nbsp;</td>
<tr>
<td>GIBCO&#x2122; Phosphate-Buffered Saline (PBS) 7.4 (10X) liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>70011044</td>
<td>&nbsp;</td>
<tr>
<td>SigmaFAST Protease Inhibitor Tablets</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Pierce Halt Phosphatase Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>78420</td>
<td>&nbsp;</td>
<tr>
<td>Retsch PM100 Ball Mill Grinder </td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>20.540.0001</td>
<td>&nbsp;</td>
<tr>
<td>Retsch 125 ml Stainless Steel Grinding Jar</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>01.462.0148</td>
<td>&nbsp;</td>
<tr>
<td>Ultramicrospin C18 column</td>
<td>&nbsp;</td>
<td>The Nest Group</td>
<td>SUM SS10</td>
<td>&nbsp;</td>
<tr>
<td>Sep-Pak Vac 500 mg C18</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT043395</td>
<td>&nbsp;</td>
<tr>
<td>TSK-Gel Amide-80 4.6 mm x 25 cm analytical column</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>13071</td>
<td>This is the HILIC chromatographic column.</td>
</tr>
<tr>
<td>Guard column 4.6 mm ID x1 cm</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>19021</td>
<td>We recommend this guard column to extend the life of your HILIC column.</td>
</tr>
<tr>
<td>PHOS-Select&trade; Iron Affinity Gel</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P9740</td>
<td>This is the IMAC resin. Aliquot and store.</td>
</tr>		</tbody>
		</table>
		</div>

quantitative phosphoproteomics in fatty acid stimulated saccharomyces cerevisiae

Ce protocole décrit la croissance et la stimulation, avec l&#39;oléate d&#39;acides gras, des isotopes lourds et légers cellules de S. cerevisiae. Les cellules sont broyées à l&#39;aide d&#39;une procédure cryolysis dans un moulin broyeur à boulets et les grindate résultant mis en solution par solubilisation d&#39;urée. Cette procédure permet la lyse des cellules dans un état métaboliquement inactives, la préservation de la phosphorylation et la prévention de la réorientation de l&#39;phosphoprotéome lors de la lyse cellulaire. Après la réduction, l&#39;alkylation, digestion par la trypsine des protéines, les échantillons sont dessalés sur des colonnes C18 et la complexité de l&#39;échantillon réduit de fractionnement par chromatographie d&#39;interaction hydrophile (HILIC). Colonnes HILIC retiennent préférentiellement des molécules hydrophiles qui est bien adapté pour phosphoprotéomique. Peptides phosphorylés ont tendance à éluer plus tard dans le profil chromatographique que leurs homologues non phosphorylées. Après fractionnement, phosphopeptides sont enrichis en utilisant la chromatographie en métal immobilisé, qui repose sur la charge basé sur des affinités pour l&#39;enrichissement phosphopeptide. A la fin de cette procédure, les échantillons sont prêtes à être analysées quantitativement par spectrométrie de masse.

Watch this Scientific Journal Video about Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae at JoVE.com

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description d&#39;une procédure de phosphorylation quantitative à l&#39;aide cryolysis, solubilziation urée, de fractionnement et l&#39;enrichissement HILIC IMAC de peptides phosphorylés.

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 ...

quantitative-phosphoproteomics-fatty-acid-stimulated-saccharomyces

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Biology

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

10.0K vues.  Institute for Systems Biology. Description d'une procédure de phosphorylation quantitative à l'aide cryolysis, solubilziation urée, de fractionnement et l'enrichissement HILIC IMAC de peptides phosphorylés.

Vidéo: Quantitative Phosphoproteomics in Fatty Acid Stimulated 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

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 ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Un test microscopie de fluorescence pour Mitophagy surveillance dans la levure<em&gt; Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

La quantification et l&#39;analyse de la formation de HO-endonucléase Stimulé par les translocations chromosomiques simple brin recuit dans Saccharomyces cerevisiae</em

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

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 ...

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 ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

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. ...

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

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

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

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

Réalisé Evolution Méthode<em&gt; Saccharomyces cerevisiae</em&gt;: Mutant Library Création et dépistage

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

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).