Ce travail a été soutenu par une subvention Joseph P. Healey de l&#39;Université du Massachusetts à Boston (LSH) et R15 GM86805 du NIH (LSH). SMP est supporté en partie par une Sanofi-Genzyme Fellowship à l&#39;Université du Massachusetts à Boston.

1. Préparation de sporulation Remarque:. Les milieux décrits dans ce protocole sont réalisés en utilisant des recettes et des méthodes standard 13,17 Le tableau 1 donne la formulation pour 1 L des différents médias utilisés dans ce protocole. <table border="1" cellpadding="0" cellspacing="0" height="180" width="469"><colgroup><col /><col /><col span="3" /><col /><col span="2" /></colgroup><tbody><tr height="15"><td height="15" style="height:20px;"></td><td style="width: 55px;"> bactopeptone </td><td style="width: 50px;"> Extrait de levure </td><td style="width: 50px;"> gélose Bacto </td><td style="width: 60px;"> Dextrose </td><td style="width: 70px;"> L&#39;acétate de potassium </td><td style="width: 60px;"> Glycérol </td><td style="width: 60px;"> ddH 2 O </td></tr><tr height="15"><td height="15" style="height:20px;"> plaques GPJ </td><td> 20 g </td><td> 10 g </td><td> 20 g </td><td> - </td><td> - </td><td> 30 ml </td><td> 970 ml </td></tr><tr height="15"><td height="15" style="height:20px;"> des boîtes de YPD </td><td> 20 g </td><td> 10 g </td><td> 20 g </td><td> 20 g </td><td> - </td><td> - </td><td> 1000 ml </td></tr><tr height="15"><td height="15" style="height:20px;"> liquide YPD </td><td> 20 g </td><td> 10 g </td><td> - </td><td> 20 g </td><td> - </td><td> - </td><td> 1000 ml </td></tr><tr height="15"><td height="15" style="height:20px;"> liquide YPA </td><td> 20 g </td><td> 10 g </td><td> - </td><td> - </td><td> 20 g </td><td> - </td><td> 1000 ml </td></tr><tr height="15"><td style="height: 20px; width: 80px;"> médias sporulation </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 10 g </td><td> - </td><td> 1000 ml </td></tr></tbody></table> Tableau 1:. Formulations des médias Les montants sont donnés pour 1 L de médias. Spécificités sur les médiasles ingrédients peuvent être trouvés dans la table des matières. <ol><li> Si toutes les cellules sporulantes sera le même Génotype, suivez cette procédure: <ol><li> la souche de levure Thaw à utiliser sur un (YPGlycerol) plaque GPJ. Cultivez levure pour 12-16 heures à 30 ° C. Fait important, les cellules SK1 sont également prêts à sporuler, donc les souches SK1 ne doivent pas être laissés trop longtemps sur des plaques de GPJ, ou ils peuvent sporuler sur la plaque. Remarque: Une souche fraîchement décongelé cultivée sur YPG est préféré, que l&#39;utilisation du glycérol comme source de carbone nécessite la phosphorylation oxydative, et sélectionne ainsi les cellules qui ont l&#39;ADN mitochondrial fonctionnel. les cellules SK1 ont tendance à perdre de l&#39;ADN mitochondrial fonctionnel et devenir petites. L&#39;ADN mitochondrial fonctionnelle est nécessaire pour la sporulation. </li><li> Streak levure à partir de la plaque de GPJ sur un YPD (YPDextrose) plaque pour des colonies isolées. Cultiver la levure sur la plaque YPD 24-28 h à 30 ° C. </li><li> Après environ 36-48 h de la croissance sur YPD milieux solides, démarrer une culture YPD 25 ml en utilisant une seule coloniede la plaque YPD. Cultivez cette culture jusqu&#39;à ce que les cellules sont sur ​​DO 600 = 4,0 à 7,0. Effectuez cette étape à 30 ° C pendant la nuit dans un incubateur à agitation, en agitant à 220 tours par minute. </li><li> De la culture YPD, utiliser une quantité appropriée de cellules pour démarrer 1000 ml de YPA (YPAcetate) culture qui est DO 600 = 0,1 pour chaque besoin plaque à 96 puits dans la section 2. Cultiver la levure dans un incubateur agitateur à 30 ° C (agitation à 220 rpm) jusqu&#39;à ce que la DO600 de la culture est compris entre 1,3 et 1,5. Note: inoculer 100 ml de culture LPJ pour chaque plaque multipuits 96 à cribler dans la section 2. Par exemple, si le dépistage quatre 96 plaques multipuits, 400 ml de culture LPJ seront nécessaires. A partir de 10% du volume supplémentaire dans la culture LPJ est nécessaire pour protéger contre la perte de volume par évaporation. En règle générale, il faudra compter entre 12-16 h pour les cellules pour atteindre la DO appropriée 600; la durée peut varier en fonction du génotype de la souche de levure. LPJ est le média de présporulation. préculturedans de l&#39; acétate aide induire l&#39; expression des gènes (comme IME1) nécessaire pour efficace et synchrone sporulation 6. </li><li> Spin à 1.800 xg dans une centrifugeuse de table pendant 5 min pour sédimenter les cellules cultivées dans LPJ. Laver les cellules 1x avec 0,5 volume d&#39;eau désionisée autoclavée stérile. Spin à nouveau, et jeter laver. Resuspendre les cellules dans les médias 1x volume de sporulation. Continuer à la section 2. </li></ol></li><li> Si l&#39; utilisation de cellules pour sporulation qui sont des génotypes différents, (c. -à- transformées avec des plasmides différents ou contenant différentes mutations, généralement obtenues et stockées sous forme de stocks congelés), des cellules de tableau des différents génotypes dans un format 96 multipuits. Ensuite, suivez cette procédure: <ol><li> Utilisez un 96 broches frogger stérile pour transférer des cellules à partir de stocks congelés décongelés dans 96 plaque (s) multipuits sur YPG milieux solides. Cultiver la levure pendant la nuit à 30 ° C. </li><li> Utilisation d&#39;un 96 broches frogger stérile, transférer les cellules de YPG milieux solides sur YPD milieux solides ou sélectiveun milieu solide ( par exemple, un milieu minimal synthétique dépourvu des acides aminés, si les cellules contiennent des plasmides qui nécessitent une sélection). Cultiver la levure à 30 ° C jusqu&#39;à ce que les colonies sont formées pour chaque souche, typiquement 1 jour sur YPD et 2 jours si des milieux sélectifs est nécessaire. </li><li> Aliquoter 1,2 ml de milieu liquide YPD ou des milieux liquides sélectifs dans chaque puits d&#39;une plaque multipuits 96 profondeur de 2 ml. Transfert des cellules à partir de supports solides dans des milieux liquides en utilisant un frogger, couvrir avec un couvercle en plastique maintenu en place par un ruban, et de croître pendant une nuit dans 30 ° C shaker, agitation à 220 rpm. Remarque: Pour minimiser la contamination, des plaques de couverture en utilisant les couvercles en plastique à partir d&#39;une boîte de Pétri rectangulaire. Tenir les paupières en place au moyen d&#39;un morceau de ruban adhésif, de sorte que la circulation de l&#39;air est très peu limité. </li><li> plaques centrifuger à 1800 xg pendant 5 min, en utilisant un seau centrifuge oscillant avec des adaptateurs pour accueillir plaques à 96 puits. Décanter médias de cellules; ajouter 500 ul LPJ à chaque puits et resuspendre cellules sédimentées dans la LPJ. Couvertureavec couvercle en plastique maintenu en place par un ruban. </li><li> Cultiver les cellules pendant 15 heures à 30 ° C dans un incubateur à agitation, en agitant à 220 tours par minute. Continuer à la section 2. </li></ol></li></ol> 2. Les cellules sporulantes dans un 96 Multiwell Format <ol><li> Ajouter un ou l&#39;autre de 3 mm perle de verre solide stérile ou un stériles 5 mm x 2 mm barre d&#39;agitation magnétique dans chaque puits d&#39;une plaque de 96 puits avec 1,3 ml de puits. (Voir résultats représentatifs ci - dessous pour une discussion sur l&#39; opportunité d&#39;utiliser un cordon ou d&#39; une barre d&#39;agitation.) </li><li> Si toutes les cellules à cribler sont du même génotype, aller à 2.2.1. Si les cellules à cribler sont de différents génotypes, aller à 2.2.2. <ol><li> Prenez des cellules de l&#39;étape 1.1.5 et aliquote de 500 pi de cellules plus les médias dans chaque puits de la plaque multipuits 96. Couvrir avec un couvercle en plastique maintenu en place par un ruban. Passez à 2.3. </li><li> les cellules de spin qui sont LPJ de 1.2.4 à 1800 g pendant 5 min, en utilisant une centrifugeuse à godet oscillant avec des adaptateurs pour recevoir des plaques à 96 puits. verser def support de cellules. Ajouter 500 ul de milieu de sporulation dans chaque puits et les remettre en suspension les cellules sédimentées dans le milieu de sporulation. Couvrir avec un couvercle en plastique maintenu en place par un ruban. Passez à 2.3. </li></ol></li><li> Si vous utilisez une perle de verre, placer les échantillons dans un incubateur sous agitation à 30 ° C et agiter à 220 rpm pour la sporulation. Si vous utilisez une barre d&#39;agitation magnétique, placer les échantillons sur une plaque d&#39;agitation à l&#39;intérieur d&#39;un 30 ° C incubateur pour sporulation. Remarque: La vitesse d&#39;agitation exacte pour les barres d&#39;agitation magnétique est pas très important, aussi longtemps que toutes les barres se déplacent énergiquement et la culture est aéré. </li><li> Pour surveiller la progression à travers la sporulation, prélever des échantillons de la culture sporulée pour la visualisation, Ajouter 6 ul de cellules à partir des cultures sporulées à 24 pi de milieu de sporulation dans une plaque à 96 puits lamelle couvre-objet (1: 5 de dilution). Laisser les cellules se contenter de 5 minutes avant d&#39;examiner les cellules en utilisant un microscope inversé. Remarque: Dans les cellules SK1 de type sauvage, les spores matures compacts formeront par 36 heures après le transfert aux médias de sporulation. La progression à travers la sporulation peut être contrôlée par microscopie en utilisant des marqueurs fluorescents, comme un marqueur nucléaire (c. -à -HTB2 mCherry 18) pour examiner la méiose ou un marqueur de la membrane ProSpore (c. -à- G20 19) pour examiner la morphogenèse des spores. En variante, la formation de spores réfringents peut être vu à l&#39;aide de DIC Nomarski microscopie à partir d&#39;environ 12 heures après le transfert au milieu de sporulation. </li></ol>

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The authors have no competing financial interests.

Nous présentons ici un protocole de sporulation SK1 levure dans un format de 96 multipuits. Aération est la clé pour la sporulation efficace, ce qui nécessite l&#39;utilisation soit d&#39;un barreau d&#39;agitation ou une perle de verre dans chaque puits. Lorsque les cellules sont sporulés dans une plaque multipuits 96 dans un incubateur à agitation sans soit une perle ou une barre d&#39;agitation, les cellules ne sporulation pas efficacement. Seule une petite augmentation de l&#39;efficacité de la sporulation es...

La sporulation chez la levure en herbe a été étudié pour donner un aperçu de nombreux aspects de la biologie, y compris le contrôle de la ségrégation des chromosomes lors de la méiose 1, les mécanismes de recombinaison génétique 2, le contrôle du développement par la cellule de signalisation 3, contrôle nutritionnel du développement 4, la transcription régulation du développement 5, et l&#39;examen de la formation de spores 6. La formation de spores comprend un événement de division cellulaire nouvelle impliquant la formation de nouveaux compartiments à l&#39; intérieur de la membrane de la cellule mère , suivie par le dépôt d&#39;une paroi protectrice 6 spores. Ces études qui examinent les cellules sporulantes profitent souvent de la souche de levure sporulation rapidement SK1, qui peut subir le processus de sporulation dans environ 24 heures de façon relativement efficace 7,8. Bien que l&#39; optimisation des conditions de sporulation de la levure bourgeonnante ont été décrits 13/09, ces expériencess examiné sporulation sur des supports solides ou dans les grandes cultures liquides à grande échelle où sporulation est effectuée en utilisant des tubes de culture ou flacons. Nous décrivons ici un procédé pour la sporulation de la levure dans un format de plaque multipuits 96. Nous constatons que, pour ce procédé, l&#39;aération est essentielle pour la sporulation synchrone et efficace, et ont mis au point des techniques pour assurer une sporulation suffisante d&#39;un format multi-puits de faible volume. Sporuler dans un format de plaque multipuits 96 permet de cellules devant être criblés en utilisant des techniques à haut débit et des réactifs optimisés pour un format de plaque multipuits, un tel criblage pour des suppresseurs de copies élevé en utilisant une bibliothèque de tuiles 14-16.

<table><tbody><tr><td>Nunc 1.3 ml DeepWell Plates</td><td>ThermoScientific</td><td>260251</td><td>Used for sporulation</td></tr><tr><td>Nunc 2.0 ml DeepWell plates</td><td>ThermoScientific</td><td>278743</td><td>Used for presporulation growth, step 1.2.3</td></tr><tr><td>3 mm glass bead</td><td>Fisher</td><td>11-312A</td><td>Used for sporulation</td></tr><tr><td>5 mm x 2 mm stir bar, pack of 12</td><td>Fisher</td><td>14-511-82</td><td>Used for sporulation</td></tr><tr><td>96 well frogger</td><td>V&amp;amp;P Scientific</td><td>VP407</td><td>needed for step 1.2</td></tr><tr><td>library copier</td><td>V&amp;amp;P Scientific</td><td>VP381</td><td>needed for step 1.2; to be used with the frogger</td></tr><tr><td>rectangular Petri dish</td><td>ThermoScientific</td><td>264728</td><td>needed for step 1.2</td></tr><tr><td>Bacto Peptone</td><td>BD</td><td>211677</td><td>needed for media</td></tr><tr><td>Yeast Extract</td><td>BD</td><td>212750</td><td>needed for media</td></tr><tr><td>Bacto Agar</td><td>BD</td><td>212750</td><td>needed for media</td></tr><tr><td>Dextrose</td><td>Fisher</td><td>D16-3</td><td>needed for media</td></tr><tr><td>Potassium Acetate</td><td>Fisher</td><td>P171-500</td><td>needed for media</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>G33-500</td><td>needed for media</td></tr><tr><td>Black 96 well glass bottom plate</td><td>MatTek</td><td>PBK96G-1.5.5-F</td><td>needed for step 2.4</td></tr></tbody></table>

efficient sporulation of saccharomyces cerevisiae in a 96 multiwell format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

<html> Pour évaluer ce protocole, l&#39; efficacité de sporulation obtenus à partir de cellules sporulantes dans des plaques multipuits (comme décrit ci - dessus) ont été comparées à des cellules sporulées en utilisant des volumes plus importants dans des flacons (tableau 2). L&#39;utilisation de plaques multipuits n&#39;a pas atteint le rendement élevé vu quand sporulation dans des flacons, où ~ 80% d&#39;efficacité peut être régulièrement vu. Sporulation...

Watch this Scientific Journal Video about Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format at JoVE.com

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

Sporulation efficace de<em&gt; Saccharomyces cerevisiae</em&gt; Dans un 96 Multiwell Format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

efficient-sporulation-saccharomyces-cerevisiae-96-multiwell

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Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

10.1K vues.  University of Massachusetts Boston. Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

Vidéo: Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

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 ...

Recherche

Biologie

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Une méthode à haut débit pour étudier la morphologie globalement organite chez S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

Les tests ACT1-CUP1 déterminent les sensibilités spécifiques au substrat des mutants splicéosomaux chez la levure bourgeonnante

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

Génétique

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Métabolomique quantitative de Saccharomyces Cerevisiae par chromatographie liquide couplée à la spectrométrie de masse en tandem

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Biochimie

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Préparation et micro-usinage Cryo-FIB de Saccharomyces cerevisiae pour la cryo-tomographie électronique

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

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 ...

Competitive Genomic Screens of Barcoded Yeast Libraries

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

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

Écrans concurrentiel des bibliothèques génomiques de levure Barcoded

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

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Cytométrie en flux à base de purification de<em&gt; S. cerevisiae</em&gt; Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

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 ...

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Here we present a protocol to estimate the respiratory and fermentative metabolism by fitting the exponential growth of Saccharomyces cerevisiae to the exponential growth equation. Calculation of the kinetic parameters allows for the screening of influences of substances/compounds on fermentation or mitochondrial respiration.

Saccharomyces cerevisiae Cinétique de croissance exponentielle en Culture pour analyser le métabolisme respiratoire et fermentatif

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The ...

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