Questo lavoro è stato sostenuto da una sovvenzione Joseph P. Healey presso la University of Massachusetts Boston (LSH) e R15 GM86805 dal NIH (LSH). SMP è sostenuto in parte da una Sanofi-Genzyme Fellowship presso l&#39;Università del Massachusetts di Boston.

1. Preparazione per Sporulazione Nota:. I media descritti in questo protocollo sono realizzati con ricette e metodi standard di 13,17 La tabella 1 riporta la formulazione per 1 L di vari supporti utilizzati in questo protocollo. <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;"> Bacto Peptone </td><td style="width: 50px;"> Estratto di lievito </td><td style="width: 50px;"> Bacto Agar </td><td style="width: 60px;"> Destrosio </td><td style="width: 70px;"> Acetato di potassio </td><td style="width: 60px;"> glicerina </td><td style="width: 60px;"> DDH 2 O </td></tr><tr height="15"><td height="15" style="height:20px;"> piastre YPG </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;"> piastre YPD </td><td> 20 g </td><td> 10 g </td><td> 20 g </td><td> 20 g </td><td> - </td><td> - </td><td> 1.000 ml </td></tr><tr height="15"><td height="15" style="height:20px;"> YPD liquido </td><td> 20 g </td><td> 10 g </td><td> - </td><td> 20 g </td><td> - </td><td> - </td><td> 1.000 ml </td></tr><tr height="15"><td height="15" style="height:20px;"> YPA liquido </td><td> 20 g </td><td> 10 g </td><td> - </td><td> - </td><td> 20 g </td><td> - </td><td> 1.000 ml </td></tr><tr height="15"><td style="height: 20px; width: 80px;"> mezzi sporulazione </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 10 g </td><td> - </td><td> 1.000 ml </td></tr></tbody></table> Tabella 1:. Formulazioni media importi vengono forniti 1 l di media. Specifiche su supportiingredienti possono essere trovati nella Tabella Materials. <ol><li> Se tutte le celle sporulanti sarà lo stesso genotipo, seguire questa procedura: <ol><li> Scongelare ceppo di lievito da utilizzare su un (YPGlycerol) Piastra YPG. Grow lieviti per 12-16 ore a 30 ° C. È importante sottolineare che le cellule SK1 sono anche pronti a sporulare, in modo da ceppi SK1 non dovrebbero essere lasciati troppo a lungo su piatti YPG, o possono sporulare sul piatto. Nota: Un ceppo appena scongelati coltivate su YPG è preferito, come l&#39;utilizzo di glicerolo come fonte di carbonio richiede fosforilazione ossidativa, e sceglie quindi per cellule che hanno DNA mitocondriale funzionale. cellule SK1 hanno una tendenza a perdere DNA mitocondriale funzionale e diventare petites. DNA mitocondriale funzionale è necessario per sporulazione. </li><li> Streak lievito dalla piastra YPG su un (YPDextrose) piastra YPD per singole colonie. Crescere lievito sulla piastra YPD 24-28 ore a 30 ° C. </li><li> Dopo circa 36-48 ore di crescita su YPD supporto solido, avviare una cultura di 25 ml YPD utilizzando una singola coloniadalla piastra YPD. Crescere questa cultura fino a quando le cellule sono circa 600 OD = 4,0-7,0. Eseguire questo passaggio a 30 ° C per una notte in un incubatore agitazione, agitazione a 220 giri al minuto. </li><li> Dalla cultura YPD, utilizzare una quantità appropriata di cellule per iniziare a 1.000 ml di YPA (YPAcetate) cultura che è OD 600 = 0,1 per ogni esigenza 96 pozzetti nella sezione 2. crescere il lievito in una agitazione incubatore a 30 ° C (agitazione a 220 rpm) fino a quando la coltura OD 600 è compreso tra 1,3 a 1,5. Nota: Seminare 100 ml di cultura YPA per ogni piatto pozzetti 96 a proiettato nella sezione 2. Per esempio, se lo screening quattro 96 piastre a pozzetti multipli, saranno necessari 400 ml di cultura YPA. A partire con il 10% in più di volume nella cultura YPA è necessario per proteggere contro la perdita di volume per evaporazione. Tipicamente, ci vorranno tra 12-16 ore per le cellule per raggiungere il diametro esterno adeguato 600; la quantità di tempo può variare a seconda del genotipo del ceppo di lievito. YPA è il supporto presporulation. Pregrowthin acetato aiuta inducono l&#39;espressione di geni (ad esempio IME1) necessario per sporulazione efficiente e sincrona 6. </li><li> Spin a 1.800 xg in una centrifuga da tavolo per 5 minuti per far sedimentare le cellule coltivate in YPA. Lavare le cellule 1x con 0,5 volume di acqua deionizzata sterile in autoclave. Spin di nuovo, e scartare lavare. Risospendere le cellule in 1x supporto del volume sporulazione. Continuare alla Sezione 2. </li></ol></li><li> Se si utilizza cellule per sporulazione che sono di diversi genotipi, (vale a dire, trasformate con diversi plasmidi o contenenti diverse mutazioni, in genere ottenuti e memorizzati come scorte congelate), le cellule matrice dei diversi genotipi in un formato pozzetti 96. Quindi, seguire questa procedura: <ol><li> Utilizzare uno sterile Frogger 96 poli per trasferire le cellule provenienti dalle scorte congelate scongelati a 96 pozzetti piastra (s) sul YPG terreni solidi. Crescere durante la notte di lievito a 30 ° C. </li><li> Utilizzando uno sterile frogger 96 poli, trasferire le cellule dal YPG terreni solidi su supporti solidi YPD o selettivomezzi solidi (cioè, terreno minimo sintetico privo aminoacidi, se le celle contengono plasmidi che richiedono la selezione). Grow lievito a 30 ° C fino colonie si formano per ogni ceppo, in genere almeno 1 giorno in YPD e 2 giorni se terreni selettivi è necessario. </li><li> Aliquotare 1,2 ml di terreno liquido YPD o supporti liquidi selettivi in ​​ciascun pozzetto di una piastra multipozzetto 96 profonde 2 ml. Trasferire le cellule da supporti solidi in liquidi utilizzando una Frogger, coprire con un coperchio di plastica tenuta in posizione da nastro adesivo, e crescere durante la notte in 30 ° C shaker, agitando a 220 giri al minuto. Nota: Per ridurre al minimo la contaminazione, piastre di copertura utilizzando i coperchi di plastica da una piastra di Petri rettangolare. Tenere coperchi in posto con un pezzo di nastro, in modo che la circolazione dell&#39;aria è minimamente limitata. </li><li> piastre centrifugare a 1.800 xg per 5 minuti, utilizzando un secchio centrifuga oscillante con gli adattatori per accogliere 96 pozzetti. Eliminare i media dalle cellule; aggiungere 500 microlitri YPA a ciascun pozzetto e sospendere nuovamente le cellule pellet nel YPA. Copertinacon coperchio in plastica trattenuta da nastro. </li><li> Crescere le cellule per 15 ore a 30 ° C in un incubatore agitazione, agitazione a 220 rpm. Continuare alla Sezione 2. </li></ol></li></ol> 2. Le cellule sporulanti in un multiwell formato 96 <ol><li> Aggiungere uno dei due 3 millimetri perle di vetro solido sterile o un sterili 5 mm x 2 mm ancoretta magnetica in ciascun pozzetto di una piastra ben 96 con 1,3 ml di pozzi. (Vedere Rappresentante Risultati al di sotto per una discussione sull&#39;opportunità di usare una perlina o un ancoretta.) </li><li> Se tutte le celle da schermati sono dello stesso genotipo, vai a 2.2.1. Se le cellule per essere schermati sono di diversi genotipi, andare a 2.2.2. <ol><li> Prendere le cellule dal punto 1.1.5 e un&#39;aliquota 500 ml di cellule più supporti in ciascun pozzetto della piastra di pozzetti 96. Coprire con il coperchio di plastica tenuta in posizione da nastro. Procedere alla 2.3. </li><li> cellule Spin che sono YPA da 1.2.4 a 1800 xg per 5 minuti, utilizzando un secchio centrifuga oscillante con adattatori per ospitare piastre da 96 pozzetti. versare dif supporti dalle cellule. Aggiungere 500 ml di mezzi sporulazione in ciascun pozzetto e sospendere nuovamente le cellule pellet nei media sporulazione. Coprire con il coperchio di plastica tenuta in posizione da nastro. Procedere alla 2.3. </li></ol></li><li> Se si utilizza un perle di vetro, luogo campioni in un incubatore agitazione a 30 ° C e agitare a 220 rpm per sporulazione. Se si utilizza un ancoretta magnetica, I campioni su un piatto mescolare all&#39;interno di un incubatore a 30 ° per la sporulazione. Nota: La velocità esatta mescolate per i bar mescolare magnetiche non è molto importante, a patto che tutte le barre si muovono con energia e la cultura è aerato. </li><li> Per monitorare la progressione attraverso la sporulazione, prelevare campioni dalla cultura sporulanti per la visualizzazione, Aggiungere 6 ml di cellule dalle culture sporulanti a 24 ml di media sporulazione in una piastra da 96 pozzetti coprioggetto (diluizione 1: 5). Lasciare le cellule accontentarsi di 5 minuti prima di esaminare le cellule utilizzando un microscopio invertito. Nota: In cellule SK1 wild-type, compatti spore mature formeranno da 36 ore dopo il trasferimento su un supporto sporulazione. La progressione attraverso sporulazione può essere monitorato tramite microscopia utilizzando marcatori fluorescenti, come ad esempio un marcatore nucleare (cioè, Htb2-mCherry 18) per esaminare la divisione meiotica o un marcatore di membrana prospore (vale a dire, G20 19) per esaminare spore morfogenesi. In alternativa, la formazione di spore rifrangenti può essere visto utilizzando Nomarski microscopia DIC partire da circa 12 ore dopo il trasferimento sul supporto sporulazione. </li></ol>

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The authors have no competing financial interests.

Qui vi presentiamo un protocollo per sporulanti SK1 lievito in un formato di pozzetti 96. L&#39;aerazione è la chiave per la sporulazione efficiente, che richiede l&#39;utilizzo della propria ancoretta o una perla di vetro in ogni pozzetto. Quando le cellule sono sporulata in una piastra multipozzetto 96 in un incubatore agitazione senza né una perlina o ancoretta, le cellule non sporulino in modo efficiente. Solo un piccolo aumento dell&#39;efficienza sporulazione è visto quando le cellule sono sporigeni senza né u...

Sporulazione nel lievito erba è stata studiata per fornire approfondimenti molti aspetti della biologia, compreso il controllo della segregazione cromosomica durante la meiosi 1, meccanismi di ricombinazione genetica 2, il controllo dello sviluppo da cellule segnalazione 3, controllo nutrizionale di sviluppo 4, il trascrizionale regolazione dello sviluppo 5, e l&#39;esame di formazione di spore 6. Formazione di spore include un nuovo evento divisione cellulare coinvolge la formazione di nuovi compartimenti di membrana all&#39;interno della cellula madre seguita dalla deposizione di un muro di spore di protezione 6. Questi studi che esaminano le cellule sporulanti spesso approfittano della rapida sporulanti ceppo di lievito SK1, che può subire il processo di sporulazione in circa 24 ore in modo relativamente efficiente 7,8. Anche se l&#39;ottimizzazione delle condizioni di sporulazione per gemmazione di lievito sono state descritte 9-13, questi esperimentis esaminato sporulazione su mezzi solidi o in colture liquide scala più ampia in cui è effettuata la sporulazione utilizzando tubi di coltura o flaconi. Qui si descrive un metodo per sporulanti lievito in un formato piatto pozzetti 96. Troviamo che per questo metodo, aerazione è fondamentale per sporulazione sincrona ed efficiente, e hanno ideato tecniche per assicurare sporulazione sufficiente un formato multipozzetto piccolo volume. Sporulanti in un formato 96 pozzetti piatto permette alle cellule di essere sottoposti a screening utilizzando tecniche high-throughput e reagenti ottimizzati per un formato piatto multipozzetto, tale screening per soppressori di alta copia utilizzando una libreria di piastrelle 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> Per valutare questo protocollo, efficienze sporulazione ottenuti da sporulanti cellule in piastre a pozzetti multipli (come descritto sopra) sono stati confrontati con cellule sporigeni utilizzando volumi maggiori in beute (Tabella 2). L&#39;uso di piastre a pozzetti multipli non ha raggiunto l&#39;alta efficienza visto quando sporulanti in fiaschi, in cui l&#39;efficienza ~ 80% può essere visto di routine. Sporulanti in piastre a pozzetti multipli con una corrett...

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.

Sporulazione efficiente di<em&gt; Saccharomyces cerevisiae</em&gt; In un pozzetti formato 96

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

Video: 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.

Dissezione di Saccharomyces cerevisiae Asci

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

Ricerca

Biologia

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.

Un high-throughput metodo per studiare la morfologia globale organello in 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.

I saggi ACT1-CUP1 determinano le sensibilità specifiche del substrato dei mutanti spliceosomi nel lievito in erba

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

Genetica

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.

Metabolomica quantitativa di Saccharomyces Cerevisiae mediante cromatografia liquida accoppiata con spettrometria di massa tandem

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

Biochimica

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.

Preparazione e microlavorazione Cryo-FIB di Saccharomyces cerevisiae per Tomografia Crioeletnico

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.

Microarray analisi per Saccharomyces cerevisiae</em

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

Competitive Genomic Screens of Barcoded Yeast Libraries

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

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

Schermi competitivo genomica delle Biblioteche di lievito con codice a barre

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

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.

Flusso Cytometry basata Purificazione<em&gt; S. cerevisiae</em&gt; Zigoti

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.

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

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

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 Cinetica di crescita esponenziale nella cultura di Batch per analizzare il metabolismo fermentativo e respiratorio

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

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