Bu çalışma NIH Massachusetts Boston (LSH) ve R15 GM86805 Üniversitesi (LSH) bir Joseph P. Healey hibe ile desteklenmiştir. SMP Massachusetts Boston Üniversitesi&#39;nde Sanofi-Genzyme Kardeşliği tarafından kısmen desteklenmektedir.

1. Sporulationun için hazırlanıyor Not:. Bu protokol açıklanan medya standart reçeteleri ve yöntemleri kullanılarak yapılan olan 13,17 Tablo 1 Bu protokolde kullanılan çeşitli medya 1 L formülasyonu verir. <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 Pepton </td><td style="width: 50px;"> Maya Özü </td><td style="width: 50px;"> Bacto Agar </td><td style="width: 60px;"> üzüm şekeri </td><td style="width: 70px;"> Potasyum Asetat </td><td style="width: 60px;"> gliserin </td><td style="width: 60px;"> GKD 2 O </td></tr><tr height="15"><td height="15" style="height:20px;"> YPG plakalar </td><td> 20 g </td><td> 10 g </td><td> 20 g </td><td> - </td><td> - </td><td> 30 mi </td><td> 970 mi </td></tr><tr height="15"><td height="15" style="height:20px;"> YPD plakaları </td><td> 20 g </td><td> 10 g </td><td> 20 g </td><td> 20 g </td><td> - </td><td> - </td><td> 1000 mi </td></tr><tr height="15"><td height="15" style="height:20px;"> YPD sıvı </td><td> 20 g </td><td> 10 g </td><td> - </td><td> 20 g </td><td> - </td><td> - </td><td> 1000 mi </td></tr><tr height="15"><td height="15" style="height:20px;"> YPA sıvı </td><td> 20 g </td><td> 10 g </td><td> - </td><td> - </td><td> 20 g </td><td> - </td><td> 1000 mi </td></tr><tr height="15"><td style="height: 20px; width: 80px;"> sporülasyon ortam </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 10 g </td><td> - </td><td> 1000 mi </td></tr></tbody></table> Tablo 1:. Ortam formülasyonlar edilmiştir ortam 1 ​​L için verilmiştir. medya konusunda Özelliklerimaddeler Malzeme Tablo bulunabilir. <ol><li> Tüm sporlanan Hücreler aynı genotip olacaksa, bu yordamı uygulayın: <ol><li> Çözülme maya suşu, bir YPG (YPGlycerol) plaka kullanılır. 30 ° C&#39;de 12-16 saat boyunca maya büyümek. Önemlisi, SK1 hücreleri de o kadar SK1 suşları YPG plakalar üzerinde çok uzun bırakılmamalıdır, sporlanmalanna gurur duyuyoruz, ya da plaka üzerinde sporlanmalanna olabilir. Not: YPG üzerinde büyümüş bir taze çözülmüş soyu, bir karbon kaynağı olarak gliserol kullanımı gibi oksidatif fosforilasyon gerektirir tercih edilir ve bu durumda işlevsel mitokondriyal DNA sahip hücreler için seçim yapmaktadır edilir. SK1 hücreler fonksiyonel mitokondriyal DNA kaybetmek ve petites olma eğilimi var. Fonksiyonel mitokondriyal DNA, sporlanma için gereklidir. </li><li> tek koloniler için YPD bir (YPDextrose) plaka üzerine YPG plakasından Streak maya. 30 ° C&#39;de YPD plaka 24-28 saat üzerinde maya büyür. </li><li> YPD katı ortam üzerinde büyüme yaklaşık 36-48 saat sonra, tek bir koloni ile 25 ml&#39;lik bir YPD kültürünü başlatmakYPD plaka dan. Hücreler 7.0 = 4.0 OD 600 hakkında kadar bu kültürü büyütün. 220 rpm&#39;de çalkalanarak, bir çalkalama inkübatöründe gece boyunca 30 ° C &#39;de bu adımı gerçekleştirir. </li><li> YPD kültüründen (220 çalkalanarak 30 ° C&#39;de ve çalkalama inkübatöründe maya büyümek 2. Bölüm, her 96 yuvalı plaka ihtiyacı için OD 600 = 0.1 olduğunu YPA (YPAcetate) kültür 1000 ml başlangıç ​​hücre, uygun bir miktarda kullanımı OD 600 1,5-1,3 arasında kültür kadar rpm). Not: Dört 96 çukurlu levhalar tarama, her 96 çukurlu levha YPA kültürü 100 ml inoküle Örneğin, Bölüm 2&#39;de elenecek, YPA kültürü 400 ml gereklidir. YPA kültür içinde% 10 ekstra hacim başlayarak buharlaştırıcı birim kaybına karşı korumak için gereklidir. Hücreler uygun OD 600 ulaşması için Tipik olarak, 12-16 saat arasında sürer; süre maya suşu genotipine bağlı olarak değişebilir. YPA presporulation ortamıdır. Pregrowthasetat içinde gen ekspresyonunu (örneğin IME1 gibi) etkili ve eş zamanlı sporlanma 6 için gerekli neden olur. </li><li> 5 dakika boyunca masa üstü bir santrifüj 1.800 x g&#39;de Spin YPA yetiştirilen hücreler pelet. Yıkama Hücreler, steril otoklavlanmış deiyonize su, 0.5 hacim 1x. Tekrar Spin ve yıkama atın. 1x hacim sporlanma medyada yeniden süspanse hücreleri. Bölüm 2 devam ediyor. </li></ol></li><li> Farklı genotipleri, vardır sporlanma için hücreleri kullanarak (farklı plazmidler ile transforme, yani veya farklı tipik elde dondurulmuş stoklar olarak saklanır mutasyonları içeren) 96 çukurlu bir biçime farklı genotip, dizi hücrelerini edin. Sonra, bu yordamı izleyin: <ol><li> YPG katı ortam üzerinde 96 oyuklu bir plaka (lar) içinde çözülmüş, dondurulmuş stoklar hücreleri aktarmak için bir steril 96 çatal Frogger kullanın. 30 ° C de bir gece boyunca, maya büyümek. </li><li> steril 96 diş Frogger kullanarak, YPD katı ortam veya seçici üzerine YPG katı ortam hücreleri aktarmakKatı ortam (hücre seçimini gerektirir plazmidler içeriyorsa, yani sentetik minimal medya, amino asitler eksik). Seçici ortam gerekirse koloniler 2 gün YPD tipik haliyle 1 gün, her bir suş için oluşturulur ve kadar 30 ° C &#39;de maya büyütün. </li><li> Kısım 96, derin 2 mi çukurlu plakanın her oyuğuna YPD sıvı ortam veya seçici sıvı ortam 1.2 mi. Bir Frogger kullanılarak sıvı ortam içine bir katı ortam transfer hücreleri, bant ile yerinde tutulan plastik bir kapak ile kapatın ve 220 rpm&#39;de çalkalanarak, 30 ° C&#39;de çalkalayıcı içinde gece boyunca büyür. Not: kontaminasyonu en aza indirmek için, dikdörtgen bir petri tabak plastik kapaklar kullanarak kapak plakaları. Bu hava sirkülasyonu minimal sınırlıdır, bu yüzden bir parça bant kullanarak yerine kapaklarını tutun. </li><li> adaptörleri ile sallanan bir kova santrifüj kullanılarak 5 dakika boyunca 1.800 xg&#39;de santrifüj plakalar, 96 gözlü plakalar karşılamak için. hücrelerden medya dökün; YPA pelet hücreleri her 500 ul Ypa de ekleme ve yeniden süspanse edin. kapakbant ile yerinde tutulur plastik kapak ile. </li><li> 220 rpm&#39;de çalkalanarak, bir çalkalama inkübatöründe 30 ° C&#39;de 15 saat boyunca hücreler büyür. Bölüm 2 devam ediyor. </li></ol></li></ol> 96 çukurlu Format 2. sporlanan Hücreler <ol><li> Bir 3 mm steril katı cam boncuk ve 1.3 mi çukurlu bir 96 oyuklu plakanın her oyuğuna steril 5 mm x 2 mm boyutlarında manyetik bir karıştırma çubuğu, ya ekleyin. (Bir boncuk veya bir karıştırma çubuğu kullanmak konusunda bir tartışma için aşağıdaki Temsilcisi Sonuçları gör.) </li><li> elenecek tüm hücreler aynı genotip ise, 2.2.1 gidin. elenecek hücreleri farklı genotip ise, 2.2.2 gidin. <ol><li> 96 çukurlu bir levhanın her çukuruna içine adım 1.1.5 ve kısım hücre ve ortam 500 ul hücreleri al. bant ile yerinde tutulur plastik kapakla örtün. 2.3 geçin. </li><li> 96 kuyucuğu karşılamak için adaptörleri ile sallanan bir kova santrifüj kullanılarak 1.2.4 den 5 dakika boyunca 1.800 xg&#39;de YPA olan Spin hücreleri. dökünhücrelerinden f medya. sporlanma medya pelet hücreleri her sporülasyon medya 500 ul iyi ekleyin ve tekrar süspansiyon. bant ile yerinde tutulur plastik kapakla örtün. 2.3 geçin. </li></ol></li><li> Cam boncuk kullanılarak, bir yerde, 30 ° C&#39;de çalkalayıcı bir inkübatör içinde, örnekler ve sporlaşma 220 rpm&#39;de çalkalanır. sporlanma için 30 ° C inkübatör içinde bir karıştırıcı plaka üzerinde bir manyetik karıştırma çubuğu, Örnekleri kullanarak. Not: tüm barlar enerjik hareket ediyor ve kültür havalandırılır olarak manyetik karıştırma çubukları için tam karıştırma hızı sürece çok önemli değildir. </li><li> , Sporulationun ilerlemeyi izlemek görselleştirme sporlanan kültüründen örnekleri çekilme, 96 iyi lamel plakada sporülasyon medya 24 ul sporlanan kültürlerinden hücrelerin 6 ul ekleyin için (1: 5 seyreltme). ters bir mikroskop kullanılarak hücrelerin incelenmesi önce 5 dakika boyunca yerleşmek için hücreleri bırakın. Not: vahşi tip SK1 hücrelerinde, kompakt olgun sporlar 3 ile oluşturacaksporlanma medyaya transfer edildikten sonra 6 saat. Sporlanma ilerleme, örneğin nükleer işaretleyici olarak flüoresan işaretçileri kullanılarak mikroskop ile izlenebilir (yani, Htb2 mCherry 18), öz değişmesine ait bölünme veya prospore membran işaretleyici incelemek için (örneğin, G-20 19) spor morfojenezi incelemek. Seçenek olarak ise, refraktil sporların oluşumunu sporülasyon ortama transferinden sonra yaklaşık 12 saat başlayan Nomarski DIC mikroskopi kullanılarak görülebilir. </li></ol>

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The authors have no competing financial interests.

Burada 96 multiwell formatta SK1 maya sporlanan için bir protokol mevcut. Havalandırma Bir karıştırma çubuğu ya da her iyi bir cam boncuk ya kullanımını gerektirir verimli sporlanma için anahtardır. hücreler, bir tane veya bir karıştırma çubuğu ya da olmayan bir çalkalama inkübatöründe 96 çukurlu bir plaka içinde sporule zaman, hücreleri verimli sporlanmalanna yoktur. Hücreler bir boncuk ya da (çalkalama olmaksızın 30 ° C de Tablo 1 olmasına kıyasla bir çalkalama inküba...

Tomurcuklanan maya sporlanma mayoz 1, genetik rekombinasyon 2 mekanizmaları, 3 sinyal hücre tarafından gelişim kontrolü, geliştirme 4 besin kontrolü, transkripsiyonel sırasında kromozom ayrımı kontrolü de dahil olmak üzere biyoloji birçok açıdan, içgörü sağlamak için çalışılmıştır gelişim 5 düzenlenmesi ve spor oluşumu 6 incelenmesi. Spor Oluşumu koruyucu spor duvar 6 birikimi, ardından ana hücreden içinde yeni zar bölümlerine oluşturulmasını içeren yeni bir hücre bölünmesi olayını içerir. Sporlanan hücrelerini inceleyen Bu çalışmalar genellikle nispeten verimli bir şekilde 7,8 yaklaşık 24 saat içinde sporülasyonun sürecinden geçmeleri için hızla sporlanan maya suşu SK1, yararlanmak. Maya tomurcuklanan sporülasyon koşullarının optimizasyonu 9-13, bu deney, tarif edilmiş olmasına rağmens katı ortam üzerinde ya da sporlaşma kültür tüpleri veya şişeleri kullanılarak gerçekleştirilir büyük ölçekli sıvı kültürlerde sporlanmasını incelenmiştir. Burada, 96 kuyucuklu bir plaka biçiminde, maya sporlanan için bir yöntemi tarif eder. Bu yöntem için, havalandırma senkron ve verimli sporülasyonun için kritik olduğunu bulmak ve küçük hacimli multiwell biçimi yeterli sporulasyonunu sağlamak için teknikler geliştirdiler. Hücreler, yüksek verimli teknikleri ve döşemeli kütüphane 14-16 kullanılarak yüksek kopya bastırma için çok oyuklu bir plaka biçiminde, bu tarama için optimize edilmiş reaktifler kullanılarak taranabilir için 96 çukurlu bir levha formatında sporlanan sağlar.

<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> Bu protokol değerlendirmek için, (yukarıda anlatıldığı gibi) çukurlu levhalarda yer alan hücreler sporlanan elde sporülasyon verimleri şişelerinde (Tablo 2) &#39;de daha büyük hacimler ile sporule hücreleri karşılaştırılmıştır. multiwell plakaların kullanımı ~% 80 verimlilik rutin görülebilir matara, sporlanan zaman görülen yüksek verim elde etmedi. (Cam boncuk veya bir karıştırma çubukları tarafından sağlanır), uygun havaland...

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.

Etkin Sporlanma<em&gt; Saccharomyces cerevisiae</em&gt; 96 çukurlu Formatında

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

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Biology

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

10.1K Görüntüleme.  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.

Diseksiyonu 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 ...

Biyoloji

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.

S. cerevisiae &quot;adlı organel morfolojisi küresel çalışma yüksek verimli bir yöntem

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.

ACT1-CUP1 Testleri, Tomurcuklanan Mayadaki Splisozomal Mutantların Substrata Özgü Hassasiyetlerini Belirler

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

Genetik

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.

Sıvı KromatografiSi Kullanan Saccharomyces Cerevisiae'nin Nicel Metabolomikleri Tandem Kütle Spektrometresi ile Birleştiğinde

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

Biyokimya

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.

Kriyo-Elektron Tomografisi için Saccharomyces cerevisiae'nin Hazırlanması ve Kriyo-FIB mikromakinesi

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.

Mikroarray Analizi için 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.

Barkodlu Maya Kütüphaneler Rekabet Genomik Ekranlar

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.

Akış Sitometri tabanlı Arıtma<em&gt; S. cerevisiae</em&gt; Zigot

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.

Evrim Yöntemi yönettiği<em&gt; Saccharomyces cerevisiae</em&gt;: Mutant Kütüphane Oluşturma ve Tarama

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 Üstel büyüme kinetik solunum ve fermantatif metabolizma analiz etmek için toplu iş kültüründe

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

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Bu videodaki bölümler

Diseksiyonu Saccharomyces cerevisiae Asci

S. cerevisiae "adlı organel morfolojisi küresel çalışma yüksek verimli bir yöntem

ACT1-CUP1 Testleri, Tomurcuklanan Mayadaki Splisozomal Mutantların Substrata Özgü Hassasiyetlerini Belirler

Sıvı KromatografiSi Kullanan Saccharomyces Cerevisiae'nin Nicel Metabolomikleri Tandem Kütle Spektrometresi ile Birleştiğinde

Kriyo-Elektron Tomografisi için Saccharomyces cerevisiae'nin Hazırlanması ve Kriyo-FIB mikromakinesi

Mikroarray Analizi için Saccharomyces cerevisiae

Barkodlu Maya Kütüphaneler Rekabet Genomik Ekranlar

Akış Sitometri tabanlı Arıtma

Evrim Yöntemi yönettiği

Saccharomyces cerevisiae Üstel büyüme kinetik solunum ve fermantatif metabolizma analiz etmek için toplu iş kültüründe