この作品は、NIH（LSH）からマサチューセッツ州ボストン（LSH）およびR15 GM86805の大学のジョセフ・P.ヒーリーの助成金によってサポートされていました。 SMPは、マサチューセッツ州ボストンの大学でサノフィ・ジェンザイムフェローシップによって部分的にサポートされています。

1.胞子形成のための準備注：このプロトコルに記載されたメディアは、標準的なレシピと方法13,17を用いて製造される。表1は 、このプロトコルで使用される様々なメディアの1 Lのための製剤が得られます。 <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;">バクトペプトン</td><td style="width: 50px;">酵母エキス</td><td style="width: 50px;">バクト寒天</td><td style="width: 60px;">ブドウ糖</td><td style="width: 70px;">酢酸カリウム</td><td style="width: 60px;">グリセロール</td><td style="width: 60px;"> ddH 2 O </td></tr><tr height="15"><td height="15" style="height:20px;"> YPGプレート</td><td> 20グラム</td><td> 10グラム</td><td> 20グラム</td><td> - </td><td> - </td><td> 30ミリリットル</td><td> 970ミリリットル</td></tr><tr height="15"><td height="15" style="height:20px;"> YPDプレート</td><td> 20グラム</td><td> 10グラム</td><td> 20グラム</td><td> 20グラム</td><td> - </td><td> - </td><td>千ミリリットル</td></tr><tr height="15"><td height="15" style="height:20px;"> YPD液体</td><td> 20グラム</td><td> 10グラム</td><td> - </td><td> 20グラム</td><td> - </td><td> - </td><td>千ミリリットル</td></tr><tr height="15"><td height="15" style="height:20px;"> YPA液体</td><td> 20グラム</td><td> 10グラム</td><td> - </td><td> - </td><td> 20グラム</td><td> - </td><td>千ミリリットル</td></tr><tr height="15"><td style="height: 20px; width: 80px;">胞子形成培地</td><td> - </td><td> - </td><td> - </td><td> - </td><td> 10グラム</td><td> - </td><td>千ミリリットル</td></tr></tbody></table> 表1：メディア製剤金額はメディアの1 Lのために与えられています。メディアに関する詳細成分は、材料表に記載されています。 <ol><li>すべての胞子形成細胞は同じ遺伝子型になる場合は、次の手順を実行します。 <ol><li>解凍酵母株はYPG（YPGlycerol）プレートに使用されます。 30℃で12-16時間、酵母を育てます。重要なことは、SK1細胞はまた、胞子形成する態勢を整えているので、SK1株はYPGプレート上に長すぎる残されるべきではない、またはそれらは、プレート上で胞子を形成することができます。 注：炭素源としてのグリセロールの使用は、酸化的リン酸化を必要とし、従って、機能的なミトコンドリアDNAを有する細胞について選択するようYPG上に成長した新たに解凍した株が好ましいです。 SK1細胞は、機能的なミトコンドリアDNAを失い、petitesのになる傾向を有します。機能的なミトコンドリアDNAは、胞子形成のために必要とされます。 </li><li>単一コロニーのためのYPD（YPDextrose）プレート上にYPGプレートからストリーク酵母。 30℃でYPDプレート24-28時間に酵母を成長させます。 </li><li> YPD固体培地上での増殖の約36〜48時間後、単一コロニーを使用して、25ミリリットルのYPD培養を開始YPDプレートから。細胞は7.0〜約OD 600 = 4.0になるまで、この文化を育てます。 220 rpmで振とう、振盪インキュベーター中で30℃で一晩、この手順を実行します。 </li><li> YPD培養物から、（220で振盪30℃振盪インキュベーター中で酵母を育てる2節の各96ウェルプレートの必要性のためにOD 600 = 0.1であることYPA（YPAcetate）文化の千ミリリットルを開始するために、細胞の適切な量を使用します培養OD 600までの回転数）は1.5から1.3の間です。 注：4 96マルチウェルプレートをスクリーニングする場合は、各96マルチウェルプレートのためのYPA培地の100ミリリットルを接種は、例えば、第2節でスクリーニングするために、YPA培地の400ミリリットルが必要になります。 YPA培養における10％の余分なボリュームで起動すると、蒸発量が消失するのを防止することが必要です。細胞は、適切なOD 600に到達するために一般的には、12-16時間の間になります。時間の量は、酵母株の遺伝子型に依存して変化し得ます。 YPAはpresporulationメディアです。 Pregrowth酢酸で効率的かつ同期胞子形成6のために必要な（例えばIME1など）の遺伝子の発現を誘導するのに役立ちます。 </li><li> 5分間テーブルトップ遠心機で1800×gでスピンはYPAで増殖させた細胞をペレット化します。洗浄細胞は、滅菌オートクレーブした脱イオン水0.5体積の1倍。再びスピン、および洗浄を捨てます。 1×ボリュームの胞子形成培地で細胞を再懸濁し。第2節に進みます。 </li></ol></li><li> 96マルチウェルフォーマットに、（ すなわち 、異なるプラスミドまたは異なる変異を含むで形質転換され、一般的に入手し、凍結ストックとして保存されている）、異なる遺伝子型の配列の細胞を、異なる遺伝子型のもので胞子形成のために細胞を使用している場合。そして、次の手順を実行します。 <ol><li> YPG固体培地上で96マルチウェルプレート（複数可）で解凍凍結ストックから細胞を転送するために、無菌の96プロングフロッガーを使用してください。 30℃での酵母の一晩を成長させます。 </li><li>滅菌96プロングフロッガーを使用して、YPD固形培地または選択的にYPG固体培地から細胞を転送固体培地（アミノ酸を欠いている、すなわち 、合成最小培地、細胞は選択を必要とするプラスミドが含まれている場合）。コロニーを各株のために形成されるまで、選択培地が必要であればYPD及び2日目に、典型的には1日、30℃で酵母を成長します。 </li><li>アリコート96深い2ミリリットルマルチウェルプレートの各ウェルにYPD液体培地または選択液体培地の1.2ミリリットル。フロッガーを使用して液体培地に固体培地から細胞を移すには、テープによって所定の位置に保持されたプラスチック蓋でカバーし、220 rpmで振とうしながら、30℃のシェーカーで一晩増殖します。 注：長方形のペトリプレートからプラスチックのふたを使用して、カバープレートの汚染を最小限にします。空気の循環が最小限に制限されているように、テープ片を使用して所定の位置に蓋を保持します。 </li><li> 96ウェルプレートを収容するためのアダプタを有するスイングバケット遠心機を用いて5分間、1,800×gで遠心分離板。細胞からの培地を捨て、各ウェルに500μlのYPAを追加し、YPAでペレット化した細胞を再懸濁します。カバープラスチック蓋をテープで適所に保持。 </li><li> 220 rpmで振とう、振盪インキュベーター中で30℃で15時間、細胞を増殖させます。第2節に進みます。 </li></ol></li></ol> 96マルチウェルフォーマット2.胞子形成細胞<ol><li> 1.3ミリリットルウェルを有する96ウェルプレートの各ウェルに1 3ミリメートル滅菌固体ガラスビーズまたは滅菌5ミリメートルのx 2ミリメートルの磁気攪拌棒のいずれかを追加します。 （ビーズまたは攪拌棒を使用するかどうかについての議論については、以下の代表的な結果を参照してください。） </li><li>スクリーニングされるすべてのセルが同じ遺伝子型である場合は、2.2.1に進みます。スクリーニングされる細胞は、異なる遺伝子型である場合は、2.2.2に進みます。 <ol><li> 96マルチウェルプレートの各ウェルにステップ1.1.5アリコート細胞+培地500μlのから細胞を取ります。テープによって所定の位置に保持プラスチック製のふたでカバーしています。 2.3に進んでください。 </li><li> 96ウェルプレートを収容するためのアダプタとスイングバケット遠心機を用いて1.2.4から5分間、1800×gでYPAであるスピン細胞、。の流動細胞からのFメディア。各ウェルに胞子形成培地500μlを添加して、胞子形成培地でペレット化した細胞を再懸濁します。テープによって所定の位置に保持プラスチック製のふたでカバーしています。 2.3に進んでください。 </li></ol></li><li>ガラスビーズを使用している場合は、場所の30℃の振盪培養器内のサンプルとは、胞子形成のための220 rpmで振とうします。胞子形成のために30℃インキュベーターの内側撹拌プレート上の磁気撹拌棒、場所のサンプルを使用している場合。 注：磁気攪拌棒の正確な撹拌速度は限りすべてのバーが精力的に動いていると文化を通気するように、非常に重要ではありません。 </li><li> （：5希釈1）可視化のために胞子形成培養物からサンプルを引き出す、胞子形成の進行を監視するために、96ウェルカバースリッププレートに胞子形成培地の24μlに胞子形成培養物から細胞の6μlを添加。倒立顕微鏡を用いて細胞を検査する前に5分間沈降細胞を残します。 注：野生型SK1細胞では、コンパクトな成熟した胞子が3によって形成されます胞子形成培地に移した後、6時間。胞子形成の進行は、例えば、核マーカー、蛍光マーカーを用いて顕微鏡検査によりモニターすることができる（ すなわち 、HTB2-mCherryを18）が減数分裂またはprospore膜マーカーを調べるために（ すなわち 、G20 19）は胞子形態形成を調べました。あるいは、屈折胞子の形成は、胞子形成培地に移した後、約12時間を開始ノマルスキー微分干渉顕微鏡を用いて見ることができます。 </li></ol>

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The authors have no competing financial interests.

ここでは、96マルチウェルフォーマットでSK1酵母を胞子形成するためのプロトコルを提示します。エアレーションは、各ウェル中の攪拌棒やガラスビーズのいずれかの使用を必要とする、効率的な胞子形成するためのキーです。細胞は、ビーズや撹拌棒のいずれかなし振盪インキュベーター中で96マルチウェルプレートに胞子形成されている場合、細胞が効率的に胞子を形成しません。細胞は...

出芽酵母における胞子形成が減数分裂1、遺伝子組換え2のメカニズム、3シグナル伝達細胞による開発のコントロール、開発4の栄養制御、転写時の染色体分離の制御を含む生物学の多くの側面、洞察を提供することが検討されています開発5の規制、および胞子形成6の検討。胞子形成は、保護胞子壁6の堆積に続いて母細胞内の新しい膜区画の形成を含む新規な細胞分裂事象を含みます。胞子形成細胞を調べるこれらの研究は、多くの場合、比較的効率的なやり方7,8で約24時間で胞子形成のプロセスを経ることができる急速に胞子形成酵母株SK1、を利用します。出芽酵母のための胞子形成条件の最適化は、9月13日 、これらの実験について説明したがSは、固体培地上または胞子形成は、培養チューブもしくはフラスコを用いて行われる大規模な液体培養物中で胞子形成を調べました。 ここでは、96マルチウェルプレートフォーマットで酵母を胞子形成するための方法を説明します。我々は、この方法のために、通気が同期および効率的な胞子形成のために重要であることを見つけると、小容量マルチウェルフォーマット十分な胞子形成を確保するための手法を考案しました。細胞は、高スループット技術とタイルライブラリ14-16を使用して高コピー抑制のためのマルチウェルプレートフォーマット、このようなスクリーニングのために最適化された試薬を用いてスクリーニングされる96マルチウェルプレート形式で胞子形成することができます。

<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> （上記のように）このプロトコールを評価するために、胞子形成効率は、マルチウェルプレート中で胞子形成細胞から得られたフラスコ（ 表2）に大きなボリュームを使用して胞子形成細胞と比較しました。マルチウェルプレートの使用は〜80％の効率が日常的に見ることができるフラスコに胞子形成時に見られる高効率を達成しませんでした。 （?...

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.

の効率的な胞子形成<em&gt;サッカロマイセス・セレビシエ</em&gt; 96マルチウェルフォーマットで

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

10.0K ビュー.  University of Massachusetts Boston. Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

ビデオ: 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.

の解剖サッカロマイセスセレビシエ ASCI

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

生物学

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.

グローバルS. cerevisiaeの細胞小器官の形態を研究するためにハイスループット法

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アッセイは、出芽酵母におけるスプライソソーム変異体の基質特異的感受性を決定

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

遺伝学

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.

液体クロマトグラフィーとタンデム質量分析法を用いたサッ カロミセス・セレビシエ の定量的メタボロミクス

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

生化学

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.

クライオ電子断層撮影のための サッカロミセス・セレビシエ の調製とクライオFIBマイクロマシニング

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.

マイクロアレイ解析のためのサッカロマイセスセレビシエ</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.

バーコード酵母ライブラリーの競争力のあるゲノム画面

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.

のフローサイトメトリーベースの精製<em&gt; S.セレビ</em&gt;受精卵

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.

指向進化方法で<em&gt;サッカロマイセス・セレビシエ</em&gt;：変異体ライブラリーの作成およびスクリーニング

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.

酵母呼吸と発酵代謝を分析するバッチ文化の指数関数的成長カイネティクス

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

の効率的な胞子形成

要約

さらに動画を探す

この動画の章

の解剖サッカロマイセスセレビシエ ASCI

グローバルS. cerevisiaeの細胞小器官の形態を研究するためにハイスループット法

ACT1-CUP1アッセイは、出芽酵母におけるスプライソソーム変異体の基質特異的感受性を決定

液体クロマトグラフィーとタンデム質量分析法を用いたサッカロミセス・セレビシエの定量的メタボロミクス

クライオ電子断層撮影のためのサッカロミセス・セレビシエの調製とクライオFIBマイクロマシニング

マイクロアレイ解析のためのサッカロマイセスセレビシエ

バーコード酵母ライブラリーの競争力のあるゲノム画面

のフローサイトメトリーベースの精製

指向進化方法で

酵母呼吸と発酵代謝を分析するバッチ文化の指数関数的成長カイネティクス

の効率的な胞子形成

要約

さらに動画を探す

この動画の章

の解剖サッカロマイセスセレビシエ ASCI

グローバルS. cerevisiaeの細胞小器官の形態を研究するためにハイスループット法

ACT1-CUP1アッセイは、出芽酵母におけるスプライソソーム変異体の基質特異的感受性を決定

液体クロマトグラフィーとタンデム質量分析法を用いたサッ カロミセス・セレビシエ の定量的メタボロミクス

クライオ電子断層撮影のための サッカロミセス・セレビシエ の調製とクライオFIBマイクロマシニング

マイクロアレイ解析のためのサッカロマイセスセレビシエ

バーコード酵母ライブラリーの競争力のあるゲノム画面

のフローサイトメトリーベースの精製

指向進化方法で

酵母呼吸と発酵代謝を分析するバッチ文化の指数関数的成長カイネティクス

液体クロマトグラフィーとタンデム質量分析法を用いたサッカロミセス・セレビシエの定量的メタボロミクス

クライオ電子断層撮影のためのサッカロミセス・セレビシエの調製とクライオFIBマイクロマシニング