筆者の研究室での作業は、保健研究のカナダの研究所、イノベーションのためのカナダ財団、自然科学と工学研究評議会とコンコルディア大学によって運営されている。

二倍体菌株の構築 交配による二倍体の生産のためのプロトコル<ol><li>必要に応じて、単一のコロニーを生成する場合、最初の株はYPD、または選択培地上で三振してください。 two半数体株は、逆の交配型（すなわち、MATAとMATα）でなければならない。 </li><li>無菌接種ループを使用しては、2つの半数体株の一つから、コロニーの小さな部分を選択。新しいYPDプレートストリークループ一回プレート間の直線の上に。プレートの底部上に矢印の付いた線の方向をマーク。 </li><li>同じYPDプレート上の2番目の歪みのためにこれを繰り返します。最初にこの時間は、ストリークコロニーの垂直を、第二の最初の連勝を横切る。第二ストリークの方向をマークと第二ストリークが最初に交差するエリアに注意してください。二倍体が生成される場所をこのエリアにはなります。 </li><li> 30℃で一晩YPDプレートをインキュベートする。 </li><li>レプリカプレートを選択新しく形成された二倍体の成長を許可する選択培地上にYPDプレート。 </li><li>単一のコロニーを生成するために、同じ選択培地上に単一コロニー可能であれば、または二倍体の縞と縞の小さな部分を選択してください。 </li></ol> ASCIの胞子形成と消化 プロトコル<ol><li>上記のプレートから単一コロニーをピックアップし、（1センチメートルX 1CM）胞子形成（SPO）プレート上にパッチを小さくすると胞子形成に3-7日のために30℃でインキュベートする。 </li><li>胞子形成を光学顕微鏡下でそれぞれのパッチからの細胞を見てチェックすることができます。水の5〜10μlので顕微鏡のスライド上の場所細胞は、液滴の上にカバースリップを置き、ASCIの存在を探します。 ASCIが簡単に見つからない場合は、インキュベーターに戻す。 </li><li>パッチのいずれかの効果的な胞子形成がある場合は、四分子​​ジュースの100μLを滅菌した15mlのコニカルチューブを準備する。 </li><li>滅菌3ミリメートル接種ループ（細胞がループの¼をカバーする必要があります）と四分子のジュースの代わりにこの胞子形成のパッチから細胞を採取する。優しくスワールループを細胞がソリューションとループとのミックスを落ちるようにする。 </li><li> 4 - 10分（ザイモリアーゼと歪み自体の濃度や活性に応じて）のために四分子ジュース（ザイモリアーゼを含む）に残す。これは、子のう胞子を周囲の細胞壁の消化を可能にする。 </li><li> YPDプレート上に接種領域の上部と下部をマーク。 </li><li>省略可能。反応物を氷冷滅菌のdH 2 Oを1mLのを加えることによって停止することができます細胞は5minsのために解決することができます。手で軽く混ぜ、氷上で維持して上清から1mLのを削除します。 </li><li>手順5から消化細胞の5μLを取り、そっと場所は、事前にマークYPDプレートの接種領域の行にドロップします。 </li><li>やっと寒天と接触しながら非常に軽くラインにドロップ1〜3回を広めるために滅菌3ミリメートル接種ループを使用してください。 </li><li>液体は寒天に吸収することを許可する（手順7が省略された場合、これは外側の細胞壁の消化が減少または停止される場合です）。解剖顕微鏡に進みます。 </li></ol> ASCIの解剖 プロトコル以下のプロトコルはシンガーMSMシステム200マイクロマニピュレーションの顕微鏡で説明されていることに注意してください。このようなツァイスマイクロマニピュレータのMRとして手動マイクロマニピュレーターを使用している場合、このプロトコルは、若干の修正を追跡することができる。 <ol><li>フィールドの半分は細胞が含まれており、半分が空であるプレートの領域に焦点を当てる。ゆっくりとそれはフィールドの空の領域に触れるように焦点に針をもたらす。これは、針が前回使用時の細胞が含まれていないことが保証されます。針と寒天との間の接触は、ゆっくりと焦点に入ってくる暗い黒い丸として見られている。 </li><li>位置A1に行列に移動し、再び寒天を貫通することでマークを付けます。これは、それが完了する前に顕微鏡をオフに実行された場合、プレートの向きを判断することです。 </li><li>検索モードに切り替えます。 ASCIを見つけます。好ましくは、細胞が十分に広がっている地域にあるASCIを選択してください。細胞のクラスターを持つ領域を避けてください。 ASCIから4子のう胞子はまだお互いに接続する必要があります。二つの子のう胞子は、他の二つよりも若干大きくなることがあります。 one子嚢胞子が緩いと子のう胞子の残りの部分に接続されていないASCIを避けます。 </li><li>針で子嚢を拾う。ゆっくりと板に針を持ち出す。マイクロニードルの影が見られる、ライ​​ン子嚢を持つこのアップがピックアップされる。円（針の先端）の黒の輪郭が見えるまで、再び針に近づく。このサークルで四分子をタッチしてからすぐに離れて針を引き抜きます。子嚢からすべての4つの子のう胞子が針に付着し、追加の細胞はこの子嚢の一部ではないマイクロニードルで収集されなかったことをしていることを確認します。 </li><li>遠く離れてプレートからマイクロニードルを引いて、マトリックス（最初の子嚢のため、に行くに行くA1の位置）。子嚢が再配置されるそれぞれの位置をメモしておいてください。マイクロニードルでプレートに触れるとすべての4つの子のう胞子は、マイクロニードルをオフに来るまで引いて、子嚢を下に置きます。ニードルアームまたはYPDプレートを保持しているプラ​​ットフォームの穏やかなタッピングは、この段階ではしばしば有用です。 </li><li> ASCIの希望数が選択されるまで、マトリックス上で連続した位置にステップ3-5と場所ASCIを繰り返します。 </li><li>各子嚢に戻って、ゆっくりとプラットフォームまたは両方の組み合わせをタップする、針でいじめが離れて4つのセルが壊れる。各子嚢の各子嚢胞子は、4つの行で区切られるまで、マトリックス内の新しい位置の背後にある一つを残して細胞を採取する。 </li><li> ° C成長パターンを観察するために数日間30℃インキュベーターでYPDプレートを置きます。 </li><li>選択培地にレプリカが板切除した細胞を必要に応じて。 </li></ol>接合体の移転 プロトコル<ol><li>単一のコロニーを得るために適切な媒体上の反対側の嵌合タイプを持つ2つの半数体系統外ストリーク。 </li><li>メイトこれら二つの菌株は、上述したように、4〜6時間（または一晩、必要に応じて）から成長することができます。 </li><li>光学顕微鏡下で受精卵を確認してください。受精卵は、芽の有無に関係なくどちらダンベル型のセルとして表示されます。 </li><li> 3ミリメートル接種ループ（細胞がループの¼をカバーしてください）で細胞をピックアップし、無菌のdH 2 Oの100μLにそっと置く</li><li> YPDプレート上に接種領域の上部と下部をマーク。 </li><li> 5μL取ると優しく場所は、事前にマークしたYPDプレートの接種領域の行にドロップします。 </li><li>やっと寒天と接触しながら非常にゆっくりとラインの低下を広めるために3ミリメートル接種ループを使用してください。 </li><li>液体は寒天に吸収することができます。解剖顕微鏡に進みます。 </li><li>フィールドの半分は細胞が含まれており、半分が空であるプレートの領域に焦点を当てる。ゆっくりとそれはフィールドの空の領域に触れるように焦点に針をもたらす。これは、針が前回使用時の細胞が含まれていないことが保証されます。針と寒天との間の接触は、ゆっくりと焦点に入ってくる暗い黒い丸として見られている。 </li><li>位置A1に行列に移動し、再び寒天を貫通することでマークを付けます。これは、それが完了する前に顕微鏡をオフに実行された場合、プレートの向きを判断することです。 </li><li>検索モードに切り替えます。受精卵を見つけます。 </li><li>針との接合子を拾う。 </li><li>場所は、行列に個別に受精卵。 </li><li>受精卵が数日間に成長することができます。 </li><li>適切な選択培地上にレプリカプレートは受精卵。 </li></ol>代表的な結果<img src="/files/ftp_upload/1146/1146fig1.jpg" alt="" /> 図1：反対の交配型の二つの半数体株の交配は、二倍体株を生成します。 <img src="/files/ftp_upload/1146/1146fig2.jpg" alt="" /> 図2：温度感受性株（LEU2栄養要求性を持つ）と野生型株（LEU2）との間の交配のTHRE結果（A）YPDプレート。消化細胞は、2つの黒線の間に厚さ成長が見られている左側の接種地域に広がっている。解剖されているASCIは、均等に、右側の行列には等間隔育つ。 （B）（A）のプレートは、SD -ロイシンドロップアウトプレート上に複製されました。検証子嚢胞子が解剖されたことを示すLEU2マーカーの午前2時02分成長に注意してください。 （C）（A）のプレートは、YPDプレート上に複製され、37℃でインキュベートした。さらに子嚢胞子が解剖されたことを検証する2：02の成長に注意してください。

<ol>
	<li>Sherman, F., Hicks, J., Guthrie, C., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+and+Dissection+of+Asci.">Micromanipulation and Dissection of Asci.</a> Guide to Yeast Genetics and Molecular Biology. , 21-37 (1991).</li></ol>

著者らは、開示することは何もない。

酵母の解離は、目的のマーカーを使用して新しい株を選択すると便利なツールです。 four子のう胞子の理論的な成長パターンを決定する際には、栄養細胞の遺伝子型を調べることが重要です。プラスミドが存在する場合、これは子嚢胞子（s）はプラスミドを受信し、操作している菌株について予測し、結論に影響を及ぼすとなる影響を与えるとして、それは、単一のローまたはハイコピーで?...

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<td>Singer MSM System 200</td>
<td>Microscope</td>
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<td>Seikagaku Corporation</td>
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<td>Yeast extract</td>
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dissection of saccharomyces cerevisiae asci

酵母は、多くの異なる細胞プロセスを研究するために使用される非常に扱いやすいモデル系である。共通の実験室株<em&gt;サッカロマイセスセレビシエ</em&gt;一倍体または二倍体の状態のいずれかに存在する。 two半数体から対立遺伝子を組み合わせることができるし、ゲノムへの変更を導入する能力は、ASCIの生産と解剖が必要になります。半数体細胞からのASCIの生産は二倍体株を生成するために互換性のある交配型を持つ2つの酵母の一倍体株の交配から始まります。これは、固体培地上または液体のいずれかのいくつかの方法で行うことができます。それは選択的に半数体株のどちらに比べて彼らの成長を促進する培地で二倍体のために選択することが有利である。二倍体は、ASCI、二倍体の減数分裂生殖から生じる4倍体の娘細胞の束を形成するために栄養の乏しい培地で胞子形成するために許可されています。栄養細胞とASCIの混合物を次いで、ASCIの子のう胞子を包む膜の嚢を離れて消化する酵素のザイモリアーゼで扱われます。一個ASCIを拾う四ascoporesを分離して再配置できます解剖顕微鏡下でマイクロニードルでマイクロマニピュレーションを使用する。解剖ASCIは、数日間増殖し、適切な選択培地にレプリカプレーティングによって興味のマーカーまたは対立遺伝子のためにテストされています。

Watch this Scientific Journal Video about Dissection of Saccharomyces Cerevisiae Asci at JoVE.com

Dissection of Saccharomyces Cerevisiae Asci

酵母細胞のマニピュレーションは、減数分裂の遺伝学的分析のために必要とされるか、二倍体の受精卵を選択する。これらmicromanipulationsは、解剖顕微鏡のマイクロニードルを用いて実施されています。マイクロニードルは、細胞を再配置するために使用され、自動化の様々な程度で使用可能なマニピュレータによって制御されます。

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 ...

dissection-of-saccharomyces-cerevisiae-asci

Research

JoVE Journal

Biology

Dissection of Saccharomyces Cerevisiae Asci

18.5K ビュー.  Concordia University. 酵母細胞のマニピュレーションは、減数分裂の遺伝学的分析のために必要とされるか、二倍体の受精卵を選択する。これらmicromanipulationsは、解剖顕微鏡のマイクロニードルを用いて実施されています。マイクロニードルは、細胞を再配置するために使用され、自動化の様々な程度で使用可能なマニピュレータによって制御されます。

ビデオ: Dissection of Saccharomyces Cerevisiae Asci

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸の定量的なPhosphoproteomicsは刺激を受けてサッカロマイセスセレビシエ</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

生物学

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 ...

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

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

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

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

酵母でMitophagyを監視するための蛍光顕微鏡アッセイサッカロマイセスセレビシエ

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

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

で嚢胞性線維症膜コンダクタンス制御因子タンパク質の発現および精製サッカロマイセスセレビシエ

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

のリフォールディング監視タンパク質を結合アッセイ<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

で表現する嚢胞性線維症膜コンダクタンス制御因子タンパク質の精製<em&gt;サッカロマイセス·セレビシエ</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 &#181;M. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

EPRは、補因子の酸化還元滴定を監視対象<em&gt;サッカロマイセス·セレビシエ</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNAの崩壊料金の測定で<em&gt;サッカロマイセス·セレビシエ</em&gt;使い方<em&gt; rpb1-1</em&gt;株

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

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

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

における化学遺伝の相互作用の迅速な同定<em&gt;サッカロマイセス·セレビシエ</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

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

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

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

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

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 ...