CJNラボの研究はNSERC、カナダの癌協会研究所（CCSRI）、およびカナダの乳がん財団（BC-ユーコン支店）から操作する補助金によってサポートされています。

注：この手順の一般的なワークフローを図2に概説されている。 成長阻害用量の1の決定<ol><li>酵母一晩培養の調製 注：このプロトコルで使用される酵母エキス-ペプトン-デキストロース増殖培地（YEPD）は、標準的なレシピ13である。 <ol><li> BY4741（ のMATa HIS3Δ1leu2Δ0met15Δ0ura3Δ0）YEPD寒天プレート上の細胞外ストリークし、30℃で、可視コロニーが形成されるまで、48時間インキュベートする。 </li><li>単一コロニーを滅菌した培養容器にYEPD液体培地の5ミリリットルを接種することによって、一晩培養を準備します。連続回転または振とうしながら30℃で一晩インキュベートする。文化は一般的に（〜2×10 8細胞/ ml）朝までに飽和状態に達するだろう。 </li></ol></li><li>様々な化学物質の投与量を含有する固体寒天培地調製<ol><li>のいくつかの株式を準備化学物質は、適当な溶媒中で100×所望の最終濃度で試験する。 注：効果的な濃度は、化学に依存し、経験的に決定されなければならない。当初は幅広い最終濃度範囲をテストすることをお勧めします。 すなわち 、低μMからハイmMまで。 </li><li>化学物質の各濃度について、いくつかの滅菌培養チューブにアリコート溶融YEPD寒天を3mlを、試験される。 55℃の水浴中で場所が固化維持する。 </li><li>試験対象の化学物質の各濃度について12ウェルプレート内の重複ウェルのそれぞれペアに溶融メディア、渦に混合する2-3秒、およびピペット1ミリリットルを100倍化合物の30μlを添加する。唯一の制御車両を含めるようにしてください。プレートを室温で一晩に設定できるようにします。余分なメディアを捨てる。 </li></ol></li><li>スプレッドプレーティング細胞<ol><li>ステップ1のように一晩増殖させ飽和酵母培養の2μL、とYEPD液体培地の10ミリリットルを接種する。1.2。 </li><li>プレートウェルあたり希釈された酵母培養液25μl、滅菌ガラスビーズやロッドを使用して均一に広げ、そしてプレートが炎の下で乾燥させる。 48時間、30℃でプレートをインキュベート。 </li><li>プレート上での酵母の増殖（コロニーの両方の合計数と大きさ）を評価する。画面を実行するために10％以上の-15％の成長を阻害しない化合物の亜致死濃度を選択する。 </li></ol></li></ol> 2.体系的化学遺伝画面<ol><li>ソースプレートの欠失変異体アレイのメンテナンス（DMA）と準備<ol><li>グリセロールストックから欠失変異体アレイを構築する上で詳細な説明についてはBaryshnikova ら 14を参照してください。欠失変異体は、プレートあたり384コロニーの密度で配列された後、DMAは4​​℃で数ヶ月間保存することができる。それぞれに成長するコロニーを防止するためにG418、必要に応じて200μgの/ mlを含むYEPD寒天上に複製するその他。 注：複数のシリアル複製が遅い成長株の損失を防止し、サプレッサー変異株の出現を最小限にするために避けるべきである。半数体としてのコレクションを維持する場合、これは特に関連する。 </li><li>ロボットシステムを配列の微生物を使用して、すべてのレプリカプレーティングの手順を実行します。また、手動のピン止めツールを使用して配列されたコロニーを操作します。 注：酵母の削除コレクションは、いくつかの商業的供給源から半数体と二倍体として利用可能で、通常は96ウェルプレートにグリセロールストックとして出荷されています。 </li></ol></li><li>欠失変異体の配列を凝縮<ol><li> G418200μgの/ mlを含むYEPD寒天培地の250ミリリットルを準備します。 G418の有効濃度は変化してもよく、各ロットは、実験的にテストする必要があります。 </li><li>プレートあたりのG418を200μg/ mlを含むYEPD寒天の50ミリリットルを注ぐことにより、5 YEPD寒天プレートを準備します。配列を凝縮する前にこの一日を行い、プレートを室温で冷却することができるようにerature一夜平らな平らな面に。 注：4つのプレートが1536 - ひずみ密度で収集に必要となる、第五のプレートが余分として注入する。 </li><li>プレート当たり384コロニーの密度で突然変異体の削除コレクションを含む16ペトリ皿を取り出し、室温（〜1時間）に来ることができます。 </li><li>繊細なタスクが拭く使用して各プレートの蓋の上に形成されたすべての結露を拭いてください。これを怠ると、配列された変異体の配列と交差汚染の上に堆積されて水滴になることがあります。 </li><li>微生物の配列ピン止めロボットを用いて、プレート当たり1536個のコロニー（4ペトリ皿）に、プレート当たり384コロニー（16ペトリ皿）の密度からDMAを凝縮。プレート1ピンから1 番目のコロニーが凝縮された配列の1列1行するように、これを実行し、プレート2の1 番目のコロニーは、1列2、2列1を行にプレート3の1 番目のコロニー行、とする行2のプレート4の1 番目のコロニーコラム2。 </li><li> 30℃で一晩連結配列をインキュベートする。 </li><li>ソースプレートからコロニーの均一な移動を確保するための配列を調べます。 DMAが正しく（ 図4A）凝縮されたために、ガイドとして役立つわざわざ配列に組み込まれ、いくつかの空白の位置が、あるでしょう。 注：これらは、試験化合物を含有する培地上にレプリカプレーティングのソースプレートになる。単一ソースプレートは、複数のpinningsのために使用することができる。また、多くのロボットは、酵母の同じような数字が毎回転送されていることを確認します相殺機能を持ちます。 </li></ol></li><li>レプリカプレート欠失変異体配列<ol><li>コントロールの700ミリリットル（車両のみ）、および実験的なメディア（化学系）の700ミリリットルを準備します。これは（条件あたり12のプレートに加え、2エクストラ）三連でアッセイを行うために十分である。 </li><li>プレート当たりの試験化学物質またはコントロールを含むYEPD寒天の50ミリリットルを注ぐ。プレートtを許可Oフラットさえ表面に室温で一晩冷やす。 </li><li>レプリカプレートにロボットを用いて、実験的に決定された濃度の化学物質を含有するプレートを3組、ならびにビヒクル対照を含有するプレートを3組にDMAを接種する。 24〜48時間室温でプレートをインキュベートする。 </li></ol></li></ol> 3.イメージングプレートとデータ解析<ol><li>インキュベーターからプレートを取り出し、それらを画像化の前に、室温（〜1時間）に来ることができます。これは、プレートを画像化しながら、結露を防ぐことができます。 </li><li>蓋を取り外し、フラットベッドスキャナ上に表を下にして置くことにより24及び48時間での画像プレート。少なくとも300dpiの解像度で画像をキャプチャします。代替的に、画像をキャプチャするデジタルカメラを使用。そうする場合は、場所のプレートは、暗い背景に表向きにして画像に蓋を取り外します。 注意：プレートのファイルフォーマットおよび位置決めを使用定量化プログラムに依存するであろう。 </li&gt; <li> Balony 15、SGAtools 16、及びScreenMill 17を含むいくつかのオープンソースプログラムのいずれかを使用してアレイコロニーの大きさの定量化と比較を行う。 </li></ol>画面の4。バリデーション注：この段階では、いくつかの酵母の欠失変異体は、対象の化学物質への過敏として獲得します。これらの化学遺伝的相互作用は、次の二つの方法で検証する必要があります。最初の感受性株の同一性は、PCRによって確認されるべきである。第二に、化学物質過敏症は、独立して採点されなければならない。以下に概説ひずみ過敏症、酵母生物学における一般的な技術を検証するために、スポッティングアッセイを行うための簡単​​なプロトコルである。 <ol><li>ストリークアウトのG418を200μg/ mlを含むYEPD寒天上に酵母削除コレクションから（過敏）変異体を希望する。コロニーが形成されるまで30℃でインキュベートする。に応じてプライマーを用いた診断PCRによって株の遺伝子型を確認してください製造業者のプロトコル。 </li><li>各菌株のためのYEPD液体の5ミリリットルを接種し、回転または振とうしながら30℃で一晩インキュベートする。 </li><li> OD 600を測定し、無菌のdH 2 O中にOD 600 = 1に各菌株を希釈これらは現在、正規化された酵母培養物である。 </li><li> 96ウェルプレートのウェルA1に正規化された野生型酵母（BY4741）培養物100μlを分注する。井戸B1-H1に最大7つの追加の株を100μl分注する。 </li><li> A6-H6を通じて井戸A2-H2に無菌のdH 2 O90μlのを分配するためにマルチチャンネルピ ​​ペッターを使用してください。最後に、正規化された酵母培養物の1:10希釈系列を用意。シリアルにマルチチャンネルピ ​​ペッターで列2に列1の10μlのピペッティングして起動し、6の希釈が行われるまで、96ウェルプレート全体で継続する（ すなわち 、10 0〜10 -5）。 </li><li>マルチチャンネルを使用してグリッドに希釈した酵母培養の2-5μlの転送化学的および車両コントロールを含むYEPD固体培地上にピペッター。 24〜48時間のための適切な温度でプレートをインキュベートする。 </li><li> （BY4741対照と比較して）プレートを点検し、化学物質に選択した変異体の感度を比較する。ステップ3.2と24と48時間での画像プレート。 注：関連遺伝子オントロジーまたは機能を有するいくつかの過敏株の同定は、スクリーンの有効性に自信を向いているが、遺伝子の野生型コピーを含有するプラスミドと過敏欠失菌株の形質転換は、正式にその遺伝子を確立するために必要とされる興味の削除は（第二部位変異を隠されていない）薬剤感受性の原因となる。 </li></ol>

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The authors declare they have no competing financial interests.

ここで説明は、化学遺伝的相互作用のプロファイルを生成するためのアプローチである。この方法は単純で、化学物質の存在下および非存在下での総合的な遺伝子欠失コレクション内の各株のコロニーのサイズを比較することにより、化学的傷害を許容するために必要なすべての遺伝子が同定されている。感受性株の結果リストの注釈濃縮分析は、アクションの薬品モードへの洞察を提供す?...

遺伝的ツールとモデル生物出芽酵母で利用可能なリソースを一括遺伝子がネットワークは、生物学的システムの要件を満たすためにとして機能する方法に新たな洞察を提供し、大規模なゲノム機能研究を可能にした。これらのツールの礎石は、酵母1,2内のすべてのオープンリーディングフレームの非必須遺伝子欠失の完全なセットの共同制作だった。顕著な観察は、標準的な実験室条件下​​で半数体として増殖させた場合の酵母遺伝子のわずか〜20％が生存に必要とされることであった。これは別の生物学的経路を利用して、ゲノムの摂動に対する緩衝する細胞の能力を強調しています。組み合わせて個別に実行可能な、しかし、致死的に接続または収束並列生物学的経路のシグナル及び生物学的機能を説明する遺伝的相互作用のネットワークを形成する遺伝的変異体。条件付きテの発展に伴い必須遺伝子のmperatureに敏感と低次形態対立遺伝子技術は、非必須遺伝子3,4の研究に限定されていません。この概念は、類似の細胞プロセスに関与する遺伝子が5が一緒にクラスタ化方法を説明するための公平な遺伝的相互作用マップを生成するゲノム規模で適用されている。 遺伝子ネットワークの化学摂動模倣遺伝子欠失（ 図1）6。過敏症の削除株の高密度アレイに対して増殖抑制化合物を問い合せると、化学的ストレスに耐えるために必要とされる遺伝子のリスト、つまり 、化学遺伝的相互作用プロファイルを識別します。遺伝的相互作用と同様に、化学ライブラリーの大規模スクリーニングは、一緒に行動クラスタの類似した様式を有する化合物7が示されている。したがって、化合物の化学遺伝的相互作用のプロファイルを確立することによって、作用機序は、LARと比較することによって推測することができるGE-規模合成遺伝的および化学的な遺伝的相互作用のデータセット8,9。 化合物のスコアは、尋問されている大規模な化学遺伝学的スクリーニングでは、バーコードの競合アッセイにより行われてきた。この手法では、欠損株のプールされたコレクションは、化学物質を含有する培地の少量の数世代にわたって一斉に成長させる。各欠失変異体は、固有の遺伝的バーコードを保有するので、欠失株のプール内の個々の突然変異体の生存/増殖は、マイクロアレイまたは高スループット配列10によって追跡される。 生物活性化合物を含む固体寒天上で増殖させた物理的に配列された変異体のコロニーの大きさを監視することにより、適合度を推定することは、化学遺伝的相互作用11,12を識別するための有効な方法である。このアプローチは、競合ベースのスクリーニングの費用対効果の高い代替物を提供し、化学物質の小さなライブラリーをアッセイするために適している。ここで概説S.における化学遺伝的相互作用のリストを生成するための簡単な方法論である分子生物学操作またはインフラストラクチャに依存しない。セレビシエ。のみ酵母削除コレクション、ロボットまたは手動のピン止め装置、及び自由に利用できる画像解析ソフトウェアを必要とする。

<table border="0" cellpadding="0" cellspacing="0" width="987">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Name of Material/ Equipment&#160;</td>
			<td style="width:161px;">Company&#160;</td>
			<td style="width:156px;">Catalog Number&#160;</td>
			<td style="width:457px;">Comments/Description&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:213px;">Yeast Extract</td>
			<td style="width:161px;">BioBasic</td>
			<td>G0961</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 1% final concentration (w/v)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:213px;">Tyrptone Powder</td>
			<td style="width:161px;">BD Biosciences</td>
			<td>211820</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 2% final concentration (w/v)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Dextrose</td>
			<td style="width:161px;">Anachemia</td>
			<td style="width:156px;">31096-380</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 2% final concentration (w/v) - Do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Agar A</td>
			<td style="width:161px;">Bio Basic</td>
			<td>FB0010</td>
			<td style="width:457px;">For YEPD solid media add to 2% final concentration (w/v)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:213px;">G418</td>
			<td style="width:161px;">A.G. Scientific Inc.</td>
			<td style="width:156px;">G-1033</td>
			<td style="width:457px;">Prepare 1000x stock at 200mg/ml in dH2O and filter sterilize.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">12 well plate</td>
			<td>Greiner Bio One</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">5ml culture tubes</td>
			<td>Evergreen Scientific</td>
			<td>222-2376-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">10cm Petri Dish</td>
			<td style="width:161px;">VWR</td>
			<td>25384-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROTOR HDA</td>
			<td>Singer Instruments&#160;</td>
			<td>ROT-001</td>
			<td>high-throughput microbial array pinning robot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PLUSPLATE&#169; Petri Dish</td>
			<td>Singer Instruments&#160;</td>
			<td>PLU-001</td>
			<td>Box of 200 dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384 Short-Pin RePad</td>
			<td>Singer Instruments&#160;</td>
			<td>RP-MP-384</td>
			<td>Box of 1000 pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1536 Short-Pin RePad</td>
			<td>Singer Instruments&#160;</td>
			<td>RP-MP-1536</td>
			<td>Box of 1000 pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alternative Pinning Tools:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fully Automated Robtic Systems</td>
			<td>S&#38;P robotics</td>
			<td>http://www.sprobotics.com</td>
			<td>Several automated colony handling robitic and imagining systems available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manual Pinning Tools</td>
			<td>V&#38;P Scientific</td>
			<td>http://www.vp-scientific.com</td>
			<td>Handheld replication tools and accessories.&#160;</td>
		</tr>
	</tbody>
</table>

rapid identification of chemical genetic interactions in saccharomyces cerevisiae

出芽酵母は、生物活性化学物質の作用機序を学術、製薬、幅広い、および産業科学者に興味がある。決定、または出芽酵母、〜6000の遺伝子の欠失変異体と低形質必須遺伝子のための完全なコレクションのモデル真核生物である変異体は、市販されている。変異体のこれらのコレクションは、系統的に化学物質を許容するために必要な化学-遺伝子の相互作用、 すなわち遺伝子を検出するために用いることができる。この情報は、順番に、化合物の作用の可能性が高いモードに報告します。ここでは、出芽酵母における化学遺伝的相互作用の迅速な同定のためのプロトコルについて説明します。我々は、アクションの明確に定義された機構を有する化学療法剤5-フルオロウラシル（5-FU）を用いた方法を実証する。我々の結果は、核TRAMPのRNAエキソソームおよびDNA修復酵素は、以前メートルと一致する5-FUの存在下で増殖のために必要とされることを示すベースのバーコード化学遺伝学的アプローチおよび5-FUに悪影響RNA及びDNA代謝に影響を与えることを知ってicroarray。これらのハイスループットスクリーニングの必要な検証プロトコルも記載されている。

このアプローチの妥当性確認として、我々は、上記で概説したプロトコル次の化学療法剤5-フルオロウラシル（5-FU）の代表的な化学遺伝的相互作用の画面を行った。 5-FUは、チミジル酸シンターゼ、ならびにDNAおよびRNA代謝18を破壊することが知られている。 5-FUの化学遺伝的相互作用は十分に研究されており、ヘテロ接合およびホモ接合性欠失コレクション8,19の両方を使用?...

Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

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. ...

rapid-identification-chemical-genetic-interactions-saccharomyces

Research

JoVE Journal

Biology

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

10.2K ビュー.  University of Victoria. 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.

ビデオ: Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

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

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiaeの定量的プロテオミクスとタンパク質の複合体の同定

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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 ...

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 ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

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 ...