This work was supported by NIH grant GM50936 to DKB.

注：以下のプロトコルのいくつかのステップは、クリーンドラフト内での作業が必要です。さらに、この方法は、スフェロプラストを監視するために10X長作動距離対物レンズを装備した酵母四分子解剖顕微鏡を必要とします。顕微鏡は、事前にフード内に設定する必要があります。範囲からマイクロマニピュレータアームとプレートホルダーを外し、作業エリアから離れ、安全な場所にこれらの項目を配置します。 スフェロプラストの調製<ol><li>所望の条件下で酵母を成長させます。 OD600 1.4 = 5×10 7細胞/ mlで各試料について、約2×10 8細胞、例えば培養の4ミリリットルを使用してください。試料の即時の処理がある場合には好ましいかもしれないが、ほとんどの用途のために、細胞のアリコートを、処理される前に、8時間まで氷上で保存することができます。 </li><li>細胞をペレットに3分間5（2345 RPM / 857.5 XG）の設定で臨床遠心分離機中で15mlの遠心管とスピンに細胞懸濁液を転送します。 </lI&gt; <li>ペレットから細胞を失わないように注意しながら培地をデカント。静かに1ミリリットルのZKバッファーで細胞を再懸濁した後、40μlの1 Mジチオスレイトール（DTT）を追加します。穏やかに混合しながら2分間インキュベートします。以前のように細胞をペレット化する（ステップ1.2を参照してください）​​。 </li><li> 1ミリリットルのZKバッファで再懸濁ペレット。完全に混合されたザイモリアーゼ100Tの新たに調製した溶液5μLを加えます。細胞壁を除去し、スフェロプラストを製造するために、30℃で20〜30分間インキュベートします。 </li><li>スフェロプラストは、完全であるかどうかを決定するために顕微鏡スライド上の細胞懸濁液の10μlの液滴をアッセイ。カバーガラスなしで解剖顕微鏡下で観察した細胞。細胞が肥大化し、ラウンドではなく、わずかに楕円形に表示されますと、水20μlを添加した後溶解する必要があります。細胞が溶解するために失敗した場合、水誘導される溶解が容易に観察されるまで10分毎に、30℃にサンプルを返し、再分析。以前のように細胞をペレット化する（ステップ1.2を参照してください）​​。 </li><li>静かに再懸濁し、細胞ペレットを洗浄Inは2.5ミリリットルの冷MES /ソルビトールバッファ。以前のように細胞をペレット化する（ステップ1.2を参照してください）​​。ゆっくり300-400μlの冷MES /ソルビトール緩衝液に細胞ペレットを再懸濁します。細胞を、数時間、この段階で氷の上に保持することができます。 </li></ol>拡散2.染色体注：染色体が拡散さのいずれかのスライドやされる際にガラス表面カバースリップは、必要があります事前に用意すること。各スライドまたはカバースリップを水に沈めなければならず、その後エタノールは、その後、乾燥させ、レンズペーパーで研磨。染色体がカバーガラスに分散されます場合は、カバースリップは、スコッチテープやゴムセメントでスライドに貼付する必要があります。特に言及しない限り、プロトコルの残りはスライドまたはカバースリップ上に広げる説明します<ol><li>きれいなスライドの表面上にP20ピペッター、ピペット細胞懸濁液の20μlのを使用。 </li><li> P200ピペッターを使用して、3％PFA /スクロース溶液40μlを添加し、穏やかにsolutioを混ぜますシュリーレン線が消えるまで手でスライドを「旋回」されることによりn。顕微鏡下でスライドを置き、細胞が焦点になっていることを確認します。 PFA溶液は、使用日のドラフト内で新たに準備する必要があります。 注：目的のタンパク質は、以前の方法により検査されていない場合、それは、タンパク質の保持は、固定条件に敏感であるかどうかを決定するために2及び4％PFAを含む溶液を用いて、追加のスライドを用意することが望ましいです。 4％PFA /スクロース溶液は、それが関連付けられたチューブリンの微小管を可視化することが望ましい場合に使用されます。高い固定剤濃度の欠点は、染色体が「underspread、「2％PFAであろうこともまた「underspread」染色体を生じる傾向があります。 </li><li> P200ピペッターを使用して、できるだけ完全に溶液を混合する前のように旋回、タイマーを開始、1％Lipsolの80μlを添加します。 Lipsolが使用できない場合は、80μlの1％NP40または80μlののdH 2 Oであってもよいです置換された（代表的な結果、図2を参照）。 </li><li>慎重に細胞を見て、ゆっくりと15秒毎のスライドを旋回。細胞の約80％が溶解したら、すぐに顕微鏡からスライドを削除し、タイマーを停止PFA /スクロース溶液80μlを添加し、渦が混合する（消えました）。溶解は、タイマーを起動した後30〜90秒から発生する必要があります。 注：いくつかのスライドがほぼ同時に溶解を示した場合は、1は、最終的には、スライドの残りの微細なモニタリングを省略し、単に慎重lipsolと固定剤の最終アリコートの添加の添加の間の期間を時間を計ることができます。同じサンプルから複数のスライドを調製する場合しかし、この短いカットが唯一の信頼性があります。それは別のスライドを異なる時間に、または異なる培養物から採取した試料から調製されたときに、顕微鏡で各サンプルの溶解を監視するのが最善です。 </li><li>きれいな平らな面にスライドを置き、全体unfrosted surfac全体に液体を広げます「水たまり」のメニスカスの下にはなく、スライド自体の表面上に保持清潔、使い捨て牧草ピペットの側面を使用して、スライドの電子、 すなわち 。ピペットでスライドの表面を「熊手」はありません。サンプルの汚染を避けるために別のスライドにピペットを再使用しないでください。 </li><li>ドラフト内で一晩乾燥さスライドにしておきます。不溶性の核成分は、スライド表面上に定着し、それに結合します。大規模な実験のために、乾燥工程のためのスペースの使用を計画することが重要です。 </li><li>乾燥した後、最適な結果は、同じ日に免疫染色段階に進行することによって得られます。しかし、乾燥したスクロース溶液は、サンプルの凍結を可能にする「蜂蜜」のスプレッド染色体を埋め込む：スライド、プラスチックスライドボックスに移し、年間-20℃で保存することができます。 </li></ol> 3.免疫染色<ol><li>ディップは、30秒間0.2％のフォトフローにスライドします。蜂蜜を除去しました。上リーンスライドエッジは、エッジはペーパータオルで休んで、残留フォトフローを除去しました。 </li><li> 5分間の1×TBSでディップスライドを洗浄します。エッジでスライドを傾斜さ、過剰な液体を除去するが、それらが乾燥させてください。 </li><li>スライドのunfrosted部分全体のスライド上にスライドつや消し面を上にし、ピペット300μlのTBS / BSAを置きます。 </li><li> 15分間、室温で湿ったチャンバー内でインキュベートします。 （飽和ペーパータオルで有孔金属板やプラスチックスライドボックスの下に水飽和ペーパータオルを敷いた大きなプラスチック容器）。 </li><li>ペーパータオル上でスライドの短辺休止して（例えば、試験管ラックなど）適切なサポートに対するスライドを傾斜さスライドを排出します。表面が乾燥しないようにしてください。 </li><li>すぐに一次抗体の適切な希釈を含むTBS / BSA緩衝液80μlのを適用します。抗体が制限されている場合は、40μlの22×22ミリメートルのカバーガラスを使用しています。粗製ウサギ血清の場合、これは、典型的には1/50および1/500希釈の間です。 注意：新しい抗体調製のための滴定を行います。バックグラウンド以上の高い信号を生成する最も希薄準備が選択されています。前免疫血清で染色されるべきバックグラウンド染色のレベルを制御するために、核は適切な欠失変異株および/または重複したスライドから準備する必要があります。 </li><li>バブルを回避スライド上の洗練された22×50mmのカバースリップを置きます。一方の短辺に近い位置での長い縁に沿って親指と人差し指の間にカバーガラスを保持することによってこれを行います。それだけで「水たまり」スライドのつや消しの領域に最も近いのエッジの内側にスライド上に載るまでスライドする30度の角度でカバーガラスを保持し、指の短辺最も遠いが低下します。そして、ゆっくりとスライドタッチベンチを保持する指まで滑らかな動きでカバースリップを下げ、その後、離します。それは着地後にカバーガラスの位置を調整しようとしないでください。 注：染色体スプレッドは上に調製した場合（スライドの表面上に直接ではなく）固定されたカバーガラス、このサンプルカバースリップを染色し、洗浄工程全体にスライドに固定されたままになります。追加のカバーガラスを均一に染色した後、廃棄の際に抗体溶液を分配するために、サンプルカバースリップの上に配置されます。 </li><li>密封された湿ったチャンバー内の場所スライド、4℃で一晩インキュベートします。 </li><li>つや消しのエッジを保持することは、スライドガラスの染色ラックにスライドを転送し、TBSを含む染色ジャーに水没します。 </li><li>静かにカバースリップを削除するには、上下にスライドを浸します。これを行うにはあまりにも多くの力を使用しないようにしてください。 TBSに浸漬することによって、スライド2×10分間洗浄します。ラックからスライドを取り外し、ペーパータオルに端をタッチすることで、過剰な液体を排出します。表面を乾燥させないでください。 </li><li>直ちに蛍光色素結合二次抗体（または22×22ミリメートルのカバーガラス40μL）の1 /千倍希釈を含む80μlのTBS / BSAを追加し、落ち着いた光の下での作業。広告以前とDのカバースリップと暗闇の中で湿ったチャンバー内で2時間4℃でインキュベートします。 </li><li> 、以前のようにカバースリップを削除するスライドを排出し、暗所で1〜2時間空気乾燥さ表面を可能にします。以前のようにペーパータオル上リーンスライドします。 注：拡散がマウントカバースリップ上で実行された場合は、乾燥前のスライドからカバースリップを切り離します。前のステップでのスライドで説明したようにカバーガラスを乾燥されます。 </li><li>抑えた光の下での作業、DAPI（310μlの滴）を含む培地を搭載する約30μlを添加した後、慎重にカバースリップを下げます。スプレッドがカバーガラス上にある場合、スライド上に、下に清潔なスライド上に封入剤の液滴を配置し、カバーガラスを下げ、染色体側。 </li><li>まだ落ち着いた光の下では、明確なマニキュアでカバースリップの端を解決し、密封するためにカバースリップのために2分を待ちます。フラットスライドボックス内にスライドを配置します。 注：STED顕微鏡の場合は、延長ゴールドでマウントします。延長金の30μLを使用DAPIのない、一晩硬化させます。マニキュアで封止することは不要です。 </li><li> DAPIが集中するために、適切なフィルターセットを用いて、40または100倍を目的とした広視野落射蛍光顕微鏡による表示スライド。必要に応じて数週間、4℃で暗所でスライドを保存します。凍結しないでください。 </li></ol>

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The authors declare no competing financial interests.

The appearance of spread nuclei critically depends upon the balance between fixation and lysis/detergent treatment. As discussed above, the preservation of varying cellular structures requires the use of different PFA concentrations. For most proteins, 3% PFA is optimal. However, preservation of spindles requires use of 4% PFA. Even with a single set of reagents, the timing of lysis relative to fixation can also affect the quality of spread nuclei. For the most consistent results, the time of lysis should be normalize...

出芽酵母サッカロマイセス·セレビシエは、染色体の機能を制御するタンパク質の研究を含む、生物学的過程の分子機構の研究のために多くの利点を提供しています。 、遺伝的分子、および生化学的研究のために、出芽酵母のよく知られている利点があるが、細胞の核内のタンパク質の分布の細胞学的研究は、その小さなサイズによって複雑になります。典型的な酵母核は、可視光の約5倍の解像度の限界である1ミクロン未満の直径を有します。したがって、従来の免疫染色から、または緑色蛍光タンパク質（GFP）などの蛍光タンパク質タグを使用することによって得ることができる核タンパク質の分布に関する情報の量が限られています。タンパク質の核内分布を特徴付けるに有用なアプローチは、拡散染色体面です。このアプローチは、細胞壁を除去し、細胞膜および核膜を破壊し、Aを含みますllowing核の不溶性内容は顕微鏡スライドの表面に安定します。これらの不溶性成分は、核マトリックスと染色体が含まれます。クロマチン結合タンパク質を検出する能力を高め、可溶性核内容物の除去を可能にすることに加えて、そのような拡散核が約3〜5μmの直径を有すること（二倍体減数分裂核のための）染色体の実質的な減圧でメソッド結果を拡散染色体と二倍体分裂核のために2〜3ミクロン。この減圧は無傷の核に解決することが比較的困難または不可能である核部分構造の検出を可能にします。 染色体拡散に明らかな欠点は、拡散手順が部分的にまたは完全に目的の構造を破壊する可能性です。特に懸念されるのは、特定の染色体に結合したタンパク質は、拡散処理の結果として失われる可能性があるということです。この潜在的な合併症の寿データを解釈する際にLDが念頭に置いておきます。拡散手順に敏感なタンパク質の一例は、β-チューブリンです。いくつかの条件の下では、主にチューブリンから構成されているスピンドルは、3を広げる時に保存されています。スピンドルの可視化は、多くの場合、目的の核をステージに便利です。しかし、チューブリンを可視化することは、高濃度の固定剤を用いた治療を必要とします。スピンドルは、以下に記載される標準的な条件の下で失われます。この例では、以前に特徴付けられていないタンパク質の分布を分析する場合、それは、タンパク質は、そのような変化に敏感であるかを決定するために、固定剤の濃度を変化させることが重要であることを示しています。染色体構造上の拡散条件の影響に関する懸念にもかかわらず、拡散方式の有用性とパワーは、多くの状況で実証されており、有糸分裂と、特に減数分裂のセル4-7の特性に広い有用性を有します。 TWO拡散方法が広く使用されてきました。ドレッサーとジルー8によって開発されたこれらの方法の第一は、洗剤の使用を回避し、DNA特異的色素DAPIについて染色したときに、比較的よく保存された染色体の形態を有するように見えるスプレッドの準備を得ることができます。しかし、この方法は、完全に比較的困難であり、スライドの一方の領域を他の領域と比較されたときにスプレッド核の品質が、劇的に変化します。この問題は、データ収集の偏りを回避するために、1つのスライドから多くの選択されていない核画像化伴う定量的アプローチを複雑にすることができます。 Loidlとクライン1によって開発された方法を、2次拡散染色体は、洗剤溶液によって促進溶解およびクロマチン解凍して、パラホルムアルデヒドによる固定をバランスすることを含みます。適切に実行すると、この方法は、ドレッサーとジルーの方法に比べて少ない地域への領域の変動と非常に再現性のある結果が得られます。このプレゼンテーションでは、modifieに焦点を当てて理由は、その信頼性とシンプルさのLoidlとクラインの方法、のDバージョン。 拡散染色体は複雑で時間のかかるではありません。最大100のスライドを一日で免疫染色のために調製することができます。また、スプレッド製剤は、免疫染色の前に年間の冷凍庫に保存することができ、したがって、ラボでは新たな生物学的疑問が生じたり、新しい染色試薬が利用可能になったときに使用することができる冷凍染色体スプレッドのリポジトリを開発することができます。 染色体拡散方法は、最も一般的に免疫染色し、広視野蛍光顕微鏡法と組み合わせて使用​​されるが、それは、誘導放出の枯渇（STED）顕微鏡法などの超解像光学顕微鏡法のためにスライドを調製することも可能です。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Zymolyase</td>
			<td>US Biological&#160;</td>
			<td>Z1004</td>
			<td>Prepare 20 mg/mL solution in 50 mM Tris pH 7.5 supplemented with 2% glucose. Prepare fresh each experiment and store at 4&#176;C until ready for use.</td>
		</tr>
		<tr>
			<td>Lipsol&#160;</td>
			<td>L.I.P. Ltd&#160;</td>
			<td>no longer commercially available</td>
			<td>Prepare 1% (v/v) solution in water. Store on ice.</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>USB</td>
			<td>19628</td>
			<td>Prepare 1% (v/v) solution in water. Store on ice.</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Corning</td>
			<td>2948-75x25</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard coverslip</td>
			<td>Fisher</td>
			<td>12-544-E or 12-540-B</td>
			<td></td>
		</tr>
		<tr>
			<td>High resolution coverslips</td>
			<td>Fisher</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo-Flo 200 solution</td>
			<td>Kodak</td>
			<td>P-7417</td>
			<td>Prepare 0.2% (v/v) solution in water.&#160;</td>
		</tr>
		<tr>
			<td>TBS</td>
			<td></td>
			<td></td>
			<td>137 mM NaCl, 2.7 mM KCl, 24.7 mM Tris, pH 8</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td>Prepare a 1% (w/v) solution in TBS. Store at 4&#176;C for up to a month.</td>
		</tr>
		<tr>
			<td>Primary antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Rabbit</td>
			<td>Invitrogen</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG (H+L) Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting media with DAPI</td>
			<td>Vector 
			Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic slide box</td>
			<td>Fisher</td>
			<td>03-448-1</td>
			<td>Store slides containing dried spreads in slots at -20&#176;C. Also, use as a wet chamber.</td>
		</tr>
		<tr>
			<td>Cardboard slide box</td>
			<td>Fisher</td>
			<td>12-587-10</td>
			<td>Use to conveniently transport stained/sealed slides or store at 4&#176;C.</td>
		</tr>
		<tr>
			<td>Coplin jar</td>
			<td>Fisher</td>
			<td>08-816</td>
			<td>Use as a wash basin for slides.&#160;</td>
		</tr>
	</tbody>
</table>

surface spreading and immunostaining of yeast chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

スプレッド核の出現は、批判的に染色体固定およびデコンパクションとの間のバランスに依存します。試薬が適切にバランスしている場合であっても、染色体の脱圧縮の程度の変化および/または別のスライド間の同じスライドの異なる領域で起こり得ます。画像が解釈される前にこのように、スライドの所定の領域に広がるの品質が評価されるべきです。 「overspreading &...

Watch this Scientific Journal Video about Surface Spreading and Immunostaining of Yeast Chromosomes at JoVE.com

Surface Spreading and Immunostaining of Yeast Chromosomes

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

表面拡散と酵母染色体の免疫染色

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

surface-spreading-and-immunostaining-of-yeast-chromosomes

Research

JoVE Journal

Biology

9.8K ビュー.  University of Chicago. A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

ビデオ: Surface Spreading and Immunostaining of Yeast Chromosomes

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

ハイスループット酵母プラスミドの過剰発現の画面

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

生物学

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 ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母のリボソームのクロマトグラフィー精製

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

ディテールに秘められたゼブラフィッシュ胚心臓の免疫染色

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

低親和性細胞外受容体 - リガンド相互作用の検出のためのスケーラブルな親和性ベースの細胞外相互作用のスクリーニング（AVEXIS）

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

自動化を使用して染色体のハイスループット物理マッピング<em&gt;その場で</em&gt;ハイブリダイゼーション

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖や成虫の免疫染色から<em&gt;キイロショウジョウバエ</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

蛍光DNAための簡単​​な方法<em&gt;その場で</em&gt;ぺしゃんこ染色体へのハイブリダイゼーション

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

分裂酵母セクシャルライフサイクルの顕微鏡観察

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

分裂酵母における細胞質分裂のイベントの時空間解析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...