著者は、博士に感謝したい。最初に蛍光顕微鏡でLASを訓練するためのPhilina S.リーとアランD.グロスマン。著者らはまた、博士に感謝します。ヘルプとイメージングのためのヒントについてはメラニーBerkmenと肇小林。この作品は、文学、科学芸術の大学から、ミシガン大学、分子細胞、および発生生物学の部門からの資金投​​入を開始することによってサポートされていました。

顕微鏡の細胞の成長文化 1。一または二日には、イメージングの前に、Bを用意する枯草菌では、可視化したい翻訳融合タンパク質を含む菌株。ステップ1Aおよび1Bの試験のラウンドは、成長条件があなたの株の最高の画像を提供しているかを判断するために必要となります。ほとんどの場合、細胞は対数増殖期の間に撮像することがあります。 <ol class="ualpha"><li>いくつかのBの枯草菌の菌株（特に、不完全にその内因性遺伝子座での関心とGFPの遺伝子との間の翻訳融合の統合による成長株）、画像への2日前の初期成長は、高品質の画像のために必要です。初日に、ストリークB.枯草菌は、抗生物質（複数可）を含むLB寒天培地上に結像されるひずみと30℃で一晩インキュベート二日目に、単一のコロニーを0.85％生理食塩水100μlを接種し、30℃で一晩インキュベートした各希釈液100μLをめっきLB寒天プレート上で3つの10倍段階希釈液（選択的な抗生物質）、、℃を実行三日目に、細胞の光密集成長による希釈プレートを選択します。板上に光密集成長が潜在的に（ステップ4を参照）、優れた画像処理結果を得るために、健康、指数関数的に成長しているスターター接種した培地を提供します。このステップでは、染色FM4 - 64で最高品質の膜のイメージングのために特に有用である。 </li><li>他のB.枯草菌の菌株は、単に無菌スティックイメージングの前に一日で、単一のコロニーのための選択LB寒天培地に塗布され、30℃で一晩インキュベートすること翌日、このプレート上の細胞は、（ステップ4を参照）イメージングのための接種物として使用することは十分になります。 注：Bの枯草菌は 、液体培地で一晩培養では起因する遅滞期の成長 1 の長期間の原因となる胞子形成とクォーラムの応答、の組み合わせに適しているわけではありません 。 </li></ol> 2。イメージング、125mLの滅菌三角フラスコにS7 50 1から3培地の代わりに10mLの日の朝。 3。接種を得るために、単一コロニーまたは光コンフルエントの細胞を含むLB寒天プレートからS7 50培地2 mLまでの細胞を再懸濁します。 4。 0.1〜〜0.08の600nmで測定された初期の光学密度（OD 600）との文化のための10mLのS7 50培地を入れた三角フラスコに十分なS7 50中期細胞接種を追加。このステップでは、それは少なくとも10倍空気媒質の比にしておくことが重要です。従って、我々は125 mLの三角フラスコに接種した培地を10〜12.5mlのを使用してください。 5。文化は、OD 600を 〜0.5から0.8に達するまで30℃でそれぞれの文化を育てる。揺れ水浴を150から200までから毎分回転数で好ましい。 DNA損傷または不整合誘導剤で攻撃の文化は（表1参照）初期の指数増殖期細胞は、治療のために必要とされる成長の追加の期間でOD 600のターゲットに到達するようにOD 600になっているはず 例えば、2 -アミノプリンは、治療の一時間を必要とするため、2 -アミノプリンで治療された文化は、OD 600になっているはず〜0.3から0.4まで、このようなOD 600の治療/成長の結果の一時間いる〜0.5から0.8、それによって細胞がイメージングのための準備が整いました。 サンプル準備 1。ピペット1.5 mlマイクロ遠心チューブに培養細胞を300μl。 2。細胞膜のイメージングが必要な場合は、1 mg / mLの原液から各サンプルに汚れFM4 - 64膜（Invitrogen社）の1:1000希釈を追加。 FM4 - 64の滴定は、所望の蛍光信号を達成するために必要な場合があります。典型的な滴定の範囲は1:100、1:1000、および1:10,000です。 3。サンプルは最大10分間、室温で座ることを許可またはスライドは以下に述べるように培地1X Spizizensの1％アガロース（&quot;スライドの準備&quot;​​のステップ1を参照）を用いて調製されている間。 4。細胞の濃度が必要な場合、我々は、真空装置と、0.2μmフィルターを用いてフィルタリングセルを好む。その後、細胞を穏やかにPBS、別の適切な緩衝液または可視化に先立って培地とフィルターの表面から洗浄することができます。 スライドの準備 1。 1X Spizizensを1％アガロースを準備します。 5 0.5 Gアガロースと共に10X SpizizensストックのmLの45ミリリットルddH2Oに、そして溶融にマイクロ波を追加。ホット溶融アガロースは、ピペッティングすることは困難であり、アガロースがピペットの先端を入力しません固化。アガロースが〜55から65までの温度° Cまで、それは内に固化せずにピペットチップを入力するための水浴中で平衡化してください<html>先端。 2。 1％はピペットの先端にアガロースを融解とSpizizensの20μlを描く。ピペット浅いアガロースパッドを形成するために、各スライドにアガロース溶液の一滴（約1〜2μL）も（我々は15ウェルスライドを使用して、表2を参照）。アガロースは、すぐに固化される。スライドは、細胞のアプリケーションの直前に行​​う必要があります。アガロースパッドを単独で室温で放置されている場合、それらは数分で脱水し、使用できなくなることがあります。 アガロースパッドが中央にしても、それぞれを埋めるがカバースリップの中断を回避するために最小の高さを持っている必要があります。、液滴が高すぎる（バブル的）またはサンプルはうまく境界を越えて広がる場合キムワイプでよくきれいなだけでスワイプして、適用新しいアガロースパッド。 3。各アガロースパッドの上に分散し、スライド全体を300μlのサンプルを（ステップ6を参照）を適用します。細胞のこの量は、スライド上の各ウェルをカバーする十分なはずです。 4。約10分間（23℃）、室温で座っているセルを搭載したスライドを許可する。このステップでは、アガロースパッドが撮像のために動かないで準備に落ち着くために細胞が可能になります。特別なニーズのためにこの温度は、温度シフト4を必要とする実験を行う際に、例えば、変更が必要な場合があります。 5。パッド自体を削除せずに、各ウェルからの過剰なメディアを離れて吸引除去する。 6。スライドの領域を越え、しっかりと均等に、かつ慎重に押して、スライドにカバースリップを適用します。カバーガラスがスライドに対して、カバースリップがスライド上に上げていないことが確認してください。 細胞の可視化 1。多くの異なる蛍光顕微鏡で問題なく動作します。それは、100X油浸レンズを持つことが重要です。シモンズラボは、使用しています。 <ul><li> 1,45 NA TIRFM 100Xの油浸対物レンズを搭載したオリンパスBX61顕微鏡</li><li>浜松ORCAR 2 CCD冷却カメラ</li><li>ルーメン200アークの金属（前）光源。 </li><li> GFP（FITC）、フィルタ励起460から500と放射510から560を検出するための</li><li> FM4 - 64の検出（TRITC）に対して、フィルタ励起510〜560および発光572〜648。 </li><li>画像はSlideBook 4.2（図1）を使用して捕獲された</li></ul> 2。白色光を使用してフォーカスに細胞をもたらす。 3。各蛍光体のために適切なフィルタを使用してイメージをキャプチャします。 代表的な結果代表的なイメージが（図1）が表示されます。 FM4 - 64染色4-7を明るくクリアになるはずですがGFP病巣は、明確に定義する必要があります。 GFPまたは膜の画像は、データを失うことなく、提示される最高品質の画像を可能にするために、任意の色を持つ擬似着色することができます。 <img src="/files/ftp_upload/1736/1736fig1.jpg" alt="図1" /> 図1：Bの代表的なGFPの画像 FM4 - 64膜の染色が赤で表示されている間、 枯草菌の翻訳融合タンパク質を。GFPタンパク質は、緑色で表示されます。白いスケールバーは3μmを示している（）MutS - GFP [関連する遺伝子型：mutS - GFP（SPC）、amyE：：Pspac mutL（猫 ）] 600μg/ mLの2 -アミノプリンおよび1mMのIPTGの存在下で行われる。 FM4 - 64信号がより明確にMutS - GFP病巣を示すために提示されない（B）MutL - GFP [関連する遺伝子型：mutL：：mutL - GFP（SPC）]。。2 -アミノプリンの存在下で（C） DnaX - GFP [関連する遺伝子型：dnaX：：dnaX - GFP（SPC）]。 （D）のRecA - GFP [関連する遺伝子型：recAの：：recAの- GFP（SPC）] 20 ng / mLのマイトマイシンCの（E）TAGC - GFP [関連する遺伝子型の存在下で：TAGC：：TAGC - GFP（SPC） 】1μg/ mLのマイトマイシンCの存在下で<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td>治療/濃度または量</td><td>機能</td><td>インキュベーションの時間</td></tr><tr><td>マイトマイシンC（MMC）[20〜150 ng / mLの] </td><td> DNAアルキル化剤</td><td> 1時間</td></tr><tr><td> 2 - アミノプリン（2 - AP）[600μg/ mLの] </td><td>ミスマッチの誘導</td><td> 1時間</td></tr><tr><td>ヒドロキシウレア（HU） </td><td> dNTPを枯渇させる</td><td> 3時間</td></tr><tr><td>紫外線（UV）20 J/m2 </td><td>チミン - チミンダイマーと6-4光産物</td><td> 20秒通常は、ソースによって異なります</td></tr><tr><td>グレー（電離放射線）5〜100 Gyの</td><td>二本鎖切断、一本鎖切断及び塩基損傷部位</td><td>ソースによって異なります</td></tr></tbody></table>DNA損傷とミスマッチ誘導治療の &quot;&gt; 表1。サマリー <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能） </td></tr><tr><td> Multitest社スライド、15ウェル</td><td> MPのバイオメディカル</td><td> 6041505E </td><td></td></tr><tr><td>顕微鏡のカバーガラス</td><td>フィッシャー</td><td> 12から544 - B </td><td></td></tr><tr><td>アガロース</td><td>フィッシャー</td><td> BP160 - 500 </td><td></td></tr><tr><td>マイトマイシンC </td><td>シグマ</td><td> M0503 - 2mgを</td><td>変異原性、摩耗の手袋</td></tr><tr><td> 2 - アミノプリン</td><td>シグマ</td><td> A3509 - 250mgの</td><td>手袋</td></tr><tr><td>ヒドロキシウレア</td><td>シグマ</td><td> H8627 - 25G </td><td>手袋</td></tr><tr><td> FM4 - 64 </td><td>インビトロジェン</td><td> T13320 </td><td>敏感な光</td></tr></tbody></table> 表2。特定の試薬 ​​と機器のリスト：

<ol>
	<li>Hardwood, C. R., Cutting, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biological Methods for Bacillus. , (1990).</li><li>Berkmen, M. B., Grossman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+organization+of+the+Bacillus+subtilis+replication+cycle.">Spatial and temporal organization of the Bacillus subtilis replication cycle.</a> Mol. Microbiol. 62, 57-71 (2006).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clp+and+Lon+proteases+occupy+distinct+subcellular+positions+in+Bacillus+subtilis.">Clp and Lon proteases occupy distinct subcellular positions in Bacillus subtilis.</a> J. Bacteriol. 190, 6758-6768 (2008).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+is+required+for+the+RecA+localization+response+to+DNA+damage+in+Bacillus+subtilis.">Replication is required for the RecA localization response to DNA damage in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U S A. 104, 1360-1365 (2007).</li><li>Simmons, L. A., Davies, B. W., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=b+Clamp+Directs+Localization+of+Mismatch+Repair+in+Bacillus+subtilis.">b Clamp Directs Localization of Mismatch Repair in Bacillus subtilis.</a> Mol. Cell. 29, 291-301 (2008).</li><li>Simmons, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+responses+to+double-strand+breaks+between+Escherichia+coli+and+Bacillus+subtilis+reveals+different+requirements+for+SOS+induction.">Comparison of responses to double-strand breaks between Escherichia coli and Bacillus subtilis reveals different requirements for SOS induction.</a> J. Bacteriol. 191, 1152-1161 (2009).</li><li>Smith, B. T., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+mismatch+repair+in+bacterial+cells.">Visualization of mismatch repair in bacterial cells.</a> Mol. Cell. 8, 1197-1206 (2001).</li></ol>

試行錯誤は、各株の最高品質の画像の露光条件を見つけるために必要とされ、我々は100〜2000ミリ秒のエクスポージャーはGFP（FITC）とFM4 - 64（TRITCに適している間に1ミリ秒は、白色光の画像に適していることを見つける）のイメージ。露光時間は、使用するイメージング機器によって異なります。複数の系統が同じスライド上に存在する場合、パッドの国境から細胞のパッドの品質と拡散は、...

特定のソリューションのレシピ1： 10倍S7 50塩 0.5 MのMOPS 100mMの硫酸アンモニウム 50mMのリン酸二水素カリウム 滅菌フィルター処理、および保管前にラップS7箔で50メディア 100xの金属 0.2 MのMgCl 2 70 mMのCaCl 2を 5mMのMnCl 2を 0.1mMのZnCl 2を 100μg/ mlのチアミン塩酸 2mMの塩酸 0.5mMのFeCl 3の * 最終的なボリュームへのdH 2 O *のFeCl 3を沈殿を防ぐために、最後に追加する必要があります。 箔のフィルターの滅菌後、ラップ100 ×メタルソリューション。 S7 50メディア 1X S7 50塩 1X金属 1％のグルコース 0.1％グルタミン酸 40μg/ mLのトリプトファン 40μg/ mLのフェニルアラニン 最終的なボリュームに蒸留H 2 O 10倍Spizizens（グラム/ L） 151.4 mMの硫酸アンモニウム（20グラム/ L） 803.8 mMのリン酸二水素カリウム（140グラム/ L） 440.9 mMリン酸カリウム二塩基性（60グラム/ L） 34.0 mMクエン酸ナトリウム（10グラム/ L） 16.6 mMのMgSO 4を （2G / L） 最終的なボリュームへのdH 2 O 滅菌フィルタリング

imaging mismatch repair and cellular responses to dna damage in bacillus subtilis

原核生物と真核生物の両方の生理的変化の複雑なセットを介してDNA損傷に応答する。遺伝子発現、既存の蛋白質の再分配、及び新たなタンパク質複合体のアセンブリの変化は、DNAの損傷とミスマッチDNAの塩基対の種々の方法によって刺激することができる。蛍光顕微鏡は、DNA損傷にと生きている細胞の複雑な細胞内のアーキテクチャの中でDNAの複製の状態を監視するために、これらおよび他の応答を可視化し定量化するための強力な実験ツールとして用いられている。蛍光レポータータンパク質とDNA複製と修復機構のコンポーネント間の翻訳融合は、手がかりを決定するために使用されているその同族病変の標的DNA修復タンパク質<em&gt; in vivoで</em&gt;そしてこれらのタンパク質が細菌細胞内で編成されているかを理解する。さらに、DNA損傷誘導性プロモーターに連結された転写と翻訳融合は、集団内の細胞は遺伝毒性ストレス応答を活性化されているいるを明らかにした。このレビューでは、我々は、画像にDNAの修復と、単一の細菌細胞のDNA複製複合体のアセンブリを蛍光顕微鏡を使用するための詳細なプロトコルを提供しています。特に、この作品は、イメージングのミスマッチ修復タンパク質、相同組換え、DNA複製とSOS誘導性蛋白質の焦点を当て<em&gt;枯草菌</em&gt;。ここで説明する手順のすべては、細菌種の様々なイメージングのタンパク質複合体に対して容易に適している。

Watch this Scientific Journal Video about Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis at JoVE.com

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

詳細なプロトコルは、イメージングのためのDNA修復複合体のリアルタイムの形成を説明しています<em&gt;枯草菌</em&gt;細胞。

イメージングのミスマッチ修復とのDNA損傷に対する細胞応答枯草菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

imaging-mismatch-repair-cellular-responses-to-dna-damage-bacillus

Research

JoVE Journal

Biology

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

10.9K ビュー.  University of Michigan-Ann Arbor. 詳細なプロトコルは、イメージングのためのDNA修復複合体のリアルタイムの形成を説明しています枯草菌細胞。

ビデオ: Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA損傷応答タンパク質の2次元および3次元生細胞イメージング

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

生物学

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

レシオメトリック二分子ビーコンを使用して、生細胞における単エンジニアのRNA転写物のリアルタイムイメージング

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

の全配列のNGS解析のためのトランスポゼースベースの技術によるgDNAのエンリッチメント<em&gt; BRCA1</em&gt;<em&gt; BRCA2</em&gt;、およびDNA損傷修復に関与する遺伝子9

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

電子分光イメージングと組み合わせたminiSOGタグ付きのDNA修復タンパク質の可視化（ESI）

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

慢性後のマウス脾細胞におけるDNA損傷と修復の測定<em&gt;インビボ</em&gt;β-およびγ-放射線の非常に低い用量への暴露

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

ラットの肝臓におけるDNA鎖切断および酸化的DNA損傷を測定するためのインビボアルカリコメットアッセイおよび酵素修飾されたアルカリコメットアッセイで

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

有害な効果の低圧プラズマ滅菌を使用して住んでいる携帯顕微鏡枯草菌胞子の生存に調査

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

枯草菌の単一細胞マイクロ流体解析

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

エンドヌクレアーゼベースの部位特異的DNA損傷の可視化と定量化

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

イン・ヴィトロ E3ユビキチンリガーゼ関数の解析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...