私たちは、透過型電子顕微鏡で助けステファンウィルキンスに感謝。この作品は、健康グラントR01AG023731の国立研究所によってサポートされていました。

これまでの研究では、Mgm101の最初のアミノ末端22残基がミトコンドリア19へのインポート後に切断されることが示されている。 大腸菌における発現のために、最初の22コドンを欠くMGM101オープンリーディングフレームをPCRで増幅された発現ベクター物pMAL-C2Eの修正版でマルトース結合タンパク質（MBP）をコードする雄配列の下流に配置された。これは、プレシジョンプロテアーゼ（ 図3）のための切断部位を含むリンカーとMBP-Mgm101融合を生成します。プラスミドは、第E.を選択することにより構成されているIPTGおよびXgal白/青選択せずに形質転換体。得られたプラスミド物pMAL-C2E-MGM101その後、E.に導入され、アンピシリンとクロラムフェニコール耐性コロニーを選択することにより、 大腸菌株BL21-CodonPlus（DE3）-RIL。 1。表現、誘導、細胞溶解とのDNase I処理<ol><li> INOCグルコース（0.2％）、アンピシリン（100μg/mlの/ ml）およびクロラムフェニコール（50μgの/ ml）を添加したLB培地10ml（1％バクトトリプトン、0.5％酵母エキス、1％NaCl）中に新鮮な形質転換体をulate。 200 rpmで振とうしながら37℃CO / Nでインキュベートする。 </li><li>上記のように添加したLB培地を2リットルに10ミリリットルの前培養に接種。 OD 600が 0.5に達するまで振とうしながら37℃で細胞を成長させる。 </li><li> 0.3mMの最終濃度イソプロピルβ-D-1-チオガラクトピラノシド（IPTG）を添加することによりMBP-Mgm101融合タンパク質の発現を誘導する。 5時間30℃で振とうしながら細胞を育てる。 </li><li>ベックマンJA-10ローター（5,500×gで、4℃、10分）を使用して、遠心分離により細胞を回収。上清を廃棄した後に、プロテアーゼ阻害剤を含む溶解緩衝液（20mMのトリス-HCl、pHが7.4および1mM EDTA、pH7.4）中（25μMロイペプチン、1μMのペプスタチン及び1mM PMSF）30ml中に細胞ペレットを再懸濁。 </li><li> 20氷上で細胞懸濁液を超音波処理秒超音波プロセッサ（熱機関;モデルW-385）を用いて50％のデューティサイクルで、30秒間、氷上で冷却し、そして2×繰り返すことができる。 </li><li> 2 mg / mlのDNアーゼで1株を1 mlを加える。 2時間4℃で細胞溶解物を揺する。 </li><li> 500mMの最終濃度のNaClを調整します。 </li><li> 30分間、4℃で10,000×gで遠心分離することにより細胞の破片を取り除きます。 </li></ol> 2。アミロースアフ​​ィニティークロマトグラフィーによる精製<ol><li>溶解緩衝液中のアミロース樹​​脂を1.5ml（50％スラリー）を平衡化する。細胞ライセートに平衡化アミロース樹​​脂を追加します。 4℃のCO / Nで優しく揺らし</li><li>寒い部屋に設置エコノカラムクロマトグラフィーカラムにライセート樹脂ミックスをロードします。非結合タンパク質は重力によって列を渡すことができます。 </li><li>洗浄バッファーI（; 400のNaCl、1mMのEDTA、pHが7.4、および0.2 mMのPMSF、20mMのトリス-HCl、pH7.4）で300mlでアミロース樹​​脂を洗ってください。 </li><li>洗浄バッファーII（20mMのT 150mlで洗浄を繰り返し立上り - 塩酸、pHが7.4、NaClが200 mM、1 mMのEDTA、pHは7.4であり、0.2 mMのPMSF）。 </li><li>カラムに、4℃で10分間インキュベートする、収集溶出緩衝液5mlの（; 200mMのNaClを、図1 mMのEDTA、pHが7.4および0.2 mMのPMSF、10mMのマルトース20mMのトリス-HCl、pHが7.4）を追加もっと回溶出を繰り返し。 </li><li>溶出液を組み合わせ、クマシー染色（ 図4）に続いて、SDS-PAGEゲル上で溶出液のアリコートをロードすることにより、MBP-Mgm101の収率および純度を決定します。 </li></ol> 3。プレシジョンプロテアーゼ切断及び陽イオン交換クロマトグラフィー<ol><li> MBP-Mgm101溶出液にプレシジョンプロテアーゼの50単位を追加します。 4℃での切断を行うCO / Nし、透析緩衝液に対して透析中継続することができ（20mMのトリス-HCl、pH7.4の、100mMのNaCl、2mMのDTT、1mMのEDTA、pHは7.4であり、0.2 mMのPMSF） </li><li> SDS-PAGEゲル（ 図5A）の切断の効率を確認してください。 </li><li>複数の注入によって切断MBP-Mgm101を読み込むバイオスケールミニマクロプレップハイSカートリッジのイオンは、Bio-Rad社の生物学的DuoFlow FPLCシステムに接続。 </li><li> 300mMの、5mMの工程の塩勾配を適用することによって、陽イオン交換クロマトグラフィーを開始し、500mMの、混合液（5 mMの塩化ナトリウムによって作成され750mMのNaClを、1,000 mMの、10mMのリン酸ナトリウム、pH7.2の図1 mMのPMSF）とバッファーB（1MのNaCl、10mMのリン酸ナトリウム、pHは7.2、1mMのPMSF）。 0.5ml /分で、流量を設定してください。 Mgm101画分を回収し、SDS-PAGEゲル（ 図5B）上のタンパク質の純度を確認してください。 </li></ol> 4。サイズ排除クロマトグラフィー<ol><li>陽イオン交換クロマトグラフィーからのタンパク質画分を兼ね備えています。 GF平衡緩衝液（; 150mMのNaCl、1mMのEDTA、pH7.4の、5mM 2 - メルカプトエタノール、0.2mMのPMSFを20mM MOPS、pH7.0）中でのタンパク質の透析。 </li><li> 4℃で3,000 xgで回転させて〜1ミリリットルの容積を減らすためにビバスピン15Rの限外ろ過スピンカラムにタンパク質を集中</li><li> Mgm101 OのロードNAはクロマトグラフィバッファー（; 150mMのNaCl、5mMのβ-メルカプトエタノール、1mMのEDTA、pH7.4の、0.2 mMのPMSFを20mM MOPS、pH7.0）で平衡化したスーパーロース6プレップグレードカラムをキャリブレーション。 </li><li> 0.5ml /分の流速でクロマトグラフィーを実行する緩衝液を用いてサイズ排除を開始する。 </li><li>精製Mgm101ピークの画分を集め。タンパク質の最終品質をチェックするための12％SDS-PAGE上の画分のアリコートをロードします。 </li><li>ビバスピン6タンパク質を集中し、その後速やかに-80℃で液体窒素とストアでサンプルを凍結℃に</li><li>一本鎖DNA結合アッセイ（ 図8）および透過型電子顕微鏡（ 図9）による構造可視化のための6-12カ月以内にMgm101サンプルを使用。 </li></ol>

<ol>
	<li>San Filippo, J., Sung, P., Klein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+eukaryotic+homologous+recombination.">Mechanism of eukaryotic homologous recombination.</a> Annu. Rev. Biochem. 77, 229-257 (2008).</li><li>Bishop, D. K., Park, D., Xu, L., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMC1:+a+meiosis-specific+yeast+homolog+of+E.+coli+recA+required+for+recombination,+synaptonemal+complex+formation,+and+cell+cycle+progression.">DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression.</a> Cell. 69, 439-456 (1992).</li><li>Shinohara, A., Ogawa, H., Ogawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad51+protein+involved+in+repair+and+recombination+in+S.+cerevisiae+is+a+RecA-like+protein.">Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.</a> Cell. 69, 457-470 (1992).</li><li>Passy, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Dmc1+protein+binds+DNA+as+an+octameric+ring.">Human Dmc1 protein binds DNA as an octameric ring.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 10684-10688 (1999).</li><li>Story, R. M., Weber, I. T., Steitz, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+E.+coli+recA+protein+monomer+and+polymer.">The structure of the E. coli recA protein monomer and polymer.</a> Nature. 355, 318-325 (1992).</li><li>Yu, X., Jacobs, S. A., West, S. C., Ogawa, T., Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+structure+and+dynamics+in+the+helical+filaments+formed+by+RecA+and+Rad51+on+DNA.">Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 8419-8424 (2001).</li><li>Conway, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+Rad51+filament.">Crystal structure of a Rad51 filament.</a> Nat. Struct. Mol. Biol. 11, 791-796 (2004).</li><li>Bai, Y., Davis, A. P., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+allele+of+RAD52+that+causes+severe+DNA+repair+and+recombination+deficiencies+only+in+the+absence+of+RAD51+or+RAD59.">A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59.</a> Genetics. 153, 1117-1130 (1999).</li><li>Bai, Y., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rad52+homolog+is+required+for+RAD51-independent+mitotic+recombination+in+Saccharomyces+cerevisiae.">A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae.</a> Genes Dev. 10, 2025-2037 (1996).</li><li>Paques, F., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathways+of+recombination+induced+by+double-strand+breaks+in+Saccharomyces+cerevisiae.">Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae.</a> Microbiol. Mol. Biol. Rev. 63, 349-404 (1999).</li><li>Lopes, A., Amarir-Bouhram, J., Faure, G., Petit, M. A., Guerois, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+novel+recombinases+in+bacteriophage+genomes+unveils+Rad52,+Rad51+and+Gp2.5+remote+homologs.">Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs.</a> Nucleic Acids Res. 38, 3952-3962 (2010).</li><li>Poteete, A. R., Sauer, R. T., Hendrix, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+structure+and+quaternary+organization+of+the+bacteriophage+P22+Erf+protein.">Domain structure and quaternary organization of the bacteriophage P22 Erf protein.</a> J. Mol. Biol. 171, 401-418 (1983).</li><li>Passy, S. I., Yu, X., Li, Z., Radding, C. M., Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rings+and+filaments+of+beta+protein+from+bacteriophage+lambda+suggest+a+superfamily+of+recombination+proteins.">Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 4279-4284 (1999).</li><li>Ploquin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+basis+for+a+bacteriophage+homolog+of+human+RAD52.">Functional and structural basis for a bacteriophage homolog of human RAD52.</a> Curr. Biol. 18, 1142-1146 (2008).</li><li>Chen, X. J., Guan, M. X., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MGM101,+a+nuclear+gene+involved+in+maintenance+of+the+mitochondrial+genome+in+Saccharomyces+cerevisiae.">MGM101, a nuclear gene involved in maintenance of the mitochondrial genome in Saccharomyces cerevisiae.</a> Nucl. Acids Res. 21, 3473-3477 (1993).</li><li>Meeusen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mgm101p+is+a+novel+component+of+the+mitochondrial+nucleoid+that+binds+DNA+and+is+required+for+the+repair+of+oxidatively+damaged+mitochondrial+DNA.">Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA.</a> J. Cell Biol. 145, 291-304 (1999).</li><li>Mbantenkhu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mgm101+is+a+Rad52-related+protein+required+for+mitochondrial+DNA+recombination.">Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.</a> J. Biol. Chem. 286, 42360-42370 (2011).</li><li>Nardozzi, J. D., Wang, X., Mbantenkhu, M., Wilkens, S., Chen, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+properly+configured+ring+structure+is+critical+for+the+function+of+the+mitochondrial+DNA+recombination+protein.">A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein.</a> Mgm101. J. Biol. Chem. 287, 37259-37268 (2012).</li><li>Zuo, X., Xue, D., Li, N., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+core+of+the+mitochondrial+genome+maintenance+protein+Mgm101p+in+Saccharomyces+cerevisiae+determined+with+a+temperature-conditional+allele.">A functional core of the mitochondrial genome maintenance protein Mgm101p in Saccharomyces cerevisiae determined with a temperature-conditional allele.</a> FEMS Yeast Res. 7, 131-140 (2007).</li><li>Itoh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+packaging+proteins+Glom+and+Glom2+coordinately+organize+the+mitochondrial+nucleoid+of+Physarum+polycephalum.">DNA packaging proteins Glom and Glom2 coordinately organize the mitochondrial nucleoid of Physarum polycephalum.</a> Mitochondrion. 11, 575-586 (2011).</li><li>Janicka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+RAD52-like+single-stranded+DNA+binding+protein+affects+mitochondrial+DNA+repair+by+recombination.">A RAD52-like single-stranded DNA binding protein affects mitochondrial DNA repair by recombination.</a> Plant J. 72, 423-435 (2012).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

これは、Eから安定した、ネイティブ換えMgm101タンパク質を産生するために課題となっている細菌細胞におけるその不溶性が原因の可能性大腸菌 。本稿では、MBP融合戦略はタンパク質が適度に高いレベルで発現されることができることを示している。ネガティブ染色透過型電子顕微鏡およびサイズ排除クロマトグラフィーを用いて、我々は以前にMBP-融合タンパク質は、インビ...

相同組換えは、二本鎖切断（DSBの）と鎖間架橋の修復のための、および縮小複製フォーク1からDNA複製の再開始のために重要である。従来の相同組換えでは、中央の反応は、原核生物においてRecAタンパク質、及びRad51の1-3および真核生物におけるDMC1含むATP依存性リコンビナーゼにより触媒される。これらのリコンビナーゼは、二本鎖DNAテンプレート（ 図1、左パネル）4-7内の相同性検索と鎖侵入を開始するために不可欠である一本鎖DNA上で核タンパク質フィラメントを形成。従来方式に加えて、相同組換えはまたRecA/Rad51-independent方法（ 図1、右パネル）で行うことができる。例えば、酵母RAD52とRad59タンパク質は直接二本鎖DNA切断の切除によって公開され、相補鎖DNA鎖のアニーリングを触媒することができます。歌うと呼ばれるこの再結合過程、ル鎖がアニーリング、一般的に相同な二本鎖DNAテンプレートとのペアリングは含まれません。アニール後、異種尾はエキソヌクレアーゼとニックゲノム継続8-10を復元するために連結されることにより除去される。一本鎖アニーリング機構により修復は、しばしば直接繰り返さ地域間のゲノム配列の欠失を伴っている。 RAD52はバクテリオ11の間で普及している組換えタンパク質の多様なグループに属しています。これらのタンパク質はまた、相同な一本鎖DNA分子のアニーリングを促進するそれらの活性に基づいて、一本鎖アニーリングタンパク質（SSAPs）として知られている。最高の特徴バクテリオファージSSAPsはRedβとラクトコッカスファージul36からプロファージRACおよびサックタンパク質からバクテリオファージλとP22、RecTをからErfにある。類似性が事実上undetectですがSSAPsは、構造的、典型的なβ-β-β-α倍によって特徴付けられる彼らの主要なシーケンスでできる。 10彼らは、すべてのフォーム大ホモオリゴマーリング- 体外 12-14 で 14回対称。この特徴的な高次構造、組織の機能的な意味はよく理解されていない。 私たちは、ミトコンドリアゲノムに相同組換えのメカニズムを理解することに興味を持っています。我々は以前に、サッカロミセス·セレビシエ 15内のmtDNAの維持に必須であるMGM101遺伝子を同定した。MGM101は 、その後、ミトコンドリア核様体と関連することが見出され、DNA損傷剤に対するミトコンドリアの許容16に必要とされる。しかし、Mgm101の研究は、組換えMgm101を生産する難しさによって10年で戻って開催されています。我々は最近、E.から大量に可溶Mgm101を生産することに成功しましたMBP融合戦略を使用して大腸菌 。これは、Mgm101株式を発揮することができましたタンパク質17,18のRAD52-家族と生化学的および構造的類似性。本報告では、三段階精製法は均質Mgm101for生化学的および構造解析します（ 図2）を生成し、これに記載されています。

<table><tbody><tr><td>Expression vector pMAL-c2E</td><td>New England Biolabs</td><td>#N8066S</td><td></td></tr><tr><td>PreScission Protease</td><td>GE Healthcare Life Sciences</td><td>#27-0843-01</td><td></td></tr><tr><td>BL21-CodonPlus(DE3)-RIL cells</td><td>Strategene</td><td>#230245</td><td></td></tr><tr><td>Leupeptin</td><td>Roche Applied Science</td><td>#11034626001</td><td></td></tr><tr><td>Pepstatin</td><td>Roche Applied Science</td><td>#11359053001</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Roche Applied Science</td><td>#10837091001</td><td></td></tr><tr><td>DNAse I</td><td>Sigma</td><td>#DN25-1G</td><td></td></tr><tr><td>Isopropyl &amp;#946;-D-1-thiogalactopyranoside (IPTG)</td><td>Roche Applied Science</td><td>#11411446001</td><td></td></tr><tr><td>Amylose resin</td><td>New England Biolabs</td><td>#E8021L</td><td></td></tr><tr><td>Econo-Column chromatography column</td><td>BIO-RAD</td><td>#7372512</td><td></td></tr><tr><td>Bio-Scale Mini Macro-Prep High S cartridge (1 ml)</td><td>BIO-RAD</td><td>#732-4130</td><td></td></tr><tr><td>VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO)</td><td>Sartorius Stedium</td><td>#VS15RH02</td><td></td></tr><tr><td>Superose 6 prep grade column</td><td>Amersham Bioscirnces</td><td>#17-0489-01</td><td></td></tr><tr><td>VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO)</td><td>Sartorius Stedium</td><td>#VS0611</td><td></td></tr></tbody></table>

preparation of the mgm101 recombination protein by mbp-based tagging strategy

MGM101遺伝子はミトコンドリアDNAの維持におけるその役割のために20年前に同定された。いくつかのグループからの研究がMgm101タンパク質は、ミトコンドリアDNAの組換え修復に関与することが示唆されている。最近の研究は、Mgm101がRAD52型組換えタンパク質ファミリーに関連することが示されている。これらのタンパク質は、大きなオリゴマーの環を形成し、相同な一本鎖DNA分子のアニーリングを促進する。しかし、Mgm101の特徴は、組換えタンパク質を産生することが困難によって妨げられてきた。ここでは、組換えMgm101の調製のための信頼性のある手順を説明する。マルトース結合タンパク質（MBP）タグ付きMgm101は、第大腸菌で発現される。融合タンパク質は、最初アミロースアフ​​ィニティークロマトグラフィーにより精製する。タンパク質分解切断によって放出された後、Mgm101は、陽イオン交換クロマトグラフィーによってMBPから分離される。単分散Mgm101その後、得られたサイズ排除クロマトグラフィーによる。細菌培養のリットルあたりMgm101の〜0.87ミリグラムの収量は日常的に得ることができる。換えMgm101は、DNAの最小の汚染されています。調製したサンプルが正常にMgm101の、生化学的構造及び単粒子像分析のために使用される。このプロトコルは、ミスフォールドおよび細菌細胞に対して毒性することができる他の大きなオリゴマーDNA結合タンパク質の調製のために使用することができる。

Mgm101はミトコンドリアにおけるRAD52関連の組換えタンパク質である。 RAD52が広くミトコンドリアDNA組換え（ 図1）におけるその役割のために研究されてきた。組換えMgm101は、3段階の手順（ 図2）により調製することができる。これは、Mgm101が可溶型で発現することができ、その後、タンパク質分解的切断（ 図3）により、タグから放出MBP-タグ付け戦略の?...

Watch this Scientific Journal Video about Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy at JoVE.com

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

酵母ミトコンドリア核様体タンパク質、Mgm101は、大きなオリゴマーの環を形成するRAD52型組換えタンパク質である。プロトコルは、陽イオン交換およびサイズ​​排除クロマトグラフィーと連結マルトース結合タンパク質（MBP）タギング方式を使用して可溶性組換えMgm101を調製するために記載されている。

MBPベースタギング戦略によってMgm101組換えタンパク質の調製

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

preparation-mgm101-recombination-protein-mbp-based-tagging

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.0K ビュー.  State University of New York Upstate Medical University. 酵母ミトコンドリア核様体タンパク質、Mgm101は、大きなオリゴマーの環を形成するRAD52型組換えタンパク質である。プロトコルは、陽イオン交換およびサイズ​​排除クロマトグラフィーと連結マルトース結合タンパク質（MBP）タギング方式を使用して可溶性組換えMgm101を調製するために記載されている。

ビデオ: Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

具体的にはヒト細胞における選択的スプライシングを操作するために工学人工要因

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

遺伝学

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

誘導性T7 RNAポリメラーゼ媒介マルチ遺伝子発現システム、pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9 仲介の対象となる統合生体内で相同性を介した終わり参加ベースの戦略を使用して

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

の遺伝的操作<em&gt;Δku80</emの機能ゲノム解析のための&gt;株<em&gt;トキソプラズマ</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

免疫・感染学

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

神経芽細胞腫転移のゼブラフィッシュモデル

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

がん研究

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

二分子蛍光相補性を持つ光活性化ローカライゼーション顕微鏡（BIFC-PALM）

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

生物工学

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

マイコバクテリアの天然および組換え細胞外小胞の濃縮

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

サブクローニングプラス挿入（SPI） - ジーンターゲティングベクターの迅速な構築のための新しい遺伝子改変方法

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

生物学

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that allow the tagging of endogenous genes to produce fluorescent fusion proteins. These methods enable researchers to visualize protein behavior in real time, providing valuable insights into their functions and interactions within the cellular environment. Many current gene tagging studies employ a two-step process where visible markers, such as eye color changes, are used to identify genetically modified organisms in the first step, and the visible marker is excised in the second step. Here, we present a one-step protocol to perform precise and rapid endogenous gene tagging in Drosophila melanogaster, which enables screening for engineered lines without the visible eye marker, offering a significant advantage over past methods. To screen for successful gene-tagging events, we employ a PCR-based technique to genotype individual flies by analyzing a small segment from their middle leg. Flies that pass the screening criteria are then used to produce stable stocks. Here, we detail the design and construction of CRISPR editing plasmids and methods for screening and confirmation of engineered lines. Together, this protocol improves the efficiency of endogenous gene tagging in Drosophila significantly and enables studies of cellular processes in vivo.

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Here, we present a simplified endogenous gene tagging protocol for Drosophila, which utilizes a PCR-based technique for marker-free identification of successful genetic modifications, facilitating the development of stable knock-in lines.

ショウジョウバエのメラノガスターにおける内在性遺伝子タグ付けのためのワンステップCRISPRベースの戦略

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that ...

神経科学

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

MBPベースタギング戦略によってMgm101組換えタンパク質の調製

要約

さらに動画を探す

この動画の章