我々は、議論や助言のためにトロント大学のロンデイビス、アダムDeutschbauer、および全体のHIP HOPの研究室に感謝します。 CNは、米国立ヒトゲノム研究所（助成番号HG000205）、RO1 HG003317、CIHR MOP - 84305、およびカナダの癌協会（＃020380）からの補助金によってサポートされています。 JOは、スタンフォードゲノムのトレーニングプログラム（米国立ヒトゲノム研究所から助成番号T32 HG00044）と国立衛生研究所（助成番号P01 GH000205）によってサポートされていました。 GGは、博士へのイノベーションのためのカナダの財団からの助成金によって部分的にサポートされているNHGRI RO1 HG003317とカナダの癌協会、グラント＃020380、TDとドネリーシーケンシングセンターによってサポートされています。ブレンダアンドリュースとジャックグリーンブラット。 AMSは、トロントオープンフェローシップの大学によってサポートされています。

<ol>
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利害の衝突は宣言されません。

ここでは、簡単にタグ付けされた変異体のコレクションを作成するためにさまざまな微生物のバーコード変異体コレクションの既存のコレクションの広い範囲に適合させることができる、ささやかな変更で、プロトコルを概説。我々は病原性酵母C.のタグトランスポゾン突然変異誘発のプロトコルを報告している間、我々はそれを強調するアルビカンス 、非常によく似たプロトコルは、単細胞の菌類の多様なに適応することができます。変更された、このプロトコルは、細菌13には適切に動作し、現在追加の真菌や細菌ゲノムの数のコレクションが建設中です。現時点では、このアッセイは、遺伝子-小分子の相互作用のための唯一の包括的な、ゲノムワイドな画面を提供します。アッセイの一つ、特に魅力的な機能は、遺伝子や低分子化合物の事前知識を必要としないことです。これらのアッセイの範囲とパワーにもかかわらず、他のラボへの譲渡は、結果の分析のための初期設備投資と情報ツールによって幾分妨げられてきた。分析のための堅牢なツールと組み合わせて次世代シーケンスの読み出しの採用により、我々は彼らの採用が増加すると予想。

<table border="1"><tbody><tr><td> 抗生物質 </td><td> ベンダーとカタログ番号 </td></tr><tr><td>カルベニシリン</td><td>シグマ、一部＃C1613 </td></tr><tr><td>カナマイシン</td><td>シグマ、一部＃K1876 </td></tr><tr><td>スペクチノマイシン</td><td>シグマ、一部＃S0692 </td></tr><tr><td>クロラムフェニコール</td><td>シグマ、一部＃C0378 </td></tr><tr><td> DNAクリーンアップと濃縮キット </td><td></td></tr><tr><td> QIAprep Spin Miniprepキット</td><td>キアゲン、一部＃27106 </td></tr><tr><td>ハイスピー​​ドプラスミドマキシキット</td><td>キアゲン、一部＃12663 </td></tr><tr><td> QIAクイックPCR精製キット</td><td>キアゲン、一部＃28106 </td></tr><tr><td> QIAquickゲル抽出キット</td><td>キアゲン、一部＃28704 </td></tr><tr><td> PCRと電気泳動試薬 </td><td></td></tr><tr><td> Taq DNAポリメラーゼ（MG -フリー）バッファ</td><td>ニューイングランドバイオラボ、一部＃M0320L </td></tr><tr><td>デオキシヌクレオチド溶液のミックス</td><td>ニューイングランドバイオラボ、一部＃N0447L </td></tr><tr><td> 25 mMのMgCl 2の </td><td>シグマ、一部＃63036 </td></tr><tr><td>アガロース、ローディング色素、および核酸は、ゲル電気泳動に適した染色</td><td>様々な</td></tr><tr><td> 10X TAEバッファー</td><td>シグマ、一部＃T8280 </td></tr><tr><td> 1 KBプラスDNAラダー</td><td>インビトロジェン、一部＃10787026 </td></tr><tr><td> YPD培養液 </td><td></td></tr><tr><td>酵母エキス10 gの</td><td>シグマ、一部＃Y1625 </td></tr><tr><td> 20グラムバクトペプトン</td><td> BDバイオサイエンス、一部＃211677 </td></tr><tr><td>デキストロース20gの</td><td>シグマ、一部＃D9434 </td></tr><tr><td> 実験機器 </td><td></td></tr><tr><td>マルチチャンネルピペット（1000、200、および20μL） </td><td>様々な</td></tr><tr><td>使い捨てピペット貯水池</td><td>様々な</td></tr><tr><td> 15〜50 mLの遠心管</td><td>様々な</td></tr><tr><td> 96 - と384ウェルディープウェルプレート</td><td> Axygen科学、一部＃P - 2ML - SQ - CS＆P - 384240SQCS </td></tr><tr><td> 96ウェルおよび384ウェルPCRプレートとシールのフィルム</td><td>様々な</td></tr><tr><td>マルチウェルプレートをシールするためのプレートのローラー</td><td>シグマ、一部＃R1275 </td></tr><tr><td> 30℃と37℃のプレート上に細菌や酵母の成長のために、チューブのインキュベーターを揺すって</td><td>様々な</td></tr><tr><td> in vitroでトランスポゾン変異誘発の </td><td></td></tr><tr><td> EZ - Tn5が知られトランスポザーゼ</td><td>シャーロットのバイオテクノロジー、一部＃TNP92110 </td></tr><tr><td> ハイスループット変換 </td><td></td></tr><tr><td> Seqprep 96 HTプラスミドプレップキット</td><td>エッジバイオシステムズ、一部＃84359 </td></tr><tr><td>ポリエチレングリコール、分子量3350 </td><td>様々な</td></tr><tr><td>リチウム酢酸</td><td>シグマ、一部＃517992 </td></tr><tr><td> 6ウェルプレート、滅菌</td><td>コー​​ニング、一部＃3335 </td></tr><tr><td> 50 mg / mLのウリジン</td><td>シグマ、一部＃U3750 </td></tr><tr><td> 100Xトリス- EDTA緩衝液</td><td>シグマ、一部＃T9285 </td></tr><tr><td> 1X TE/0.1M LiOAc </td><td>様々な</td></tr><tr><td>サケ精巣DNA </td><td>シグマ、一部＃1626 </td></tr><tr><td> バーコードのコレクションの成長 </td><td></td></tr><tr><td> 48ウェルプレート、プレート中で培養を成長した場合</td><td>グレイナー、一部＃M9437 </td></tr><tr><td>粘着板のシール</td><td> ABgene、一部＃AB - 0580 </td></tr><tr><td> 200mLの培養フラスコ</td><td>様々な</td></tr><tr><td>吸光度が可能な分光光度計</td><td>様々な</td></tr><tr><td> 250mlのフラスコや揺れ分光光度計用の温度制御シェーカー</td><td>様々な</td></tr><tr><td>セイフ - ロック微量遠心チューブに、2 mLの</td><td>エッペンドルフ、一部＃0030 120.094 </td></tr><tr><td> ハイブリダイゼーション装置 </td><td></td></tr><tr><td>ハイブリダイゼーションオーブン640 </td><td>アフィメトリクス、一部＃800138 </td></tr><tr><td>搭載されたGeneChip流体駅450 </td><td>アフィメトリクス、部分＃00から0079 </td></tr><tr><td> GeneArrayスキャナ3000 </td><td>アフィメトリクス、部分＃00から0212 </td></tr><tr><td>フローティングラックと沸騰水浴</td><td>様々な</td></tr><tr><td> ハイブリダイゼーションの消耗品 </td><td></td></tr><tr><td> Genflexタグ16K配列v2の</td><td> Affymetrix、一部＃511331 </td></tr><tr><td>デンハルト溶液、50倍濃縮</td><td>シグマ、一部＃D2532 </td></tr><tr><td>ストレプトアビジン、R -フィコエリスリン共役（SAPE） </td><td>インビトロジェン、一部＃S866 </td></tr><tr><td>セイフ - ロックマイクロ遠心チューブ、0.5 mLの</td><td>エッペンドルフ、一部＃0030 123.301 </td></tr><tr><td>小さなタフスポット</td><td>多様なバイオテック、一部＃LTTM - 1000 </td></tr><tr><td> 0.5 M EDTA </td><td>バイオラッド、一部＃161-0729 </td></tr><tr><td> 10％のTween </td><td>シグマ、一部＃T2700 </td></tr><tr><td> MES遊離酸一水和物</td><td>シグマ、一部＃M5287 </td></tr><tr><td> MESのナトリウム塩</td><td>シグマ、一部＃M5057 </td></tr><tr><td> 5 M NaClを</td><td>シグマ、一部＃71386 </td></tr><tr><td> 20X SSPE </td><td>シグマ、一部＃S2015 </td></tr><tr><td>分子生物学グレードの水</td><td>シグマ、一部＃W4502 </td></tr><tr><td> ハイブリダイゼーションのプライマー </td><td> 様々なサプライヤー（標準脱塩） </td></tr><tr><td> Uptag </td><td> 5&#39;GATGTCCACGAGGTCTCT 3&#39; </td></tr><tr><td> Buptagkanmx4 </td><td> 5&#39;ビオチン- GTCGACCTGCAGCGTACG 3&#39; </td></tr><tr><td> Dntag </td><td> 5&#39;CGGTGTCGGTCTCGTAG 3&#39; </td></tr><tr><td> Bdntagkanmx4 </td><td> 5&#39;ビオチン- GAAAACGAGCTCGAATTCATCG 3&#39; </td></tr><tr><td> B213 </td><td> 5&#39;ビオチン- CTGAACGGTAGCATCTTGAC 3&#39; </td></tr><tr><td> Uptagkanmx </td><td> 5&#39;GTCGACCTGCAGCGTACG 3&#39; </td></tr><tr><td> Dntagkanmx </td><td> 5&#39;GAAAACGAGCTCGAATTCATCG 3&#39; </td></tr><tr><td> Uptagcomp </td><td> 5&#39;AGAGACCTCGTGGACATC 3&#39; </td></tr><tr><td> Dntagcomp </td><td> 5&#39;CTACGAGACCGACACCG 3&#39; </td></tr><tr><td> Upkancomp </td><td> 5&#39;CGTACGCTGCAGGTCGAC 3&#39; </td></tr><tr><td> Dnkancomp </td><td> 5&#39;CGATGAATTCGAGCTCGTTTTC 3&#39; </td></tr><tr><td> シーケンシングプライマー：イルミナのプラットフォーム </td><td> 様々なサプライヤー </td></tr><tr><td> UpTagフォワード（100uM） </td><td> 5&#39;CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC T GAT GTC CAC GAG GTC TCT 3&#39; </td></tr><tr><td> UpTagリバース（100uM） </td><td> 5&#39;AAT GAT ACG GCG ACC ACC GAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T NNNNN GTC GAC CTG CAG CGT ACG 3&#39; </td></tr><tr><td> DownTagフォワード（100uM） </td><td> 5&#39;CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC T GAA AAC GAG CTC GAA TTC ATC G 3&#39; </td></tr><tr><td> DownTagリバース（100uM） </td><td> 5&#39;AAT GAT ACG GCG ACC ACC GAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T NNNNN CGG TGT CGG TCT CGT AG 3` </td></tr><tr><td> 1を読んUP -タグ配列のプライマー（100uM） </td><td> 5&#39;GTC GAC CTG C​​AG CGT ACG 3&#39; </td></tr><tr><td> 1 DOWN -タグ配列のプライマー（100uM）をお読みください</td><td> 5&#39;CGG TGT CGG TCT CGT AG 3&#39; </td></tr><tr><td> 2インデックスシーケンスプライマー（標準イルミナR1のプライマー）（100uM）をお読みください</td><td> 5&#39;AC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T 3&#39; </td></tr><tr><td> 追加のシークエンシング試薬/装置 </td><td></td></tr><tr><td>キアゲンアプライ96 UF PCR精製キット</td><td>キアゲン、一部＃29051 </td></tr><tr><td>真空ポンプ</td><td>任意のベンダー</td></tr><tr><td>マシュレ-ナーゲル真空マニホールド</td><td>マシュレ-ナーゲル、一部＃740 681 </td></tr><tr><td>インビトロジェンクワント- ITのdsDNA BRアッセイキット</td><td>インビトロジェン、一部＃Q32853 </td></tr><tr><td>インビトロジェン量子ビットのアッセイチューブ</td><td>インビトロジェン、一部＃Q32856 </td></tr><tr><td> 40％アクリルアミドプラス1％N、N&#39; -メチレン - ビス - アクリルアミド、37.5:1 </td><td>バイオラッド、一部＃161-0148 </td></tr><tr><td>トリスベース</td><td>シグマ、一部＃T1503 - 1KG </td></tr><tr><td>ホウ酸</td><td>シグマ、一部＃B6768 - 500G </td></tr><tr><td> 0.5M EDTA、pH8.0の</td><td> Teknova、一部＃E0306 </td></tr><tr><td>過硫酸アンモニウム</td><td>シグマ、一部＃A3678 - 25G </td></tr><tr><td> TEMED </td><td> Bioshop、一部＃TEM001.25 </td></tr><tr><td>臭化エチジウム溶液</td><td> Bioshop、一部＃ETB444.10 </td></tr><tr><td> 0.5M酢酸アンモニウム</td><td> Teknova、一部＃A2000 </td></tr><tr><td> 10mMの酢酸マグネシウム四水和物</td><td>シグマ、一部＃M0631 - 100G </td></tr><tr><td> 1mMのEDTA、pH8.0の</td><td> 0.5M EDTA、pHが8.0を参照してください。 </td></tr><tr><td>エタノール</td><td>様々な</td></tr><tr><td>酢酸ナトリウム、pH 5.2 </td><td> Teknova、一部＃S0297 </td></tr><tr><td>速度の真空</td><td>様々な</td></tr><tr><td>シングルリードクラスタ生成キット</td><td>イルミナ、一部＃GD - 300 - 1001 </td></tr><tr><td> v4の36Cシークエンシングキット</td><td>イルミナ、一部＃FC - 104 - 4002 </td></tr><tr><td></td><td></td></tr></tbody></table> 10X TBEのレシピ<table border="1"><tbody><tr><td>額</td><td>試薬</td></tr><tr><td> 108グラム</td><td>トリスベース</td></tr><tr><td> 55グラム</td><td>ホウ酸</td></tr><tr><td> 40mlの</td><td> 0.5M EDTA（pH 8.0）を</td></tr><tr><td colspan="2"> 1LのマークにのdH 2 Oを追加</td></tr></tbody></table> 12％ポリアクリルアミドゲルのレシピ<table border="1"><tbody><tr><td>ボリューム</td><td>試薬</td></tr><tr><td> 5.8ミリリットル</td><td> 40％アクリルアミドプラス1％N、N&#39; -メチレン - ビス - アクリルアミド、37.5:1 </td></tr><tr><td> 12ミリリットル</td><td>のdH 2 O </td></tr><tr><td> 2ミリリットル</td><td> 10X TBE </td></tr><tr><td> 140μL </td><td> 10％の過硫酸アンモニウム</td></tr><tr><td>総量：20ミリリットル</td><td></td></tr></tbody></table>プライマーミックスをUptag： その後再懸Uptagと100μMでのddH 2 OでBuptagkanmx4それぞれ、50μMの各終濃度に対して1:1の比率で混合する。 -20℃で保存プライマーミックスをDowntag： 再懸​​DntagとBdntagkanmx4100μMでのddH 2 Oのそれぞれは、その後50μMの各終濃度に対して1:1の比率で混ぜる。 -20℃で保存混合オリゴヌクレオチド： 100μMでのddH 2 Oで次の8つのオリゴ（標準脱塩）のそれぞれを再懸濁します。 Uptag、Dntag、Uptagkanmx、Dntagkanmx、Uptagcomp、Dntagcomp、Upkancomp、Dnkancomp。 12.5μM各終濃度8つのオリゴヌクレオチドの等量を混ぜる。 12X MES在庫： 10ミリリットルの場合は、8ミリリットル分子生物学グレードの水に0.7グラムMES遊離酸一水和物と1.93グラムMESのナトリウム塩を溶かす。よく混合した後、pHをチェックし、pHを6.5から6.7に、必要に応じて調整します。 10ミリリットルの合計体積に水を追加。滅菌濾過し、4℃での光（例えば、ホイルで管を包む）から保護。解決策が目に見え黄色または6ヶ月後になった場合に交換してください。 2Xハイブリダイゼーション緩衝液： 50ミリリットルの場合は、12X MES株式の8.3ミリリットル（2.9.14から）、5 M NaClを、0.5 M EDTA、10％Tween 20を0.1 mlの（vol / vol）の4.0 mlおよび19.9ミリリットルのddHフィルタリングの17.7 mlを混ぜて2 O滅菌フィルタします。 ミックス300ミリリットル20X SSPE、1 mlの10％トゥイーン（vol / vol）の、699ミリリットルのddH 2 O：ウォッシュ滅菌フィルタします。 ウォッシュB：ミックス150ミリリットル20X SSPE、1 mlの10％トゥイーン（vol / vol）の、849ミリリットルのddH 2 O滅菌フィルタします。 バーコードシーケンシングプライマー UP -タグのプライマー配列の5&#39;末端には、（ 太字 ）FとRプライマーに組み込まイルミナ特定のアダプターの配列である。変数の配列（ イタリック体）インデックス/多重読み出しに使用される5 - merのインデックス作成のタグを表します。 3&#39;末端には（下線）uptagのバーコードを隣接の一般的なプライマーを表しており、酵母のバーコードを増幅するために必要です。 DOWN - tag内のプライマーは5しかし、3&#39;末端が（下線）を増幅するために使用される共通のプライマーで置き換えられる、UP -タグプライマー（イルミナ特定のシーケンス）の尾&quot;尾は5と同一である&#39;配列DOWNをタグのバーコード。

competitive genomic screens of barcoded yeast libraries

次世代シーケンシング技術の進歩のおかげで、我々は、ほぼ毎日新しいゲノム配列へのアクセス権を持っている。これらの進歩のテンポがより大きな深さと幅広さを約束し、加速しています。これらの飛躍的進歩を踏まえて、遺伝子の機能を定義するために、高速、並​​列メソッドの必要性がますます重要になります。酵母やE.におけるゲノムワイドな欠失変異体のコレクション大腸菌の遺伝子の機能の機能解析のための主力を務めているが、この方法はスケーラブルではない、現在の遺伝子欠失のアプローチは、ゲノムを構成する数千の遺伝子のそれぞれを削除し、検証する必要があります。この作業が完了した後にのみ、我々は、高スループットの表現型解析を挟むこともできます。過去10年間で、私たちの研究室では競争力のある、小型化、ハイスループットのゲノムワイドな並列実行できるアッセイのポートフォリオを改良しました。この並列化では、バーコードは、突然変異のプロキシとして機能しているためDNA&quot;タグ&quot;、または各変異体に&quot;バーコード&quot;を含めることが可能であり、1は変異型フィットネスを評価するためにバーコードの存在量を測定することができます。本研究では、我々はDNA配列やバーコード変異体コレクションの間のギャップを埋めるために求めている。これを達成するために我々は、新たに配列決定するまでのパラレルバーコードのアッセイを開きますが、悪い微生物を特徴とする複合トランスポゾン混乱 - バーコードのアプローチを紹介する。我々は新しいカンジダバーコード混乱のコレクションを発表し、どのように両方のマイクロアレイベースの、次世代シーケンシングベースのプラットフォームを記述するこのアプローチを説明するために10,000を集めるために使用することができます-一回の実験で1,000,000遺伝子-遺伝子と薬剤遺伝子の相互作用。

Watch this Scientific Journal Video about Competitive Genomic Screens of Barcoded Yeast Libraries at JoVE.com

Competitive Genomic Screens of Barcoded Yeast Libraries

我々は包括的、公平なゲノムワイドな遺伝子薬剤と遺伝子 - 環境相互作用を理解するために画面を開発しました。これらの変異体のコレクションをスクリーニングするための方法が提示されます。

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 ...

competitive-genomic-screens-of-barcoded-yeast-libraries

Research

JoVE Journal

Biology

18.1K ビュー.  University of Toronto. 我々は包括的、公平なゲノムワイドな遺伝子薬剤と遺伝子 - 環境相互作用を理解するために画面を開発しました。これらの変異体のコレクションをスクリーニングするための方法が提示されます。

ビデオ: Competitive Genomic Screens of Barcoded Yeast Libraries

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

メタゲノムライブラリの大規模スクリーン

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

生物学

Isolation of Genomic DNA from Mouse Tails

マウスの尾からのゲノムDNAの単離

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大規模な挿入環境ゲノムライブラリーの製造

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

酵母細胞からミトコンドリアの精製

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

ゼブラフィッシュの化学スクリーンのための自動化された画像と解析の開発。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

酵母のコロニーの埋め込み法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

ゲノムMRI - ゲノムDNA内の配列パターンを研究するためのパブリックリソース

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Picoeukaryoteのゲノム形質転換<em&gt; Ostreococcusタウリ</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

ゲノムDNAから5  - ヒドロキシの選択的捕獲

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

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Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...