著者らは、 シロイヌナズナ植物のメンテナンスのために氏ジェニファーM.ロンメルとタラN. Rognanに感謝。この作品は、健康補助1R15GM086846 - 01とW ·シャオに3R15GM086846 - 01S1のセントルイス大学と国立研究所からの起動資金によってサポートされていました。

I.遺伝クロッシング 1。女性の親をEmasculating DNA配列の多型を使用して母親と父親の対立遺伝子を区別するために、二つの異なる生態型、例えば、コロンビア- 0（COL - 0）とLERは、女性と男性の両親として選択されます。植物は、若くて健康でなければなりません。一つは、解剖顕微鏡、拡大鏡のバイザー、または肉眼を使用して女性の親を骨抜きにすることができます。ステージ- 12花を見つけて（スミスら 、1990）とハサミと小花柄のベースクリッピングによるあらゆる花やsiliques上下それらを削除します。鉗子上の任意の花粉を除去し、同様に花粉を殺す95％エタノール、のビーカーに静かに先端の基盤を浸漬することで鉗子を滅菌する。静かに鉗子を使用して花芽をこじ開ける、ゆっくりと雌しべがむき出しとそのままにして、4がく片、4枚の花弁、6雄しべを取り除く。このプロセスの間に心皮への損傷を避けるようにしてください。 2。花粉ドナーをピッキングし、受粉を行っ最高の花粉のドナーは、花粉の多くが脱落している雌ずい（スミスら 、1990）、90 °の角度で伸びる花びらでステージ14で開いている花をanthesedされています。ベースで花をつかむと、ちょうど小花柄の上に、これは花が開いて広がることになります。葯と準備雌しべの柱頭ほこりを取り除きます。受粉後、汚名は簡単に顕微鏡下で観察することができる黄色の花粉で覆われる。 3。クロスのラベリング植物のすべての去勢雌しべを受粉した後、我々は、宝石のタグと、女性と男性の両親と日付の情報をクロスにラベルを付けます。工場に近い土壌​​の株式を置く、株式への花序の茎を結ぶために文字列を使用して、ビニール袋で受粉雌しべをカバー。 II。 シロイヌナズナにおける胚乳組織の分離 1。材料の準備液体窒素タンク、液体窒素、解剖顕微鏡、精密チップピンセット（デュモン生物学、スイスで5 INOX FST）、顕微鏡のスライドガラス（の二つの新しいペア：我々は通常、胚乳および胚組織を収穫する前に準備が必要な材料を得る3&quot;X 1&quot;X 1.0 mm）を、ソルビトール0.3 MのpH 5.7溶液及び5mMのMES。 2。収集siliques 7時 - または8日目の後の受粉（DAP）、種子は胚発生の後期魚雷段階半ばまでに収穫し、胚乳および胚のために解剖する準備が整いました。去勢花は若すぎる場合、時に、それは9〜10 DAPがかかる場合があります。 3。胚乳および胚の隔離 シロイヌナズナの種子は小さいですので、我々は、胚乳および胚を分離するために解剖顕微鏡を使用してください。顕微鏡下で8 DAPの​​長角果を入れ、長角果柄を保持するためにピンセットを使って二心皮のヒューズマージンで開いて長角果をスライドさせて鉗子の別のペアの先端を使用してください。種皮から胚乳と別の胚乳を押し出すためにカットされていない端を絞る、胚を引き出すために卵門端に小さなカットを行う、ソルビトール0.3 Mおよび5mM MESのpHは5.7ソリューションにつのシードをピックアップしてピンセットを使用して、 （木下ら 、2004）。液体窒素で別々のマイクロチューブに胚と胚乳を置く。 -80℃の冷凍庫に管を保管し、我々は10〜15 siliquesから胚や胚乳を蓄積するまで、これを続けると。いくつかのケースでは、胚乳は種皮、すなわちから分離する必要がない、人は遺伝子刷り込みの分析のための胚乳と種子コートの混合物からトータルRNAを分離することができます。 III。バイサルファイトシークエンシング 1。必要な試薬 、：ゲノムDNA、制限酵素、3 M水酸化ナトリウム（たて）、6.42 Mの尿素/ 4 Mの亜硫酸水素ナトリウム（190 2 Mのメタ重亜硫酸ナトリウム、Sigma - Aldrichは、S9000、Na2S2O5、分子量）を調製するためのセチルトリメチルアンモニウムブロマイド（CTAB） 10mMのヒドロキノン、DNA精製キット（Promega社ウィザードDNAクリーンアップシステム、カタログ番号A7280）、TEバッファー、6.3 M水酸化ナトリウム（たて）、10 M NH 4 OAC、20μg/μLのtRNAは、100％エタノール。 2。亜硫酸水素塩の治療プロトコルの詳細<ol><li> CTABの手順を（ロジャーズとBendich、1988）を用いたゲノム胚乳や胚のDNAを分離します。 </li><li>ダイジェストは100 ng - 分析する領域の外側に切断する制限酵素で100μL全量を20μLのゲノムDNA2μgの。 MEAのプロモーターの場合は、我々はXhoIで、NdeIで、とPstIまたはHindIII部位を使用してください。 </li><li> 5分間DNAを煮沸して制限酵素を変性し、氷上で急冷する。 </li><li> 15分間37℃で3 M NaOHおよびインキュベートの1 / 9量（20μL消化されたDNA 2.2μL）° Cを追加。 </li><li> 250μLのPCRチューブにソリューションを転送する。 </li><li>滅菌蒸留水10mlに尿素7.5 gを溶かし、ゆっくりと1〜2時間にわたってメタ重亜硫酸ナトリウムの7.6 gを追加し、加熱は、通常、溶解することができます。焼きたての10 M NaOHで5にpHを調整し、最終的に滅菌蒸留水を追加します。 20mlに容積。これは、6.24 Mの尿素/ 4 Mの亜硫酸水素ナトリウムのソリューションです。 </li><li>それぞれ、5.36 Mおよび3.44 Mの最終濃度に6.24 Mの尿素/ 4 Mの亜硫酸水素ナトリウム溶液を加える（ポーリンら 、1998）。例えば、変性したゲノムDNAの上に22.2μL（小ら 、2003）に6.42 Mの尿素/ 4 Mの亜硫酸水素ナトリウム溶液を208μLを加える。 </li><li> 0.5mMの（20μL消化は12μL）の最終濃度でDNAを10 mMヒドロキノンを追加。 </li><li> 55 30サイクル℃で15分間と30秒間95 ° C：PCRのマシンで亜硫酸水素塩処理を行う。 </li><li>プロメガからウィザードのDNAクリーンアップシステムを用いてBisulfite処理したDNAを脱塩し、（ヤコブセンら 、2000）プロトコルをフォローアップ。 </li><li>脱塩後にカラムから回収TEの正確な体積を測定し、37℃0.3 Mインキュベートの最終濃度は6.3 M NaOHを加える℃で15分間。 </li><li> 3 Mの最終濃度10 M NH 4 OAC液（pH7.0）、20μg/μLのtRNAの2μL、および100％エタノールとしてミックスの3倍量を追加。 14,000 rpmで15分間遠心。 </li><li> 、70％エタノールで1回ペレットを洗浄短い遠心を行い、余分なエタノールを除去します。 </li><li> 25の5-10分を再懸濁します用speedvacのドライペレット - DNAの量を始めるに応じて、100μlのTEバッファー。ナトリウム、亜硫酸水素塩処理したDNAは、現在のPCR解析の準備ができています。 </li></ol> 3。 PCRの増幅<ol><li>非メチル化シトシンをウラシルに変換されるので、テンプレートとして、亜硫酸水素塩処理したDNAを用いて大規模な断片を増幅することは困難です。従って、我々は通常500 bpよりもはや製品を増幅するプライマーを設計する。シーケンス4 KB MEAのプロモーターをするために、我々は、プライマーの多くのセットを設計し、14重複したフラグメントは、地域全体をカバーするために増幅（小ら 、2003）。 </li><li>シーケンスに上部の鎖は、フォワードプライマー、I）の設計でプライマー内に追加可能なエキストラ長いヌクレオチドことなく、より高いアニーリング温度を持つために、G（グアニン）に富んだ領域を選択し、II）の変更C（シトシン）Yへ（ CGとCNGコンテキストにおけるピリミジン）とT（チミン）に、残りのCを変更してください。リバースプライマーを設計する上で、I）C -リッチ領域を選択する、II）は、CGでのR（プリン）とCNGコンテキストにGを変更し、A（アデニン）に残りのGに変更。 </li><li>フォワードプライマーを設計する上でシーケンスボトムストランドを、、私に）C -リッチ領域を選択する、II）は、CGでのRおよびCNGコンテキストにGを変更し、リバースプライマー、私の設計ではAに、残りのGを変更） G -リッチ領域、II）CGでのYのC（ピリミジン）とCNGコンテキストを変更し、T.、残りのCを変更する</li><li>我々は通常、各PCR増幅（小ら 、2003）のためのテンプレートとしてナトリウム、亜硫酸水素塩処理したDNAの1〜2μLを使用してください。 PCR産物は、ゲルインサートとして精製し、TOPO TAクローニングベクターpCR2.1（Invitrogen）にクローニングすることに、フラグメントの正しいサイズを確認するために、ゲル電気泳動によって分析する必要があります。単一のコロニーを拾い、培養され、プラスミドDNAを抽出し、配列決定のために送信されます。 </li></ol> 4。シーケンスの解析バイサルファイトシークエンシングの原理は、非メチル化シトシンがPCR産物にチミンとして増幅されるpH5.0での亜硫酸水素ナトリウムの高濃度で加水分解的脱アミノによるウラシルに変換されますです、5 - メチルシトシンは亜硫酸水素ナトリウムによって変更されることはありませんが、とPCR増幅後のシトシンに残ります（。クラークら 、1994;海外ホテルら、1992。）。シーケンシングの結果を得た後、我々は、PCR増幅に使用される鎖特異的なテンプレートと比較します。テンプレート内のシトシン残基は配列決定の結果でチミンとして読み取る場合、それはシトシンがメチル化されていないことを示しています。テンプレート内のシトシン残基はシーケンス中のシトシンのままである場合、それはシトシンがメチル化されていることを意味します。 IV。代表的な結果<img src="/files/ftp_upload/2327/2327fig1.jpg" alt="図1 /" /> 図1。

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それは、胚乳と種皮から胚を分離することは比較的容易ですが、それは胚発生の初期または中期魚雷の段階で、特に種子のために、種皮から胚乳を分離することは面倒です。種皮のみを組織のごく少量を寄与するため、いくつかの遺伝子については、例えば、MEAとFWAは 、我々は種皮から胚乳を分離する必要はありません。我々は胚乳と種皮の組織の混合物からRNAを分離できるこ...

電源<ul><li>解剖顕微鏡</li><li>はさみ</li><li>ファインチップ鉗子</li><li>ジュエリータグ</li><li>植物ステークス</li><li>文字列またはツイストネクタイ</li><li> 4&quot;X 2&quot;X 8&quot;ポリエチレン袋</li><li> 3&quot;X 1&quot;X 1.0 mmの顕微鏡用スライド</li><li>液体窒素</li><li>液体窒素容器</li><li>ヒートブロック</li><li> PCRチューブ</li><li>クラー</li><li>マイクロ遠心チューブ</li><li>マイクロ遠心機</li><li>ゲル電気泳動の施設</li><li> シロイヌナズナコロンビア- 0植物</li><li> シロイヌナズナ Landsbergのエレクタの植物</li></ul>試薬<ul><li> 70％エタノール</li><li> 95％エタノール</li><li> 100％エタノール</li><li>ソルビトール0.3 Mおよび5mM MES - pHは5.7 </li><li>セチルトリメチルアンモニウムブロマイド（CTAB） </li><li> 100％エタノール</li><li> Cholorform </li><li>制限酵素</li><li> 3 M NaOHを</li><li> 6.3 M NaOHを</li><li> 6.24 M尿素/ 4 Mの亜硫酸水素ナトリウム</li><li>滅菌蒸留H 2 O </li><li> 10mMのヒドロキノン</li><li>ウィザードDNAクリーンアップシステム（プロメガ） </li><li> 10 M NH 4 OAC </li><li>で20μg/μLのtRNA </li><li> TEバッファー</li><li> TOPO TAクローニングキット（Invitrogen社） </li></ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

シロイヌナズナでは、エピジェネティックなメカニズムを研究するための優れたモデル生物である。理由の一つは、DNAメチルトランスフェラーゼの機能喪失型のヌル変異体であるため、ゲノムのDNAメチル化の消失は、成長と発展にどのように影響するかを研究するシステムを提供し、実行可能です。刷り込みは、母親と父親の対立遺伝子の差次的発現を意味し、哺乳類や植物の両方で再生開発において重要な役割を果たしている。 DNAメチル化はインプリント遺伝子の母性や父性の対立遺伝子が発現したり、沈黙されているかどうかを決定するための非常に重要です。顕花植物では、生殖における重複受精のイベントがあります：一つ精子細胞は、胚乳を生じさせるために、中央細胞と胚と第二精子のヒューズを形成するために卵細胞を受精。胚乳は、刷り込みが植物に発生した組織です。MEDEA、SETドメインポリコーム群遺伝子、及びFWA、開花を調節する転写因子は、胚乳およびそれらの発現にプリントにして表示された最初の2つの遺伝子であるにDNAメチル化と脱メチル化によって制御される植物。胚乳の遺伝子とメチル化パターンの刷り込みの状態を決定するために、我々は最初の胚乳を分離できるようにする必要があります。シードは、 シロイヌナズナの小さななので、それはシロイヌナズナ胚乳を分離し、そのメチル化を調べることが困難なままである。このビデオプロトコルでは、我々は、遺伝的交雑を実施するために種子から胚乳組織を分離するために、そして、重亜硫酸塩シークエンシングによりメチル化状態を判別する方法を報告する。

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Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

刷り込みは、植物と哺乳類の生殖の現象です。 DNAメチル化はインプリンティングのメカニズムに重要な役割を果たしている。胚乳を分離し、でインプリント遺伝子のメチル化状態を決定する<em&gt;シロイヌナズナ</em&gt;困難になる可能性があります。このプロトコルでは、我々は、胚乳を分離し、バイサルファイトシークエンシングによってメチル化を決定する方法について説明します。

のインプリント遺伝子のDNAメチル化の定量シロイヌナズナ胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

determination-dna-methylation-imprinted-genes-arabidopsis

Research

JoVE Journal

Biology

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

17.3K ビュー.  Saint Louis University. 刷り込みは、植物と哺乳類の生殖の現象です。 DNAメチル化はインプリンティングのメカニズムに重要な役割を果たしている。胚乳を分離し、でインプリント遺伝子のメチル化状態を決定するシロイヌナズナ困難になる可能性があります。このプロトコルでは、我々は、胚乳を分離し、バイサルファイトシークエンシングによってメチル化を決?...

ビデオ: Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNAメチル化：重亜硫酸修飾と分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

生物学

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics：同時に新規OctoChrome FISHアッセイを使用した全24ヒト染色体における数値と構造変化の検出

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

遺伝的に探索するために種皮寝具アッセイ<em&gt;体外</em&gt;どのように胚乳コントロール種子発芽<em&gt;シロイヌナズナ</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

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 ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

タンパク質アルギニンメチル化を研究するためのin vitroでメチル化アッセイで

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

塩基対解像度でDNAメチル化の評価のための強化されたの減少表現重亜硫酸塩シークエンシング

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

アストロサイトの発現および転写活性を有する遺伝子特異的DNAメチル化の変化の相関<em&gt; KCNJ10</em&gt;（Kir4.1）

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

次世代シーケンシングによりDNAメチル化解析をターゲット

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

シロイヌナズナタンパク質-DNA相互作用の同定のための免疫沈降アッセイクロマチン<em&gt;インビボ</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

結晶セルロース蓄積中の高解像度の定量化<em&gt;シロイヌナズナ</em&gt;ルーツは、組織特異的な細胞壁の変更を監視します

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...