We thank Anne Wochele for her assistance in the laboratory and Vera Augsburger for help with the automated gel sequencer. We thank the Deutsche Forschungsgemeinschaft for funding by grants BE4038/2 and BE4038/5 within the &#8220;priority programmes&#8221; SPP1316 and SPP1617.

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The authors have nothing to disclose that would present a conflict of interest.

蛍光プライマー伸長はTSP-または二次RNA処理識別のためのいずれか、RNAの5 &#39;末端を決定するための簡単​​かつ迅速な方法である。原因蛍光プライマーの使用のために、反応が設定することができ、（放射性標識したプライマーの場合と異なり）追加のセキュリティ対策なしで実行されます。サンプルは、蛍光によって検出されるように、電気泳動、X線フィルムが一般的に使用される放射?...

プライマー伸長は、1つの基地解像度までの特定のRNA分子の5 &#39;末端を決定するための分子方法である。そのような5&#39;-RACE（cDNA末端の迅速増幅）のような他の方法の利点は、高速のターンアラウンドタイムと容易にRNA分子の異なる長さの混合物を分析する能力である。 この方法は、特定の長さのcDNA断片を生成する、特異的な蛍光プライマーを用いて逆転写反応するRNA分子を供することによって動作する。これらのcDNA分子は、変性ポリアクリルアミドゲル上で従来のサンガー配列決定反応2と一緒に実行される起因蛍光標識プライマーの使用に、それらの蛍光によって検出することができる。 cDNA断片の長さは、5 &#39;RNA末端のマッピングを可能にする、シークエンシングラダーとの比較によって評価される。 伝統的に、プライマー伸長反応は、組み合わせて使用​​される放射性同位体でX線フィルム上のcDNA分子を検出することができる。それらの感度が若干低いとはいえ起因する健康被害、廃棄物処理の問題や取り扱いの容易さのために、より新しいプロトコルは、自動化されたシーケンサーを持つプライマー伸長を検出するための蛍光を利用している。蛍光プライマーは（我々の手で一年以上）、長い​​間安定しているように、蛍光標識されたプライマーを用いて、定期的な放射性標識化手順は、省略することができる。 ここで記述する方法は、自動化されたゲルシーケンサーを利用したが、若干の変更を加えて、キャピラリーシーケンサーも、cDNAの分離および検出の3のために使用することができる。ゲル分析の並列性は、RNA切断または加工の小さな量を検出することが可能となる。別の利点は、検出することができる末端切断、あるいは一つの塩基の処理として、この方法は、高解像度である。 RNA切断または処理、tは検出に関してはypicallyプライマー拡張の2つのタイプが区別される。他の場合には、処理は、生体内で行われ、得られたRNAが精製されるのに対し、あるケースでは、酵素処理は、精製されたRNAおよび精製された酵素を用いてインビトロで行われる。プライマー伸長は、in vitroで実施する両方の場合において、RNAは、RNAの供給源に依存して、しかし、供され、この方法は、in vitroまたはin vivoでのプライマー伸長と呼ばれるか。ここで提示したプロトコルでは、使いやすさのために（無精製タンパク質必要）と同時に、転写開始点と処理を決定する可能性を、in vivoでのプライマー伸長のみに焦点を当てる。しかし、in vitroでのプライマー拡張は同じように設定する原則にあり、このプロトコルは、出発点として役立つことができる。 ここに示された方法は、それらが高に適しているように、多くの細菌種に適用することができ純度および核酸の高収率製剤。 私たちの研究室での研究は、毒素-抗毒素（TA-）システム4,5、プライマー伸長法が広く使用されているフィールドの規制範囲に焦点を当てています。 TA-システムが安定しており、内因的に積極的な毒性タンパク質や毒性6,7を打ち消す主に不安定なタンパク質またはRNA抗毒素で構成され、原核生物のゲノム中に存在する小さな遺伝的要素である。毒素活性は、しばしば複製の阻害、細胞壁合成、または他の機構によって作用するが、ほとんどの場合RNase活性8,9によってれる。一般的に、RNアーゼ特異性プライマー伸長法であるそのうちの一つの異なる試験を行うことによって決定される。切断された完全長断片の混合物が同時にそれらの5 &#39;末端を決定するために分析することができるように、プライマー伸長反応は、この用途に非常に適している。 、in vitroおよびin vivoプライマーエクステンション内のミックスを使用特定の毒素のRNase切断は、 例えば、配列特異性は、10-13を決定することができる。 <img alt="図1" fo:content-width="6in" src="/files/ftp_upload/52134/52134fig1highres.jpg" width="600" /> プライマー伸長手順の概要を図1。細菌培養実験のニーズに応じてインキュベートし、処理される。全RNAを、細胞から抽出したDNAの痕跡を除去するためにDNアーゼIで処理したcDNAを得た標的特異的蛍光DNAプライマーを用いて逆転写反応に供される。ゲノムDNAまたはプラスミドを抽出し、続いてcDNA断片とサイズ比較用蛍光サンガー配列決定反応のために使用される。プライマー伸長産物は、変性尿素ポリアクリルアミドゲル上サンガーシークエンシング産物と一緒に実行して、自動化されたレーザー顕微鏡で分析する。ラcDNAのバンドとラインアップしているシーケンシングベースSTの5 &#39;cDNAの端（青い矢印）のベース。 Feketeの中でより多くの情報、 ら 3 <a href="https://www.jove.com/files/ftp_upload/52134/52134fig1highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 全体のプライマー伸長手順の概要を図1に見ることができる。簡単に説明すると、細菌細胞を培養し、収穫し、細胞ペレットを溶解し、RNAを抽出した。精製されたRNAは、逆転写酵素のためのテンプレートとして作用し得るDNA分子の痕跡を除去するためにDNアーゼIで処理する。特異的な蛍光プライマーは、RNAに付加関心領域にハイブリダイズし、続いて一本鎖の相補的DNA（cDNA）の結果として、逆転写されている。配列決定ラダーは、蛍光プライマーを用いた従来のサンガー配列決定によって作成され、プライマー伸長cDNA断片の横に変性ポリアクリルアミドゲル上で分離する。結果として生じるゲルは、対象の5 &#39;末端の同定を可能にする、蛍光バンドを比較することによって分析される。転写開始点およびプロセシング部位は、次いで配列比較によって個別に評価される。

<table border="0" cellpadding="0" cellspacing="0" width="1200">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;"><a name="RANGE!A1:D16">Name</a></td>
			<td style="width:196px;">Supplier</td>
			<td style="width:244px;">Catalog Number(s)</td>
			<td style="width:363px;">Comment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">AMV Reverse Transcriptase (20-25 U/&#181;l)</td>
			<td style="width:196px;">NEB / Roche</td>
			<td style="width:244px;">NEB: M0277-T / Roche: 10109118001</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:399px;">DNase I (RNase free)</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM2222</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">FastPrep-24 Instrument&#160;</td>
			<td style="width:196px;">MPBio</td>
			<td align="right">116004500</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:399px;">Fluorescently labeled primers</td>
			<td style="width:196px;">Biomers</td>
			<td style="width:244px;">n/a</td>
			<td style="width:363px;">5&#8217; DY-681 modification of &#8220;ordinary&#8221; DNA oligonucleotides. Compatible dyes such as the LICOR IRDye 700/800 are also available from other suppliers such as IDTdna.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Li-Cor 4200 Sequencer incl. ImagIR Data collection software</td>
			<td style="width:196px;">Li-Cor</td>
			<td style="width:244px;"></td>
			<td style="width:363px;">Product discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">NanoDrop 2000</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Nuclease free water</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM9915G</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Plasmid mini preparation kit</td>
			<td style="width:196px;">QIAGEN</td>
			<td align="right">12125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">RapidGel-XL-40% Concentrate</td>
			<td style="width:196px;">USB</td>
			<td style="width:244px;">US75863</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">RNA STORAGE BUFFER</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td>AM7000</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:399px;">Roti-Aqua-P/C/I</td>
			<td style="width:196px;">Carl Roth</td>
			<td style="width:244px;">X985.3</td>
			<td style="width:363px;">Alternative: &#8220;Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)&#8221; from Ambion (AM9720)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">SUPERase&#8226;In RNase Inhibitor</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM2696</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:399px;">Thermo Sequenase fluorescently labelled primer cycle sequencing kit with 7-deaza-dGTP</td>
			<td style="width:196px;">GE Healthcare</td>
			<td style="width:244px;">RPN2538</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">TRIzol reagent</td>
			<td style="width:196px;">life technologies</td>
			<td style="width:244px;">15596-026</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Zirconia/Silica Beads 0.1 mm</td>
			<td style="width:196px;">BioSpec</td>
			<td style="width:244px;">11079101z</td>
			<td style="width:363px;"></td>
		</tr>
	</tbody>
</table>

fluorescence based primer extension technique to determine transcriptional starting points and cleavage sites of rnases in vivo

蛍光ベースのプライマー伸長（FPE）は、転写開始点またはRNA分子のプロセシング部位を決定するための分子方法である。これは、特定の蛍光標識プライマーおよび変性ポリアクリルアミドゲル電気泳動によって得られたcDNA断片のその後の分析を使用して、目的のRNAを逆転写することによって達成される。同時に、従来のサンガー配列決定反応は、正確な対応するベースにcDNA断片の末端をマッピングするためにゲル上で実行される。生成物をクローン化しなければならず、複数の候補を配列決定5&#39;-RACE（cDNA末端の迅速増幅）とは対照的に、プライマー伸長によって生成されたcDNA断片の大部分は、同時に1つのゲルの実行を検出することができる。また、（逆転写の結果の最終分析に）全体の手順は、1営業日で完了することができる。蛍光標識されたプライマーを用いることによって、危険な放射​​性同位体の使用は、試薬を標識回避することができ、生成物は、電気泳動手順中に検出することができるように、処理時間が低減される。 以下のプロトコールでは、確実かつ迅速にS.（例えば、毒素-抗毒素システムコンポーネントによって）開始点及びRNAプロセッシング部位、転写を推定するRNAの5 &#39;末端を検出するためのin vivo蛍光プライマー伸長方法を記載球菌、E。 coliおよび他の細菌。

図6に示されるように、プライマー伸長反応は、目的の転写物の転写開始点を決定するために用いることができる（通常-10および-35要素によって識別される）のプロモーター領域を推定するのを助けることができる。一番上の（最長）のcDNA断片は、mRNAの5 &#39;末端を表し、従って、配列決定ラダーと比較した場合に容易にマッピングすることができる。 <img al...

Watch this Scientific Journal Video about Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo at JoVE.com

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

転写開始点を決定するための蛍光に基づくプライマー伸長技術とのRNaseの切断サイト<em&gt;インビボ</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

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Research

JoVE Journal

Biology

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

26.8K ビュー.  University of Tübingen. We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

ビデオ: Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.

Proboscis Extension Response PER Assay in Drosophila

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

ショウジョウバエにおけるテングザル拡張レスポンス（PER）アッセイ

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

生物学

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

のためのメゾスコピック蛍光トモグラフィー生体内</em開発の>イメージングショウジョウバエ</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

プライマー伸長キャプチャ：重縮退DNAソースからターゲットシーケンスの取得

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

遺伝子調節の1分子蛍光イメージング<emインビボ&gt;</em&gt;開裂により同時翻訳アクティベーションの使用（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

遺伝子発現のDNA相互作用、ディスカバー転写レギュレータ、および新規抗腫瘍剤を同定：タンパク質を研究するために、定量的アッセイ

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

プロテオリポソー​​ムベースの流出アッセイのClの単一分子特性を決定する<sup&gt; -</sup&gt;チャネルとトランスポーター

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

代替プロモーターの使用およびマウスBcrp1の転写調節を発見する方法

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

腸管上皮細胞の透磁率を決定する体外と体内アプローチ

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

標的下およびタグ付け下での切断を用いた植物におけるH3K4me3修飾のプロファイリング

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...