Questo lavoro è stato finanziato dal programma Human Frontier Science concessione RGP0024.

1. Preparazione del DNA template e trascrizione in vitro <ol><li> Il gene di interesse viene clonato sotto T7 e / o ad esempio SP6 promotore di trascrizione. </li><li> Per la trascrizione in vitro del DNA stampo è linearizzato con un enzima di restrizione appropriato taglio a valle del codone di stop ORF e / o mRNA 3 &#39;. Si deve verificare linearizzazione completa del DNA plasmide eseguendo il prodotto della digestione di restrizione elettroforesi su gel di agarosio. </li><li> Il plasmide linearizzato viene utilizzato per la reazione di trascrizione in vitro. Diversa concentrazione di DNA stampo può essere testato per identificare concentrazione ottimale di DNA necessarie per la trascrizione in vitro. Generalmente, con Kit MEGAscript Ambion Trascrizione di alto rendimento (Ambion / Life Technologies, Grand Island, NY), 1 mg DNA linearizzato cede 40-60 ug di mRNA. Trascrizione in vitro viene effettuata seguendo le istruzioni del fabbricante (Ambion / Life Technologies, Grand Island, NY). </li><li> Dopo la trascrizione in vitro l&#39;mRNA viene purificato mediante precipitazione litio cloruro secondo le istruzioni del produttore (Ambion di MEGAscript alta Kit Trascrizione resa). </li><li> integrità mRNA è ulteriormente verificato mediante elettroforesi su gel di acrilammide o agarosio. </li></ol> 2. Traduzione cellulare in vitro gratuito Per la traduzione in vitro utilizzando R abbit R eticulocyte lisato, RRL (Promega, Madison, WI), attenersi alla seguente procedura: <ol><li> Preparare 100 pl in istruzioni del produttore traduzione in vitro reazione successivo. In breve, in acqua priva di nucleasi aggiungere 2 microlitri miscela di aminoacidi meno metionina (1 mM), 10 unità di inibitore della RNasi (Invitrogen / Life Technologies, Grand Island, NY), 20 uCi di sostanze radioattive [35 S]-metionina (Biomedicals MP, Solon, OH), 70 microlitri di lisato di reticolociti di coniglio (Promega, Madison, WI). </li><li> Incubare °e reazione a 30 ° C per 5 min a caldo pre la reazione di traduzione. Aggiungere 2 ug di mRNA alla reazione di traduzione e incubare a 30 ° C per 5 min. </li><li> Arrestare la reazione ponendo la reazione di traduzione in ghiaccio. </li></ol> 3. Isolamento di polipeptidi nascenti dal Reaction traduzione in vitro <ol><li> Per isolare polipeptide nascente associato a ribosomi (polisomi), reazione di traduzione è sovrapposto di 4,5 ml di glicerolo 30% in 10 mM Tris-HCl pH 7,6, contenente 100 mM KCl, 10 mM MgCl 2, e centrifugata a 100.000 xg in un rotore-110 TLA (Beckman Coulter, Inc., Brea, CA) per 1 ora a 4 ° C. </li><li> Il supernatante viene rimosso attentamente ulteriormente. </li><li> Il pellet contenente polysomal polipeptidi nascenti viene risospeso in un piccolo volume (10-15 pl) di 1 mM Tris-HCl pH 7,6, contenente 0,5 mg / ml ribonucleasi A (Invitrogen / Life Technologies, Grand Island, NY), e incubate per 30 min a 37 ° C. Perper migliorare l&#39;idrolisi del peptidil-tRNA legame estere, NaOH viene aggiunto ad una concentrazione finale di 10 mM, e l&#39;incubazione è continuata per ulteriori 30 min. </li></ol> In vitro reazione traduzione assemblato senza mRNA e eseguite alle stesse condizioni (seguita da isolamento di peptidi nascenti come è stato descritto) servirebbe come controllo principale. Trattamento della reazione di traduzione (s) con puromicina prima di sottoporre l&#39;estratto di centrifugazione in gradiente di densità può servire come un controllo aggiuntivo. 4. Risolvere il polipeptide nascente sulla Tris tricina SDS-PAGE <ol><li> I polipeptidi isolati nascenti vengono risolti e analizzati in Tris-tricina SDS-PAGE. Si noti che la quantità del materiale caricato sul gel dipende dalla efficienza di etichettatura proteine, l&#39;efficienza di traduzione di proteine ​​e molti altri parametri. La quantità di materiale caricata deve essere regolato empiricamente per consentire ai BESt visualizzazione e separazione delle singole catene polipeptidiche nascenti. </li><li> Dopo gel elettroforesi sono fissati, essiccati sotto vuoto usando gel Dyer e sottoposti ad autoradiografia. La distribuzione dei polipeptidi nascenti sono osservate usando phosphoimaging. </li></ol> 5. Risultati rappresentativi Bovina Gamma-B sonoro superiore cristallino è stato tradotto nel lisato di reticolociti di coniglio così come in E. coli S30 Sistemi estratto (Promega, Madison, WI) seguito da isolamento del nascenti catene polipeptidiche. figura 1 illustra fasi coinvolte nella isolamento di ribosoma catene legate nascenti. In questo studio lo scopo era quello di testare, se modificando l&#39;ambiente del repertorio cioè traduzione di tRNA (noto per essere sostanzialmente diverso nei mammiferi (RRL) e procarioti (E. coli) sistemi a causa delle differenze di polarizzazione tra gli organismi codone) può alterare il movimento ribosoma lungo mRNA e effettuare la distribuzione dei siti di pausa di traduzione.Altered movimento ribosoma dovrebbe portare a cambiamenti nella distribuzione delle dimensioni di polipeptidi nascenti accumulano durante la traslazione, nonché la loro intensità relative. Intensità relativa delle bande riflettono la durata della pausa. I polipeptidi sono stati isolati e nascenti risolte 16,5% T, 6% C Tris-tricina SDS PAGE (Figura 2). Gamma-B sonoro superiore cristallino è una proteina di 20 kDa. L&#39;osservazione di polipeptidi nascenti di non identici lunghezze e intensità in RRL ed E. sistemi coli indica che il movimento dei ribosomi lungo mRNA in questi due sistemi segue cinetiche traduzione differenti. Per esempio, abbiamo osservato un gruppo importante di 17,7 kDa in E. coli lisato e non nel sistema RRL, suggerendo che vi sia un ulteriore sito pausa traslazionale nel terminale 3 &#39;della regione di mRNA (codifica regione C-terminale di Gamma sonoro superiore cristallino B), quando si traduce in E. coli sistema. Prendiamo atto che Gamma-B sonoro superiore cristallino porti tandem Arg codoni (AGG 153 e AGA 154) che sono frequenti nei sistemi di mammifero, ma sono noti per essere estremamente raro e lentamente tradotto in E. coli. un nuovo peptide nascente accumulando in E. coli del sistema (~ 17,7 kDa Figura 2.2) è probabilmente causato da una pausa ribosomiale a questi codoni tandem rari. Si nota che nessuna bande possono essere osservati in assenza di mRNA (Figura 2.3) e che il trattamento puromicina rimuove la maggior parte delle catene nascenti da polisomi (Figura 2.3). Il trattamento prolungato con puromicina (&gt; 15 min) rimuove tutte le catene nascenti (dati non mostrati). Ciò conferma che i prodotti osservati polipeptidiche sono ribosoma nascenti catene di associati, piuttosto che mRNPs cosedimenting con polisomi. Come menzionato sopra, è polarizzazione codone dell&#39;organismo come pure il tessuto specifico. L&#39;esempio rappresentativo qui descritto, indica chiaramente che modificando l&#39;ambiente del repertorio cioè traduzione di tRNA / codonepolarizzazione può portare a modificare il movimento del ribosoma lungo l&#39;mRNA. Evidentemente, questa semplice tecnica non può soltanto permettere l&#39;identificazione dei siti di pausa traslazione lungo mRNA, ma permette anche rapido confronto dei siti di pausa la traduzione tra diversi sistemi. <img alt="Figura 1" src="/files/ftp_upload/4026/4026fig1.jpg" /> Figura 1. Schema che spiega i passaggi necessari per isolare ribosoma polipeptidi legati nascenti. Bovina Gamma-B sonoro superiore cristallino sequenza di ORF (528 paia di basi) è stato clonato a valle del promotore T7 in pBSKS +. Per la trascrizione in vitro del DNA stampo è stato linearizzato trattandolo con EcoRI. Modello linearizzato è stato utilizzato per la trascrizione in vitro. Promotore T7 nel modello del DNA è riconosciuta da T7 RNA polimerasi che trascrive la valle Gamma-B gene sonoro superiore cristallino. mRNA viene purificato utilizzando cloruro di litio metodo di precipitazione. L&#39;mRNA purificato viene utilizzato per la traduzione in vitro. Seguitoincubazione, la reazione di traduzione è sovrapposto di soluzione di glicerolo 30% e centrifugato per 1 ora a 4 ° C a 100.000 X g per isolare ribosoma polipeptidi legati nascenti. <img alt="Figura 2" src="/files/ftp_upload/4026/4026fig2.jpg" /> Figura 2. Isolamento dei bovini Gamma-B cristallina polipeptidi nascenti tradotti in Coniglio reticolociti lisato (RRL) e E. Lysate coli. 2 mRNA mcg è stato miscelato in 100 microlitri di reazione contenente RRL o E. coli S30 estratto System (Promega, Madison, WI) secondo le istruzioni del produttore con 20 uCi [35 S]-metionina e incubate a 30 ° C per 5 min. Dopo incubazione la reazione di traduzione è stato stratificato sopra di 4,5 ml di glicerolo 30% in 10 mM Tris-HCl pH 7,6, contenente 100 mM KCl, 10 mM MgCl 2, e centrifugato per 1 ora a 4 ° C e 100.000 g in X A-110 TLA rotore (Beckman Coulter, Inc., Brea, CA). Il pellet è stato isolato polyribosome Resutrascorsa in 20 pl di 1 mM Tris-HCl pH 7,6, contenente 0,5 mg / ml ribonucleasi A (Invitrogen / Life Technologies, Grand Island, NY) seguita da trattamento con NaOH (come descritto nella sezione protocollo). Polipeptidi nascenti sono stati risolti sul 16,5% T, C 6% Tris-Tricina gel SDS PAGE. Il gel è stato essiccato ed esposto per autoradiografia. Dopo l&#39;esposizione il gel è stato analizzato utilizzando scanner Typhoon Imaging di GE Healthcare. La figura 2.1 mostra il gel con polipeptidi nascenti isolati e risolti dopo le reazioni di traduzione effettuato in due diversi sistemi (RRL ed Escherichia coli). Figura 2.2 Il gel è stato analizzato da Image Quant TL ., software v2005 e qui utilizzato come esempio per mostrare che il peso molecolare e l&#39;intensità dei polipeptidi nascenti che si accumulano nei due sistemi possono essere facilmente analizzati e confrontati La figura 2.3 mostra due serie di controlli: polipeptidi nascenti isolati e risolti sulla PAGE SDS dopo la traduzione Le reazioni sono state assemblatesenza mRNA e / o dopo le reazioni di traduzione sono stati trattati con puromicina (5 min, conc finale. 1 mM) prima della reazione è stata sottoposta a centrifugazione.

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Per risultati riproducibili, la qualità e la concentrazione dei componenti utilizzati per la trascrizione in vitro e le reazioni di traduzione sono critiche. In questo studio abbiamo usato i kit disponibili in commercio e gli estratti che forniscono dati altamente riproducibili, se maneggiato con cura. Tuttavia, traduzione-competenti estratti possono essere preparati dalla cella di propria scelta, se necessario. Qualità di mRNA può influenzare la traduzione, quindi è della massima importanza per testare l&#...

<table border="1"><tbody><tr><td> Nome del reagente / Kit </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> MEGAscript T7 alta Kit Trascrizione rendimento </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> Ribonucleasi Inhibitor </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> Trans [35 S]-Label </td><td> MP Biomedicals </td><td> 0151006 </td></tr><tr><td> Ribonucleasi-A </td><td> Invitrogen </td><td> 12091 </td></tr><tr><td> Coniglio reticolociti sistema Lysate, nucleasi trattato </td><td> Promega </td><td> L4960 </td></tr><tr><td> Sistema di E. coli per i modelli S30 estratto lineari </td><td> Promega </td><td> L1030 </td></tr><tr><td> Centrifugazione </td><td> Beckman Coulter </td><td> Optima TLX ultracentrifuga </td></tr><tr><td> Stoccaggio fosforo autoradiography </td><td> GE Healthcare </td><td> Typhoon 9410 imager modalità variabile </td></tr><tr><td> Software per l&#39;analisi nascente polipeptide </td><td> GE Healthcare </td><td> Immagine Quant TL, v2005 </td></tr></tbody></table>

isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mrna

Il tasso di allungamento traslazionale è non uniforme. mRNA struttura secondaria, l&#39;uso codone mRNA e proteine ​​associate possono alterare il movimento ribosoma sul messaggio per una rassegna vedi 1. Tuttavia, è ormai ampiamente riconosciuto che l&#39;utilizzo codone sinonimo è la causa primaria di non-uniformi tassi di allungamento traslazionali 1. Codoni sinonimo non vengono utilizzati con frequenza identica. Una polarizzazione esiste l&#39;uso di codoni sinonimo con alcuni codoni usati più frequentemente di altri 2. Codone bias è organismo così come tessuto specifico 2,3. Inoltre, la frequenza di uso dei codoni è direttamente proporzionale alla concentrazione delle affine tRNA 4. Così, un codone frequentemente usato avrà maggiore moltitudine di tRNA corrispondenti, implicando che un codone di frequente si tradurrà più velocemente di un infrequente uno. Così, regioni mRNA arricchito in codoni rari (siti pausa potenziali) saranno di regola rallentare il movimento del ribosoma Message e causa l&#39;accumulo di peptidi nascenti delle rispettive dimensioni 5-8. Questi siti di pausa può avere un impatto funzionale l&#39;espressione della proteina, la stabilità mRNA e folding delle proteine ​​per una rassegna vedi 9. Infatti, è stato dimostrato che la riduzione di tali siti di pausa può alterare il movimento ribosoma mRNA e successivamente possono influenzare l&#39;efficienza di co-traduzionale (in vivo) 1,7,10,11 ripiegamento delle proteine. Per comprendere il processo di ripiegamento delle proteine ​​in vivo, nella cella, che è accoppiato alla fine al processo di sintesi proteica è essenziale di acquisire informazioni complete sull&#39;impatto codone di utilizzo / tRNA contenuto sul movimento dei ribosomi lungo mRNA durante l&#39;allungamento traslazionale . Qui si descrive una tecnica semplice che può essere utilizzata per localizzare siti di traduzione principali pausa per un dato mRNA tradotto in vari sistemi acellulari 6-8. Tale procedura si basa sull&#39;isolamento del nascente polipeptidi accumulating sui ribosomi durante la traduzione in vitro di un mRNA bersaglio. La ragione è che a bassa frequenza codoni, l&#39;aumento del tempo di residenza dei risultati ribosomi in quantità maggiore di peptidi nascenti delle dimensioni corrispondenti. In vitro mRNA trascritti viene utilizzato per reazioni di traslazione in vitro in presenza di amminoacidi marcati radioattivamente per consentire il rilevamento delle catene nascenti. Al fine di isolare ribosoma associazione complessi polipeptide nascente la reazione di traduzione è sovrapposto di soluzione di glicerolo 30% seguito da centrifugazione. Polipeptidi nascenti in polysomal pellet sono ulteriormente trattati con ribonucleasi A e risolti mediante SDS PAGE. Questa tecnica può essere potenzialmente utilizzati per ogni proteina e permette l&#39;analisi di movimento ribosoma lungo mRNA e la rilevazione dei siti di pausa principali. Inoltre, questo protocollo può essere adattato per studiare fattori e condizioni che possono alterare il movimento ribosoma e quindi potenzialmente possono anche alterare the funzione / conformazione della proteina.

Watch this Scientific Journal Video about Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA at JoVE.com

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

Una tecnica per identificare i siti di pausa traslabile su mRNA viene descritto. Questa procedura è basata su isolamento di polipeptidi nascenti accumulano sui ribosomi durante traduzione in vitro di un mRNA bersaglio, seguita dall&#39;analisi dimensione delle catene nascenti elettroforesi utilizzando un gel denaturante.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

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

isolation-ribosome-bound-nascent-polypeptides-vitro-to-identify

Research

JoVE Journal

Biology

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

16.2K Views.  Cleveland State University. Una tecnica per identificare i siti di pausa traslabile su mRNA viene descritto. Questa procedura è basata su isolamento di polipeptidi nascenti accumulano sui ribosomi durante traduzione in vitro di un mRNA bersaglio, seguita dall'analisi dimensione delle catene nascenti elettroforesi utilizzando un gel denaturante.

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

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - un metodo per identificare trascrittoma livello i siti di legame delle proteine ​​RNA binding

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Ricerca

Biologia

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Isolamento e In vitro Attivazione di Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Utilizzando SCOPE per identificare potenziali Motivi regolamentari in geni Coregulated

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Trascrizione in vitro e tappatura mRNA Luciferase Gaussia Seguito da cellule HeLa Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

In vitro Sintesi di mRNA modificata per l&#39;induzione di espressione della proteina nelle cellule umane

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Misura Stress ossidativo Resistenza<em&gt; Caenorhabditis elegans</em&gt; In 96 pozzetti micropiastre

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Esame di proteine ​​legate al DNA Nascente in cellule di mammifero utilizzando la tecnica BrdU-chip-Slot-occidentale

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Un protocollo per l'utilizzo di Gene imposta analisi di arricchimento per identificare il modello animale adeguato per la ricerca traslazionale

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

In Vitro Saggio di SUMOilazione per studiare l'attività E3 ligasi SUMO

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...