Este trabajo fue financiado por el Programa de Ciencias Humanas de subvención RGP0024 Frontera.

1. Preparación del ADN de plantilla y la transcripción in vitro <ol><li> El gen de interés se clona bajo T7 y / o, por ejemplo SP6 promotor transcripcional. </li><li> Para la transcripción in vitro del ADN molde se linealizó con una enzima de restricción apropiada de corte aguas abajo del codón de parada ORF y / o ARNm extremo 3 &#39;. Uno tiene que verificar linealización completa del ADN de plásmido mediante la ejecución del producto de digestión de restricción en la electroforesis en gel de agarosa. </li><li> El plásmido linealizado se utiliza en la reacción de transcripción in vitro. Diferentes concentraciones de ADN molde se puede analizar para determinar las concentraciones de ADN óptima requerida para la transcripción in vitro. Por lo general, con el kit de alta Megascript Ambion de transcripción rendimiento (Ambion / Life Technologies, Grand Island, NY), 1 mg de ADN linearizado da 40-60 g de ARNm. En la transcripción in vitro se realiza siguiendo las instrucciones del fabricante (Ambion / Life Technologies, Grand Island, NY). </li><li> Tras la transcripción in vitro del mRNA se purificó por precipitación de cloruro de litio de acuerdo con las instrucciones del fabricante (Megascript Kit de alta Ambion de transcripción de rendimiento). </li><li> integridad ARNm se ve verificada por electroforesis en geles de acrilamida o agarosa. </li></ol> 2. Celular in vitro traducción libre Para la traducción in vitro usando R abbit R eticulocyte lisado, RRL (Promega, Madison, WI), siga los siguientes pasos: <ol><li> Preparar 100 l en la enseñanza de traducción in vitro de reacción fabricante siguiente. En pocas palabras, en agua libre de nucleasa añadir 2 l mezcla de aminoácidos menos metionina (1 mM), 10 unidades de inhibidor de RNasa (Invitrogen / Life Technologies, Grand Island, NY), 20 Ci de los desechos radiactivos [S 35]-metionina (MP Biomedicals, Solon, OH), 70 l de lisado de reticulocitos de conejo (Promega, Madison, WI). </li><li> Incubar ºe reacción a 30 ° C durante 5 minutos para pre caliente la reacción de traducción. Añadir 2 g de ARNm para la reacción de traducción y se incuba a 30 ° C durante 5 min. </li><li> Se detiene la reacción mediante la colocación de la reacción de traducción en hielo. </li></ol> 3. El aislamiento de polipéptidos nacientes de reacción de traducción in vitro <ol><li> Para aislar polipéptido naciente obligado a ribosomas (polisomas), la reacción de traducción se coloca sobre la parte superior de 4,5 ml de 30% de glicerol en 10 mM, pH tampón Tris-HCl 7,6, que contenía 100 mM de KCl, 10 mM de MgCI 2, y se centrifugó a 100.000 xg en un TLA-110 del rotor (Beckman Coulter, Inc., Brea, CA) durante 1 hora a 4 ° C. </li><li> El sobrenadante se retira cuidadosamente adicional. </li><li> El polysomal pastilla que contiene polipéptidos nacientes se resuspende en un volumen pequeño (10-15 l) de 1 mM, pH tampón Tris-HCl 7,6, que contiene 0,5 mg / ml de ribonucleasa A, (Invitrogen / Life Technologies, Grand Island, NY) y se incubaron durante 30 min a 37 ° C. En ordenpara mejorar la hidrólisis del enlace éster peptidil-ARNt, se añade NaOH a una concentración final de 10 mM, y la incubación se continúa durante 30 minutos adicionales. </li></ol> In vitro reacción de traducción del mRNA y montado sin realizó bajo las mismas condiciones (seguido por aislamiento de los péptidos nacientes como se ha descrito) serviría como un mayor control. El tratamiento de la reacción de traducción (s) con puromicina antes de someter el extracto a centrifugación en gradiente de densidad puede servir como un control adicional. 4. La solución del polipéptido naciente en Tris-tricina SDS-PAGE <ol><li> Los polipéptidos nacientes aislados se resuelven y se analizaron en Tris-tricina SDS-PAGE. Tenga en cuenta que la cantidad del material cargado en el gel depende de la eficiencia de etiquetado proteína, la eficiencia en la traducción de proteínas y muchos otros parámetros. La cantidad de material cargado debe ajustarse empíricamente para permitir que los BESt visualización y la separación de cadenas de polipéptidos individuales nacientes. </li><li> Después de geles de electroforesis se fijan, se secó al vacío utilizando gel de tintorero y se sometió a autorradiografía. La distribución de los polipéptidos nacientes se observan utilizando phosphoimaging. </li></ol> 5. Los resultados representativos Bovina gamma-B Cristalina se tradujo en lisado de reticulocitos de conejo, así como en E. E. S30 Sistemas Extracto (Promega, Madison, WI), seguido por aislamiento de nacientes cadenas polipeptídicas. La Figura 1 muestra los pasos implicados en el aislamiento de ribosoma cadenas nacientes consolidados. En el presente estudio el objetivo fue poner a prueba, si se cambia el entorno del repertorio de la traducción, es decir de los tRNAs (conocido por ser sustancialmente diferentes de los mamíferos (RRL) y los sistemas procariotas (E. coli) debido a las diferencias en el codón sesgo entre los organismos) puede alterar movimiento ribosoma a lo largo de ARNm y efectuar la distribución de los sitios de pausa de traducción.Movimiento ribosoma alterada debería conducir a cambios en la distribución de tamaños de polipéptidos nacientes acumulan durante la traducción, así como sus intensidades relativas. Intensidades relativas de las bandas de la duración de la pausa. Los polipéptidos nacientes se aislaron y se resolvieron en 16,5% T, 6% de C Tris-tricina SDS-PAGE (Figura 2). Gamma-B Cristalina es una proteína de 20 kDa. La observación de polipéptidos nacientes de no idénticos longitudes e intensidades en RRL y E. sistemas coli indica que el movimiento de los ribosomas a lo largo de ARNm en estos dos sistemas sigue una cinética diferentes de traducción. Por ejemplo, se observó un destacado grupo de 17,7 kDa en E. E. lisado y no en el sistema de RRL, que sugiere que hay un sitio adicional pausa traslacional en la región extremo 3 &#39;del ARNm (codificación región C-terminal de Gamma B Cristalina), cuando se traduce en E. E. del sistema. Tomamos nota de que la gamma-B puertos Cristalina tándem codones Arg (AGG 153 y 154 AGA) que son frecuentes en sistemas de mamíferos, pero se sabe que son extremadamente raros y traducido lentamente en E. coli. un nuevo péptido naciente acumulando en E. sistema de E. coli (~ 17,7 kDa Figura 2.2) es causado probablemente por una pausa en los ribosomas estos codones raros en tándem. Nótese que las bandas no se puede observar en la ausencia de ARNm (Figura 2,3) y que el tratamiento puromicina elimina la mayor parte de las cadenas nacientes de polisomas (Figura 2.3). El tratamiento prolongado con puromicina (&gt; 15 min) elimina todas las cadenas nacientes (datos no presentados). Esto confirma que los productos polipeptídicos observadas son las cadenas de ribosomas asociados nacientes, en lugar de mRNPs cosedimenting con polisomas. Como se mencionó anteriormente, el sesgo codón es organismo, así como tejidos específicos. El ejemplo representativo se describe aquí, indica claramente que cambiar el entorno del repertorio de la traducción, es decir de tRNAs / codónsesgo puede conducir a alteraciones del movimiento del ribosoma a lo largo del ARNm. Evidentemente, esta sencilla técnica no sólo puede permitir la identificación de los sitios de pausa de traslación a lo largo de ARNm, sino que también permite una rápida comparación de los sitios de traducción de pausa entre los diferentes sistemas. <img alt="Figura 1" src="/files/ftp_upload/4026/4026fig1.jpg" /> Figura 1. Esquema explicar los pasos implicados en el aislamiento de ribosoma polipéptidos nacientes consolidados. Bovina gamma-B Cristalina secuencia ORF (528 pares de bases) fue clonado aguas abajo del promotor T7 en pBSKS +. Para la transcripción in vitro del ADN molde se linealizó por tratamiento con EcoRI. Plantilla linealizado fue utilizado para la transcripción in vitro. Promotor T7 en molde de ADN es reconocida por la RNA polimerasa T7 que transcribe el aguas abajo gamma-B gen Cristalina. ARNm se purifica usando cloruro de litio método de precipitación. El mRNA purificado se utiliza para la traducción in vitro. Siguienteincubación, la reacción de traducción se coloca sobre la parte superior de solución de glicerol al 30% y se centrifuga durante 1 hora a 4 ° C a 100.000 xg para aislar ribosoma polipéptidos nacientes consolidados. <img alt="Figura 2" src="/files/ftp_upload/4026/4026fig2.jpg" /> Figura 2. El aislamiento de la especie bovina B gamma-cristalinas nacientes polipéptidos traducidos en lisado de reticulocitos de conejo (RRL) y E. Lisado coli. 2 ARNm g se mezcló en 100 l de reacción que contiene RRL o E. E. S30 Extracto System (Promega, Madison, WI) de acuerdo con las instrucciones del fabricante con 20 Ci [35 S]-metionina y se incubaron a 30 ° C durante 5 min. Después de la incubación de la reacción de traducción se estratificó en la parte superior de 4,5 ml de 30% de glicerol en 10 mM, pH tampón Tris-HCl 7,6, que contenía 100 mM de KCl, 10 mM de MgCI 2, y se centrifuga durante 1 hora a 4 ° C y 100.000 g X en un rotor TLA-110 (Beckman Coulter, Inc., Brea, CA). El aislado precipitado polyribosome fue resuspended en 20 l de 1 mM de Tris-HCl pH 7,6, que contiene 0,5 mg / ml de ribonucleasa A (Invitrogen / Life Technologies, Grand Island, NY), seguido por tratamiento con NaOH (como se describe en la sección de protocolo). Polipéptidos nacientes se resolvieron en 16,5% T, 6% de C Tris-Tricina gel de SDS PAGE. El gel se seca y se expone a autorradiografía. Tras la exposición, el gel se escanea con escáner de imágenes Typhoon de GE Healthcare. La figura 2.1 muestra el gel con polipéptidos nacientes aislado y resuelto después de las reacciones de traducción realizado en dos sistemas diferentes (RRL y E. coli). Figura 2.2 El gel fue analizado por Image Quant TL ., software v2005 y se utiliza aquí como un ejemplo para mostrar que el peso molecular y la intensidad de los polipéptidos nacientes que se acumulan en dos sistemas pueden ser fácilmente analizado y comparado la figura 2.3 se muestran dos tipos de controles: polipéptidos nacientes aislados y se resolvieron en SDS-PAGE después de la traducción Las reacciones fueron ensambladassin ARNm y / o después de las reacciones de traducción fueron tratados con puromicina (5 min, concentración final. 1 mM) antes de la reacción se sometió a centrifugación.

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No hay conflictos de interés declarado.

Para obtener resultados reproducibles, la calidad y la concentración de los componentes utilizados para la transcripción in vitro y reacciones de traducción son críticos. En el presente estudio hemos utilizado los kits disponibles comercialmente y los extractos que proporcionan datos altamente reproducibles, si se maneja con cuidado. Sin embargo, competentes traducción-extractos pueden prepararse a partir de la celda de su elección, si es necesario. Calidad de ARNm pueden afectar a la traducción...

<table border="1"><tbody><tr><td> Nombre del reactivo / Kit </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Megascript T7 Kit de Transcripción de alta rentabilidad </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> Inhibidor de ribonucleasa </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> Ribonucleasa-A </td><td> Invitrogen </td><td> 12091 </td></tr><tr><td> Sistema de lisado de reticulocitos de conejo, nucleasa tratada </td><td> Promega </td><td> L4960 </td></tr><tr><td> E. coli S30 Extracto del Sistema de las plantillas lineales </td><td> Promega </td><td> L1030 </td></tr><tr><td> Centrifugación </td><td> Beckman Coulter </td><td> Optima TLX Ultracentrífuga </td></tr><tr><td> De almacenamiento de fósforo autorradiografíay </td><td> GE Healthcare </td><td> El tifón 9410 Imager modo variable </td></tr><tr><td> Software para análisis polipéptido naciente </td><td> GE Healthcare </td><td> Image Quant TL, v2005 </td></tr></tbody></table>

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

La tasa de alargamiento de traslación no es uniforme. mRNA estructura secundaria, el uso de codones y ARNm proteínas asociadas pueden alterar el movimiento del ribosoma en el mensaje para una revisión ver 1. Sin embargo, ahora se acepta ampliamente que el uso de codones sinónimos es la principal causa de la falta de uniformidad tasa de elongación de traducción 1. Codones sinónimos no se utilizan con frecuencia idéntica. Un sesgo existe en el uso de codones sinónimos con algunos codones utilizados con mayor frecuencia que los otros 2. Codón sesgo es organismo, así como tejido específico 2,3. Además, la frecuencia de uso del codón es directamente proporcional a las concentraciones de cognado ARNt 4. Así, un codón de frecuencia utilizado tendrá mayor multitud de ARNt correspondientes, que implica, además, que un codón frecuente se traducirá más rápido que un infrecuente una. Así, las regiones en ARNm enriquecido en los codones raros (sitios potenciales de pausa), por regla general más lento movimiento ribosoma en la MessaGE y causa la acumulación de péptidos nacientes de los respectivos tamaños 5-8. Estos sitios de pausa puede tener un impacto funcional en la expresión de la proteína, la estabilidad del mRNA y plegamiento de proteínas para una revisión véase 9. De hecho, se ha demostrado que el alivio de los sitios de pausa tales puede alterar movimiento ribosoma del ARNm y, posteriormente, puede afectar a la eficiencia de la co-traduccional plegamiento de proteínas (in vivo) 1,7,10,11. Para comprender el proceso de plegamiento de proteínas in vivo, en la célula, que finalmente se acopla con el proceso de síntesis de proteínas es esencial para obtener información integrales en el impacto de uso del codón / ARNt contenido en el movimiento de los ribosomas a lo largo de ARNm durante la elongación traslacional . Aquí se describe una técnica simple que se puede utilizar para localizar los sitios principales de pausa de traducción para un ARNm dado traducido en diversos sistemas libres de células de 6-8. Este procedimiento se basa en el aislamiento de la naciente accumulati polipéptidosng en ribosomas durante la traducción in vitro de un ARNm diana. La razón es que en los codones de baja frecuencia, el aumento en el tiempo de residencia de los resultados ribosomas en mayores cantidades de péptidos nacientes de las dimensiones correspondientes. En ARNm transcrito in vitro se utiliza para in vitro reacciones de traducción en la presencia de aminoácidos marcados radiactivamente para permitir la detección de las cadenas nacientes. Con el fin de aislar ribosoma enlazados complejos polipeptídicas nacientes la reacción de traducción se coloca sobre la parte superior de solución de glicerol 30%, seguido por centrifugación. Polipéptidos nacientes en polysomal pellet se trata adicionalmente con ribonucleasa A y se resolvieron por SDS-PAGE. Esta técnica puede ser potencialmente utilizado para cualquier proteína y permite el análisis de movimiento a lo largo de ARNm al ribosoma y la detección de los sitios de pausa principales. Además, este protocolo puede ser adaptado para estudiar los factores y condiciones que pueden alterar el movimiento del ribosoma y, por tanto, potencialmente, también pueden alterar ºfunción e / conformación de la proteína.

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 técnica para identificar sitios traduccionales pausa en ARNm se describe. Este procedimiento se basa en el aislamiento de polipéptidos nacientes acumulan en los ribosomas durante la traducción in vitro de un ARNm diana, seguido por el análisis del tamaño de las cadenas nacientes utilizando una electroforesis en gel de desnaturalización.

El aislamiento de los ribosomas polipéptidos nacientes Bound<em&gt; In vitro</em&gt; Para la identificación de sitios de traslación pausa a lo largo del ARNm

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

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

16.2K vistas.  Cleveland State University. Una técnica para identificar sitios traduccionales pausa en ARNm se describe. Este procedimiento se basa en el aislamiento de polipéptidos nacientes acumulan en los ribosomas durante la traducción in vitro de un ARNm diana, seguido por el análisis del tamaño de las cadenas nacientes utilizando una electroforesis en gel de desnaturalización.

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 método para identificar transcriptoma en todo los sitios de unión de las proteínas de unión a ARN

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

Investigación

Biología

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.

El aislamiento y la In vitro La activación de Caenorhabditis elegans El esperma

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.

Usando alcance para identificar posibles motivos de reglamentación en los genes 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.

En la transcripción in vitro y la nivelación de mRNA de luciferasa Gaussia Seguido por transfección de células HeLa

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.

Usar secuencias de detención SecM como una herramienta para aislar Ribosome polipéptidos enlazados

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.

Síntesis in vitro de ARNm modificado para la inducción de la expresión proteica en las células humanas

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.

Medición de la resistencia de estrés oxidativo<em&gt; Caenorhabditis elegans</em&gt; 96 pocillos en placas de microtitulación

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.

El examen de las proteínas unidas al ADN naciente en células de mamífero utilizando BrdU-chip-Slot-occidental Técnica

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 protocolo para el uso de gen Set enriquecimiento análisis para identificar el modelo Animal adecuado para la investigación traslacional

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 Ensayo de la sumoilación estudiar SUMO E3 ligasa actividad

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