La investigación presentada aquí fue apoyado por el Departamento de Salud de Pennsylvania, la Asociación Médica Ellison, V y Los fundamentos de esmeralda.

1. Expresión de proteínas recombinantes TERT Tc <ol><li> Inocular 6 L de 2YT los medios de comunicación (6 x 2 L Desconcertado matraces Erlenmeyer que contiene 1 l de caldo de 2YT en cada uno) de seis nuevas placas de las células transformadas pLysS Rosetta (DE3) que contiene la síntesis Tc TERT plásmido con un TEV escindibles N-terminal hexahistidina- etiqueta. </li><li> Se cultivan las células a 37 ° C mientras se agita a 220 rpm a una OD 600 de 0.5-0.6. </li><li> Inducir la expresión de proteínas mediante la adición de 1 mM IPTG. </li><li> Reduzca la temperatura a 30 ° C, lenta agitación a 150 rpm, y permiten a las células expresan la proteína de 04.05 h. </li><li> Las células de la cosecha por centrifugación a 4 ° C y 3500 rpm durante 30 minutos. </li><li> Resuspender cada una L sedimento celular en 20 ml de solución tampón que contiene 25 mM Tris, pH 7,5, 10% de glicerol, 0,5 M KCl, 15 mM imidazol, 5 mM β-mercaptoetanol, y 0,1 mM de fluoruro de sulfonilo fenilmetil (PMSF) y benzamidina. </li><li> Flash congelar mezcla de células se resuspendieron goteando lentamente por las células en un tubo de plástico de 50 ml cónico lleno de nitrógeno líquido, para que cada gota se congela en el impacto en el N2 líquido. Almacenar células congeladas a -80 ° C hasta el momento de purificación de proteínas. </li></ol> 2. La purificación de Tc TERT <ol><li> Células descongele a temperatura ambiente hasta obtener una consistencia líquida que se alcanza y luego pasar a vasos de metal en el hielo de ultrasonidos. </li><li> Sonicar las células en hielo durante 2 minutos en intervalos de 30 segundos con 30 segundos hace una pausa, aproximadamente a 51 W de potencia. </li><li> Centrifugar las células lisadas a 18.000 rpm durante 20 minutos para separar las fracciones de proteína soluble e insoluble y retirar con cuidado el sobrenadante por cromatografía de afinidad de níquel. </li><li> Equilibrar el Ni-NTA (níquel-ácido nitrilotriacético) columna con un tampón que contiene 25 mM Tris, pH 7,5, 10% de glicerol, 0,5 M KCl, 15 mM imidazol, 5 mM β-mercaptoetanol, y 0,1 mM PMSF y benzamidina. </li><li> Sobrenadante de carga sobre la resina y recoger el flujo a través. </li><li> Lavar la columna con un tampón que contiene 25 mM Tris, pH 7,5, glicerol al 10%, 500 mM KCl, 15 mM imidazol, 5 mM β-mercaptoetanol, y 0,1 mM PMSF para eliminar todas las proteínas residuales y no específicamente de ruedas. </li><li> Eluir Tc TERT de la resina Ni-NTA utilizando un gradiente de imidazol de 15 - 300 mm y cobrar fracciones de 3 ml para el análisis de SDS-PAGE. </li><li> Utilizar el análisis de página para determinar qué fracciones de proteína TERT contienen Tc y concentrar estas fracciones en un Amicon 30 kDa (Millipore) de filtro. </li><li> Concentrado Tc fracciones TERT en un volumen inferior a 10 ml para una mayor purificación. </li><li> El TERT Tc se purifica aún más sobre la columna de intercambio catiónico (SA poros, Applied Biosystems) pre-equilibrada con un tampón que contiene 25 mM Tris, pH 7,5, glicerol al 10%, 500 mM KCl y 1 mM DTT. </li><li> Cargar la mezcla de proteínas en la columna SA poros y lavar para eliminar todas las proteínas y ácidos nucleicos contaminantes. </li><li> Lavar la columna con un tampón que contiene 500 mM KCl seguido por 700 mM KCl para eliminar cualquier contaminante restante. </li><li> Eluir la proteína TERT Tc de la columna SA poros con un buffer M KCl. En este punto, la proteína debe ser más del 99% de pureza. </li><li> Eliminar cualquier agregados de la proteína al pasar la proteína en una columna de Superdex-200 de tamaño (el tamaño de cromatografía de exclusión - GE Healthcare), previamente equilibrada con un tampón que contiene 25 mM Tris, pH 7,5, glicerol al 10%, 500 mM KCl y 1 mM TCEP. </li></ol> 3. Tribolium castaneum escarabajo cultura <ol><li> El T. castaneum escarabajos en un principio como un regalo por el Dr. S. Brown, de la División de Biología de la Universidad Estatal de Kansas. </li><li> Inicio T. castaneum la cultura mediante la adición de cien escarabajos adultos a un contenedor, con 28,5 g de harina y 1,5 g de levadura seca de panadería. </li><li> Tienda de las culturas del escarabajo a temperatura ambiente y en un ambiente oscuro y húmedo de tres a cuatro semanas para permitir la reproducción y crecimiento. Coloque un vaso con agua en el área de almacenamiento del escarabajo para asegurar un ambiente húmedo. </li><li> Cosecha de las larvas de escarabajos, según sea necesario para el aislamiento de ARN. Deje que el resto de las larvas a madurar a los adultos, manteniéndolos en la misma cultura. </li><li> Cada mes, la transferencia de los escarabajos más activos (no más de un centenar) en un nuevo frasco que contiene la harina y la levadura fresca para reponer el suministro de alimentos. </li></ol> 4. T. castaneum total aislamiento de ARN <ol><li> Cosecha de 20 larvas del escarabajo de la cultura (cada cultura debe contener entre 500 a 1000 larvas), separándolos de los escarabajos adultos y la cultura de harina / levadura. Eliminar las partículas de harina o la levadura unido a los escarabajos antes de molerlos. </li><li> Esterilizar la zona de banquillo, el mortero y el pilón (considerar el tratamiento de los guantes, así) que se utiliza para este procedimiento con RNaseZap (Ambion, Inc.) para eliminar todos los rastros de ribonucleasas antes de la molienda de los escarabajos. </li><li> Triturar las larvas en N2 líquido en un polvo fino utilizando un mortero y ttransferencia se entiende el polvo a un tubo de centrífuga de 50 ml, mientras que el mantenimiento de la N 2 líquido para asegurarse de que el polvo se queda congelado hasta que el tampón de extracción, se añade. </li><li> Permita que el exceso de líquido se evapore N 2 a partir del polvo. Después de que el N 2 líquido se haya evaporado, homogeneizar el polvo de escarabajo en 200 l de tampón de extracción (por las larvas del escarabajo 20) que contiene 25 mM Tris-HCl, 5 mM β-mercaptoetanol, 1 mM EGTA, 0,1 benzamidina mM, 200 mM KCl, 10 % de glicerol, 10 mM imidazol y 20U de RNasin, pH 7,5 e incubar en hielo durante 30 minutos. </li><li> Centrifugar el homogeneizado a 15.000 rpm durante 20 minutos a 4 ° C, recoger el sobrenadante y el flash congelar la muestra en N 2 líquido. Si es necesario, las muestras pueden ser almacenadas a -80 ° C durante la noche. </li><li> Extraer el ARN total utilizando el RNeasy Mini Kit (Qiagen). </li></ol> 5. Reconstitución in vitro de la telomerasa Tribolium castaneum <ol><li> Mezclar 20 g de proteínas recombinantes de etiquetado Su TERT Tc con 50 l de T. castaneum ARN total en el tampón de unión que contiene 25 mM Tris-HCl, 200 mM KCl, 10% de glicerol, 5 mM β-mercaptoetanol, y 10 mM imidazol, pH 7,5. </li><li> Incubar el TERT Tc - total mezcla de ARN durante dos horas a 22 ° C en T. castaneum lisado en la presencia de un inhibidor RNasin para evitar la degradación del ARN. </li><li> Purificar el complejo en una columna de Ni-NTA para eliminar las impurezas lisado y el ARN en exceso. </li></ol> 6. Repetir la telomerasa protocolo de amplificación (TRAP) ensayos <ol><li> Mezcle 4 mg de Ni-NTA purificado T. castaneum telomerasa en 50 l de amortiguación elongación que contiene 20 mM Tris-HCl (pH - 8,3), 7,5 mM MgCl2, 63 mM KCl, 0,05% de Tween 20, 1 mM EGTA, el 0,01% de BSA, 0,5 mM dNTPs, 1 M ATC-TS ADN primer (5&#39;-AAGCCGTCGAGCAGAGTC-3 &#39;), y se incuba la mezcla a 30 ° C durante 60 minutos para permitir el alargamiento de la cartilla de ADN ATC-TS por el reconstituido T. castaneum telomerasa. </li><li> Extracto de la alargada ADN de cadena sencilla (ssDNA) mediante la adición de un volumen igual de fenol cloroformo a la mezcla de reacción. Agite la mezcla suavemente hasta que la emulsión es homogénea. </li><li> Centrifugar la muestra a 12.000 rpm durante cinco minutos a 4 ° C para separar el ADN de cadena simple de los contaminantes. </li><li> La transferencia de la capa superior acuosa de la muestra a un tubo eppendorf de 1,5 ml, añadir una concentración final de 10 mM NaOAc (pH 5,0), 1 mM de MgCl 2, y el 66,4% EtOH y luego dejar que la muestra para precipitar la noche a -20 ° C. </li><li> Girar la muestra a 15.000 rpm durante treinta minutos a 4 ° C para que sedimenten las ssDNA precipitado. Se decanta la solución con una pipeta y luego añadir 200 l de EtOH al 70% de la muestra para lavar restantes NaOAc y MgCl 2 de la muestra. Centrifugar la muestra durante cinco minutos a la misma velocidad y temperatura para volver a pellet de la muestra. </li><li> Decantar la solución EtOH con una pipeta y eliminar los rastros restantes de etanol al permitir que el tubo eppendorf para la incubación de abrir a temperatura ambiente durante tres minutos. Contar con la presencia de etanol en la muestra va a interferir con el paso de amplificación de PCR. </li><li> Resuspender el precipitado ssDNA (que contiene el producto telomerasa alargada y el primer ATC-TS) en 50 l de tampón de reacción de PCR que contiene 10 mM Tris-HCl (pH 8), 50 mM KCl, 2 mM MgCl 2, dNTPs 100 mM (dATP, dGTP y dTTP), 10 M [32 P] dCTP, 1 M ATC-CX primer (5&#39;-GTGTGACCTGACCTGACC-3 &#39;) y 1,25 U de Taq polimerasa de ADN. </li><li> Amplificar por PCR los productos de alargamiento de la telomerasa (29 ciclos de 94 ° C durante 30 segundos, 50 ° C durante 30 segundos y 72 ° C durante 1 minuto), de modo que sean visibles en un gel de poliacrilamida al 12%. </li><li> Pre-ejecutar el gel del 12% con Tris-borato-EDTA (TBE) en funcionamiento un tampón que contiene 44,5 mM Tris, 44,5 mM de ácido bórico, 1 mM EDTA, pH 8 durante treinta minutos antes de correr las muestras de PCR. Después de pre-ejecución y la carga del gel con la reacción de PCR, ejecute el gel a 185 voltios de 1.5 a 2 horas a 4 ° C o en hielo. </li><li> Secar el gel y el uso de la placa de fósforo de imagen para visualizar la actividad. </li></ol> 7. Los resultados representativos: Un ejemplo de la TERT purificada Tc después de cromatografía de exclusión se muestra en la Figura 2. La proteína concentrada después de la purificación se ejecuta en un 12% SDS-gel de poliacrilamida y se demuestra que es más de 99% (Figura 2). La exclusión de tamaño (S200) cromatograma también se proporciona (Figura 2B) para demostrar la pureza y la falta de agregados de la muestra de proteína purificada. Un rendimiento de 5 mg de proteína después de todos los pasos de purificación se han completado, aunque generalmente se obtiene el rendimiento final puede variar. Un ensayo TRAP representante de la reconstituida T. telomerasa castaneum se muestra en la Figura 3 como se informó anteriormente por Mitchell et al. 12. La escala gradual de los productos de la telomerasa se puede ver en la calle 1, sin embargoestán ausentes tanto en la telomerasa RNasa tratados y la TERT sitio activo de las muestras de mutantes (D251A) en las calles 2 y 3, respectivamente. <img src="/files/ftp_upload/2799/2799fig1.jpg" alt="Figura 1"/> Figura 1. Diagrama de flujo de los pasos necesarios para la reconstitución de la recombinación del TERT Tc. En primer lugar, la recombinación del TERT Tc está sobre-expresado en E. coli. La proteína se purifica por el níquel, el intercambio de cationes y cromatografía de exclusión por tamaño. El T. escarabajos castaneum se cultivan en casa y sus larvas se utilizan para aislar el ARN total. El purificado Tc TERT y el ARN total se mezclan para reconstituir el complejo RNP telomerasa, y un posterior ensayo TRAP se utiliza para confirmar el complejo RNP es una telomerasa activa. <img src="/files/ftp_upload/2799/2799fig2.jpg" alt="Figura 2"/> Figura 2. Tamaño cromatograma de exclusión y el análisis de SDS-PAGE de la proteína TERT Tc. (A) 12% SDS-gel de poliacrilamida de la proteína TERT Tc después de cromatografía de exclusión. La proteína TERT Tc (70 kDa), emigra más rápido en el gel SDS-PAGE, ya que tiene un alto punto isoeléctrico (pI - 9,8). (B) Cromatograma de exclusión de tamaño (Superdex S200) de la proteína TERT Tc. <img src="/files/ftp_upload/2799/2799fig3.jpg" alt="Figura 3"/> Figura 3. Ensayos trampa de la in vitro reconstituido Tribolium castaneum telomerasa adaptado de Mitchell et al. 12. Pista 1: tipo salvaje Tc TERT y el ARN total aislado de las larvas del escarabajo. Carril 2: de tipo salvaje Tc TERT y RNasa tratados ARN total de las larvas y los extractos de células. Carril 3: sitio activo mutantes Tc TERT (D251A) y el escarabajo de ARN total de las larvas de las larvas.

<ol>
	<li>Gillis, A. J., Schuller, A. P., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+Tribolium+castaneum+telomerase+catalytic+subunit+TERT.">Structure of the Tribolium castaneum telomerase catalytic subunit TERT.</a> Nature. 455, 633-637 (2008).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+specific+telomere+terminal+transferase+activity+in+Tetrahymena+extracts.">Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.</a> Cell. 43, 405-413 (1985).</li><li>Harley, C. B., Futcher, A. B., Greider, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+shorten+during+ageing+of+human+fibroblasts.">Telomeres shorten during ageing of human fibroblasts.</a> Nature. 345, 458-460 (1990).</li><li>Hayflick, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Limited+in+vitro+Lifetime+of+Human+Diploid+Cell+Strains.">The Limited in vitro Lifetime of Human Diploid Cell Strains.</a> Exp Cell Res. 37, 614-636 (1965).</li><li>Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres:+no+end+in+sight.">Telomeres: no end in sight.</a> Cell. 77, 621-623 (1994).</li><li>Kim, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+association+of+human+telomerase+activity+with+immortal+cells+and+cancer.">Specific association of human telomerase activity with immortal cells and cancer.</a> Science. 266, 2011-2015 (1994).</li><li>Bodnar, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+of+life-span+by+introduction+of+telomerase+into+normal+human+cells.">Extension of life-span by introduction of telomerase into normal human cells.</a> Science. 279, 349-352 (1998).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telomere+terminal+transferase+of+Tetrahymena+is+a+ribonucleoprotein+enzyme+with+two+kinds+of+primer+specificity.">The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity.</a> Cell. 51, 887-898 (1987).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+telomeric+sequence+in+the+RNA+of+Tetrahymena+telomerase+required+for+telomere+repeat+synthesis.">A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis.</a> Nature. 337, 331-337 (1989).</li><li>Jacobs, S. A., Podell, E. R., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+essential+N-terminal+domain+of+telomerase+reverse+transcriptase.">Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase.</a> Nat Struct Mol Biol. 13, 218-225 (2006).</li><li>Rouda, S., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+RNA-binding+domain+of+telomerase:+implications+for+RNA+recognition+and+binding.">Structure of the RNA-binding domain of telomerase: implications for RNA recognition and binding.</a> Structure. 15, 1403-1412 (2007).</li><li>Mitchell, M., Gillis, A., Futahashi, M., Fujiwara, H., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+telomerase+catalytic+subunit+TERT+binding+to+RNA+template+and+telomeric+DNA.">Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA.</a> Nat Struct Mol Biol. 17, 513-518 (2010).</li></ol>

El método aquí presentado permite la producción de grandes cantidades de la subunidad catalítica de la T. castaneum TERT de la telomerasa en forma soluble y activa para realizar estudios estructurales y bioquímicos. El método de Tc TERT sobre-expresión es sensible a los cambios sutiles en la temperatura o la densidad de las células de las arriba indicadas y puede afectar dramáticamente los niveles de expresión de la proteína. En concreto, hemos encontrado que la inducción de las células pa...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> Rosetta (DE3) pLysS células </td><td> Novagen </td><td> 70956 </td></tr><tr><td> 2YT caldo </td><td> Teknova </td><td> Y0215 </td></tr><tr><td> IPTG </td><td> Oro Biotecnología </td><td> I2481C </td></tr><tr><td> Misonix Sonicator 3000 </td><td> Qsonica, LLC. </td><td></td></tr><tr><td> ÄKTA purificador FPLC </td><td> GE Ciencias de la Vida </td><td></td></tr><tr><td> Ni-NTA SuperFlow resina </td><td> Qiagen </td><td> 30410 </td></tr><tr><td> Amicon Ultra-15 Dispositivo de filtro centrífugo </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> POROS 50 SA intercambio de cationes fuerte embalaje </td><td> Applied Biosystems </td><td> 1-3359-06 </td></tr><tr><td> POROS 50 HQ intercambio de cationes fuerte embalaje </td><td> Applied Biosystems </td><td> 1-2559-06 </td></tr><tr><td> Superdex 200 10/300 de exclusión por tamaño de columna </td><td> GE Ciencias de la Vida </td><td> 17-5175-01 </td></tr><tr><td> Fenol: cloroformo: isoamílico 25:24:1 con 10 mM Tris, pH 8, 1 mM EDTA </td><td> Sigma </td><td> P3803-100 ml </td></tr><tr><td> RNaseZap </td><td> Ambion </td><td> AM9780 </td></tr><tr><td> Recombinante inhibidor de la ribonucleasa RNasin </td><td> Promega </td><td> N251B </td></tr><tr><td> RNeasy Mini Kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> Oligonucleótidos de ADN </td><td> Integrado las tecnologías de ADN </td><td></td></tr></tbody></table>

in vitro reconstitution of the active t. castaneum telomerase

Esfuerzos para aislar a la subunidad catalítica de la telomerasa, TERT, en cantidades suficientes para realizar estudios estructurales, han tenido un éxito limitado desde hace más de una década. En este sentido, los actuales métodos para el aislamiento de la recombinación del TERT Tribolium castaneum (Tc TERT) y la reconstitución de la T. activos telomerasa castaneum ribonucleoproteína (RNP) complejos in vitro. La telomerasa es una transcriptasa reversa especializada 1 que añade repeticiones cortas de ADN, llamados telómeros, al extremo 3 &#39;de los cromosomas lineales dos que sirven para protegerse de la fusión de extremo a extremo y la degradación. Después de la replicación del ADN, un segmento corto se pierde al final del cromosoma 3, y sin telomerasa, las células siguen dividiendo hasta que finalmente llegando a su límite Hayflick 4. Además, la telomerasa está inactiva en la mayoría de las células somáticas cinco en adultos, pero está presente en seis células de cáncer en la que promueve la inmortalidad celular 7. La enzima telomerasa mínima consta de dos componentes básicos: la subunidad de la proteína (TERT), que comprende la subunidad catalítica de la enzima y un componente integral de ARN (TER), que contiene el TERT plantilla utiliza para sintetizar telómeros 8,9. Antes de 2008, sólo las estructuras de los dominios individuales de la telomerasa se ​​había resuelto 10,11. Un gran avance en este campo llegó a la determinación de la estructura cristalina de los 12 activos, subunidad catalítica de la T. telomerasa castaneum, Tc TERT 1. Aquí, se presentan métodos para producir grandes cantidades de TERT activo, soluble Tc para estudios estructurales y bioquímicos, y la reconstitución del complejo RNP telomerasa in vitro para los ensayos de actividad de la telomerasa. Una visión general de los métodos experimentales utilizados se muestra en la Figura 1.

Watch this Scientific Journal Video about In vitro Reconstitution of the Active T. castaneum Telomerase at JoVE.com

In vitro Reconstitution of the Active T. castaneum Telomerase

Esfuerzos para aislar a la subunidad catalítica de la telomerasa, TERT, en cantidades suficientes para realizar estudios estructurales, han tenido un éxito limitado desde hace más de una década. En este sentido, los actuales métodos para el aislamiento de la recombinación del TERT Tribolium castaneum (<em&gt; Tc</em&gt; TERT) y la reconstitución de los activos<em&gt; T. castaneum</em&gt; Telomerasa ribonucleoproteína (RNP) complejo<em&gt; In vitro</em&gt;.

En la reconstitución in vitro de los activos T. castaneum telomerasa

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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In vitro Reconstitution of the Active T. castaneum Telomerase

11.6K vistas.  University of Pennsylvania. Esfuerzos para aislar a la subunidad catalítica de la telomerasa, TERT, en cantidades suficientes para realizar estudios estructurales, han tenido un éxito limitado desde hace más de una década. En este sentido, los actuales métodos para el aislamiento de la recombinación del TERT Tribolium castaneum ( Tc TERT) y la reconstitución de los activos T. castaneum Telomerasa ribonucleoproteína (RNP) complejo In vitro.

Video: In vitro Reconstitution of the Active T. castaneum Telomerase

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T-linfocitos capturar usando un chip de microfluidos desechables para el VIH

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Biología

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

La cuantificación de γH2AX focos en respuesta a la radiación ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

Evaluación de la distribución espacial de la radiación ionizante γH2AX siguientes

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

El análisis de Schwann-astrocito interacciones utilizando In Vitro Ensayos

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

La longitud del telómero y la actividad de la telomerasa, una Yin y Yang de la senescencia celular

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

Análisis de las reacciones de procesamiento del ARN que utilizan teléfonos Free Systems: 3 &#39;End división de pre-mRNA Sustratos<em&gt; In vitro</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &amp;#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

La reconstitución de la degradación de β-catenina en<em&gt; Xenopus</em&gt; Extracto de Huevo

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

La producción de embriones de pez cebra mediante haploide<em&gt; In Vitro</em&gt; Fertilización

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

En Vitro Reconstitución de complejos captadores de luz de Plantas y Algas

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...