La recherche présentée ici a été soutenu par le Pennsylvania Department of Health, The Medical Ellison, le V et le Fondations d&#39;Emeraude.

1. L&#39;expression de TERT recombinante Tc <ol><li> Inoculer 6 L de 2YT médias (6 x 2 L Perplexe erlenmeyers contenant 1 litre de bouillon 2YT dans chaque) à partir de six plaques fraîches de cellules pLysS transformé Rosetta (DE3) contenant les synthétiques Tc TERT plasmide avec une TEV clivable N-terminale hexahistidine- tag. </li><li> Cultiver des cellules à 37 ° C tout en agitant à 220 rpm pour une DO 600 de 0,5-0,6. </li><li> Induire l&#39;expression de protéines en ajoutant 1 mM d&#39;IPTG. </li><li> Réduire la température à 30 ° C, lent secouant à 150 rpm, et permettent aux cellules d&#39;exprimer la protéine pour 4-5 h. </li><li> Récolte des cellules par centrifugation à 4 ° C et 3500 rpm pendant 30 min. </li><li> Resuspendre chaque cellule 1 L culot dans 20 ml de tampon contenant 25 mM Tris, pH 7,5, 10% glycérol, 0,5 M de KCl, 15 mM imidazole, 5 mM β-mercaptoéthanol et 0,1 mM de fluorure de sulfonyle phénylméthyle (PMSF) et benzamidine. </li><li> Flash gel des remises en suspension le mélange de cellules par goutte lentement les cellules dans un tube conique de 50 ml en plastique rempli d&#39;azote liquide, afin que chaque goutte se fige sur l&#39;impact dans le liquide de N 2. Magasin de cellules congelées à -80 ° C jusqu&#39;au moment de la purification des protéines. </li></ol> 2. Purification de Tc TERT <ol><li> Cellules décongeler à température ambiante jusqu&#39;à une consistance liquide est atteint, puis passer à des béchers de métal sur la glace pendant la sonication. </li><li> Soniquer cellules sur la glace pendant 2 min dans des intervalles de 30 secondes avec 30 secondes pauses, à environ 51 W de puissance. </li><li> Isoler les cellules lysées à 18 000 rpm pendant 20 min pour séparer les fractions protéiques solubles et insolubles et retirer délicatement le surnageant pour chromatographie d&#39;affinité de nickel. </li><li> Equilibrer le Ni-NTA (nickel-acide nitrilotriacétique) colonne avec le tampon contenant 25 mM Tris, pH 7,5, 10% glycérol, 0,5 M de KCl, 15 imidazole mM, 5 mM β-mercaptoéthanol et 0,1 mM PMSF et benzamidine. </li><li> Charge surnageant sur la résine et de recueillir le flux à travers. </li><li> Laver la colonne avec le tampon contenant 25 mM Tris, pH 7,5, glycerol 10%, 500 mM de KCl, 15 mM imidazole, 5 mM β-mercaptoéthanol et 0,1 mM de PMSF pour enlever toutes les protéines résiduelles et non spécifiquement lié. </li><li> Eluer Tc TERT de la résine Ni-NTA en utilisant un gradient d&#39;imidazole de 15 - 300 mm et de recueillir des fractions de 3 ml pour l&#39;analyse SDS-PAGE. </li><li> Utilisez analyse de page pour déterminer lequel des fractions contenant la protéine TERT Tc et concentrer ces fractions dans une Amicon 30 kDa (Millipore) filtre. </li><li> Concentré Tc fractions TERT à un volume inférieur à 10 ml d&#39;une purification supplémentaire. </li><li> Le Tc TERT est purifié sur la colonne échangeuse de cations (SH Poros, Applied Biosystems) pré-équilibrée avec le tampon contenant 25 mM Tris, pH 7,5, glycerol 10%, 500 mM de KCl, et 1 mM de DTT. </li><li> Chargez le mélange de protéines sur la colonne HS Poros et le laver pour enlever tous les contaminants protéiques et d&#39;acides nucléiques. </li><li> Laver la colonne avec le tampon contenant 500 mM de KCl suivie par 700 mM de KCl à éliminer tous les contaminants restants. </li><li> Eluer la protéine TERT Tc de la colonne Poros HS avec 1 M de KCl tampon. A ce stade la protéine devrait être plus que pur à 99%. </li><li> Retirez tout agrégats de protéines en passant la protéine sur une colonne Superdex-200 dimensionnement (chromatographie d&#39;exclusion stérique - GE Healthcare), pré-équilibrée avec le tampon contenant 25 mM Tris, pH 7,5, 10% glycérol, KCl 500 mM, et 1 mM PTCE. </li></ol> 3. Coléoptère Tribolium castaneum la culture <ol><li> Le T. castaneum coléoptères, où initialement prévu comme cadeau par le Dr S. Brown, Division de la Biologie, la Kansas State University. </li><li> Démarrer T. la culture castaneum en ajoutant cent coléoptères adultes dans un récipient avec 28,5 g de farine et 1,5 g de levure sèche de boulanger. </li><li> Magasin de cultures coléoptère à température ambiante et dans un endroit sombre, à l&#39;environnement humide pendant trois à quatre semaines pour permettre la reproduction et la croissance. Placer un bécher contenant de l&#39;eau dans la zone de stockage du dendroctone pour assurer un environnement humide. </li><li> Récolter les larves de coléoptères comme nécessaire pour l&#39;isolement d&#39;ARN. Autoriser les larves restant à maturité pour adultes, en les gardant dans la même culture. </li><li> Chaque mois, le transfert des coléoptères les plus actives (pas plus d&#39;une centaine) dans un nouveau flacon contenant de la farine et la levure fraîche à renouveler leur approvisionnement alimentaire. </li></ol> 4. T. castaneum ARN total isolement <ol><li> Larves de coléoptères récolte 20 à partir de la culture (chaque culture doit contenir entre 500 à 1000 larves) en les séparant des coléoptères adultes et de la culture de la farine / levure. Éliminer la farine ou des particules de levures attaché à la coléoptères avant de les moudre. </li><li> Stériliser la zone du banc, le mortier et le pilon (envisager de traiter les gants aussi) utilisé pour cette procédure avec RNaseZap (Ambion, Inc) pour enlever toute trace de ribonucléases avant le broyage des coléoptères. </li><li> Moudre les larves dans l&#39;azote liquide 2 en une fine poudre à l&#39;aide d&#39;un mortier et un pilon et transfert de la poudre dans un tube à centrifuger de 50 ml, tout en maintenant le liquide de N 2 pour s&#39;assurer que la poudre reste gelé jusqu&#39;à ce que le tampon d&#39;extraction est ajouté. </li><li> Autoriser l&#39;excès de liquide N 2 à s&#39;évaporer de la poudre. Après que le liquide s&#39;est évaporé N 2, d&#39;homogénéiser la poudre ponderosa dans les 200 pi de tampon d&#39;extraction (par 20 larves de coléoptères) contenant 25 mM Tris-HCl, 5 mM β-mercaptoéthanol, 1 mM EGTA, 0,1 mM benzamidine, 200 mM de KCl, 10 % de glycérol, 10 mM d&#39;imidazole et 20U de RNasin, pH 7,5, et incuber sur glace pendant 30 minutes. </li><li> Centrifuger l&#39;homogénat à 15 000 rpm pendant 20 minutes à 4 ° C, de recueillir le surnageant et flash congeler l&#39;échantillon dans un liquide de N 2. Si nécessaire, les échantillons peuvent être conservés à -80 ° C la nuit. </li><li> Extrait de l&#39;ARN total en utilisant le kit RNeasy Mini (Qiagen). </li></ol> 5. Reconstitution in vitro de la télomérase castaneum Tribolium <ol><li> Mélangez 20 mg de recombinaison TERT étiquette His Tc avec 50 ul de T. L&#39;ARN total dans castaneum le tampon de liaison contenant 25 mM Tris-HCl, 200 mM de KCl, 10% glycérol, 5 mM β-mercaptoéthanol, et 10 mM imidazole, pH 7,5. </li><li> Incuber la TERT Tc - Total mélange ARN pendant deux heures à 22 ° C en T. lysat castaneum, en présence de l&#39;inhibiteur de RNasine à prévenir la dégradation d&#39;ARN. </li><li> Purifier le complexe sur une colonne Ni-NTA pour éliminer les impuretés et de l&#39;ARN lysat excès. </li></ol> 6. Protocole d&#39;amplification télomérase répétition (TRAP) des dosages <ol><li> Mélanger 4 ug de Ni-NTA purifiée T. télomérase castaneum dans 50 ul contenant du tampon d&#39;élongation à 20 mM Tris-HCl (pH - 8,3), 7,5 mM MgCl2, 63 mM de KCl, 0,05% Tween 20, 1 mM EGTA, 0,01% de BSA, 0,5 mM de dNTPs, 1 uM TCAS-TS amorce ADN (5&#39;-AAGCCGTCGAGCAGAGTC-3 &#39;), et incuber le mélange à 30 ° C pendant 60 minutes afin de permettre l&#39;allongement de l&#39;amorce d&#39;ADN TCAS-TS par le reconstituée T. télomérase castaneum. </li><li> Extrait de la forme allongée ADN simple brin (ADNsb) en ajoutant un volume égal de phénol-chloroforme au mélange réactionnel. Agiter le mélange doucement jusqu&#39;à ce que l&#39;émulsion est homogène. </li><li> Centrifuger l&#39;échantillon à 12000 rpm pendant cinq minutes à 4 ° C pour séparer les ADNss de contaminants. </li><li> Transfert de la couche aqueuse supérieure de l&#39;échantillon dans un tube eppendorf de 1,5 ml; ajouter une concentration finale de 10 mM de NaOAc (pH 5,0), 1 mM de MgCl 2, et 66,4% EtOH et ensuite l&#39;échantillon à précipiter nuit à -20 ° C. </li><li> Spin de l&#39;échantillon à 15 000 rpm pendant trente minutes à 4 ° C pour culotter les ADNss précipité. Décanter la solution à l&#39;aide d&#39;une pipette, puis ajouter 200 ul d&#39;EtOH à 70% de l&#39;échantillon à laver reste NaOAc et MgCl 2 de l&#39;échantillon. Centrifuger l&#39;échantillon pendant cinq minutes à la même vitesse et la température de re-granules de l&#39;échantillon. </li><li> Décanter la solution EtOH aide d&#39;une pipette et éliminer les traces restantes de l&#39;éthanol en permettant au tube Eppendorf à incuber ouverte à température ambiante pendant trois minutes. Ayant éthanol présent dans l&#39;échantillon va interférer avec l&#39;étape d&#39;amplification PCR. </li><li> Reprendre le culot ADNss (contenant le produit télomérase allongé et l&#39;amorce du TCAS-TS) dans 50 pl de tampon de réaction PCR contenant 10 mM Tris-HCl (pH 8), KCl 50 mM, MgCl2 2 mM, dNTP 100 uM (dATP, dGTP et dTTP), 10 pM [32 P] dCTP, 1 uM TCAS-CX amorce (5&#39;-GTGTGACCTGACCTGACC-3 &#39;) et 1,25 U de Taq ADN polymérase. </li><li> PCR amplifient les produits d&#39;élongation télomérase (29 cycles de 94 ° C pendant 30 secondes, 50 ° C pendant 30 secondes, et 72 ° C pendant 1 minute), de sorte qu&#39;ils sont visibles sur un gel de polyacrylamide 12%. </li><li> Pré-exécuter le gel de 12% avec Tris-Borate-EDTA (TBE) fonctionnant tampon contenant 44,5 mM de Tris, 44,5 mM d&#39;acide borique, 1 mM EDTA, pH 8, pendant trente minutes avant l&#39;exécution des exemples de PCR. Après le pré-course et le chargement du gel avec les réactions PCR, exécutez le gel à 185 volts pendant 1,5-2 heures à 4 ° Celsius ou sur la glace. </li><li> Sécher le gel et l&#39;utilisation de la plaque de phosphore d&#39;imagerie pour visualiser l&#39;activité. </li></ol> 7. Les résultats représentatifs: Un exemple de la TERT purifiée Tc après chromatographie d&#39;exclusion stérique est montré dans la figure 2. La protéine concentrée après purification est exécutée sur un 12% gel SDS-polyacrylamide et se révèle être plus que pur à 99% (figure 2A). L&#39;exclusion de taille (S200) chromatogramme est également fourni (figure 2B) pour démontrer la pureté et l&#39;absence d&#39;agrégats à partir de l&#39;échantillon de protéine purifiée. Un rendement de 5 mg de protéine, après toutes les étapes de purification ont été réalisées est habituellement obtenue bien que le rendement final peut varier. Un test TRAP représentant pour la reconstitution T. télomérase castaneum est montré dans la figure 3 comme indiqué précédemment par Mitchell et al. 12. L&#39;échelle progressive des produits de la télomérase peut être vu dans le couloir 1, toutefoisils sont absents dans les deux télomérase traité à la RNase et le TERT site actif mutante (D251A) échantillons dans les couloirs 2 et 3 respectivement. <img src="/files/ftp_upload/2799/2799fig1.jpg" alt="Figure 1"/> Figure 1. Organigramme des étapes impliquées dans la reconstitution de la recombinaison de TERT Tc. Tout d&#39;abord, le recombinant Tc TERT est sur-exprimé dans E. coli. La protéine est ensuite purifiée par le nickel, d&#39;échange cationique, et la chromatographie d&#39;exclusion stérique. Le T. coléoptères castaneum sont cultivées dans la maison et leurs larves sont utilisées pour isoler l&#39;ARN total. Le Tc purifiée TERT et l&#39;ARN total sont ensuite mélangés à reconstituer le complexe RNP télomérase, et un test TRAP subséquente est utilisée pour confirmer le complexe RNP est une télomérase active. <img src="/files/ftp_upload/2799/2799fig2.jpg" alt="Figure 2"/> Figure 2. Chromatogramme d&#39;exclusion de taille et de l&#39;analyse SDS-PAGE de Tc TERT protéines. (A) 12% gel SDS-polyacrylamide de la protéine TERT Tc après chromatographie d&#39;exclusion stérique. La protéine TERT Tc (70 kDa) migre plus rapidement sur ​​le gel SDS-PAGE, car il a un point isoélectrique élevé (pI - 9,8) (B). Chromatogramme d&#39;exclusion de taille (Superdex S200) de la protéine TERT Tc. <img src="/files/ftp_upload/2799/2799fig3.jpg" alt="Figure 3"/> Figure 3. Dosages piège de la reconstitués in vitro Tribolium castaneum télomérase adapté de Mitchell et al. 12. Piste 1: type sauvage Tc TERT et l&#39;ARN total isolé des larves de coléoptères. Ligne 2: type sauvage Tc TERT et traité à la RNase ARN total des larves et des extraits cellulaires. Voie 3: le site actif mutant Tc TERT (D251A) et de coléoptères ARN total de larves larves.

<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>

La méthode présentée ici permet de produire de grandes quantités de sous-unité catalytique de T. TERT télomérase castaneum sous forme soluble, actifs pour des études structurales et biochimiques. La méthode de Tc TERT sur-expression est sensible à des changements subtils dans la température ou la densité des cellules de ceux mentionnés ci-dessus et peut considérablement affecter les niveaux d&#39;expression de protéines. Plus précisément, nous avons constaté que les cellules ...

<table border="1"><tbody><tr><th> Nom de réactif </th><th> Société </th><th> Nombre de catalogue </th></tr><tr><td> Rosetta (DE3) Cellules plysS </td><td> Novagen </td><td> 70956 </td></tr><tr><td> 2YT Bouillon </td><td> Teknova </td><td> Y0215 </td></tr><tr><td> IPTG </td><td> Or la biotechnologie </td><td> I2481C </td></tr><tr><td> MISONIX Sonicator 3000 </td><td> Qsonica, LLC. </td><td></td></tr><tr><td> ÄKTA Purificateur FPLC </td><td> GE Life Sciences </td><td></td></tr><tr><td> Résine Superflow Ni-NTA </td><td> Qiagen </td><td> 30410 </td></tr><tr><td> Amicon Ultra-15 dispositif centrifuge Filtre </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> POROS 50 HS échange de cations forts d&#39;emballage </td><td> Applied Biosystems </td><td> 1-3359-06 </td></tr><tr><td> POROS 50 HQ échange de cations forts d&#39;emballage </td><td> Applied Biosystems </td><td> 1-2559-06 </td></tr><tr><td> Superdex 200 10/300 colonne d&#39;exclusion de taille </td><td> GE Life Sciences </td><td> 17-5175-01 </td></tr><tr><td> Phénol: chloroforme: isoamyle 25:24:1 avec 10mm de Tris, pH 8, EDTA 1 mM </td><td> Sigma </td><td> P3803-100mL </td></tr><tr><td> RNaseZap </td><td> Ambion </td><td> AM9780 </td></tr><tr><td> Recombinant inhibiteur de la ribonucléase Rnasin </td><td> Promega </td><td> N251B </td></tr><tr><td> RNeasy Mini Kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> Oligonucléotides d&#39;ADN </td><td> Integrated DNA Technologies </td><td></td></tr></tbody></table>

in vitro reconstitution of the active t. castaneum telomerase

Les efforts déployés pour isoler la sous-unité catalytique de la télomérase, TERT, en quantité suffisante pour des études structurales, ont été atteints avec un succès limité pendant plus d&#39;une décennie. Ici, nous présentons les méthodes pour l&#39;isolement de la protéine recombinante TERT Tribolium castaneum (Tc TERT) et la reconstitution de la T. actifs télomérase castaneum ribonucléoprotéines (RNP) complexes in vitro. La télomérase est une transcriptase inverse spécialisée qui ajoute une répétition courts d&#39;ADN, appelées télomères, à l&#39;extrémité 3 &#39;des chromosomes linéaires 2 qui servent à les protéger de bout en bout de fusion et de dégradation. Après réplication de l&#39;ADN, un court segment est perdu à l&#39;extrémité du chromosome 3, et sans télomérase, les cellules continuent divisant jusqu&#39;à ce que finalement atteint leur limite de Hayflick 4. De plus, la télomérase est en dormance dans la plupart des cellules somatiques 5 chez les adultes, mais elle est active dans six cellules cancéreuses où il favorise l&#39;immortalité cellulaire 7. L&#39;enzyme télomérase minimale se compose de deux éléments fondamentaux: la sous-unité protéique (TERT), qui comprend la sous-unité catalytique de l&#39;enzyme et une composante intégrante ARN (TER), qui contient la TERT modèle utilise pour synthétiser les télomères 8,9. Avant 2008, seules les structures pour les domaines télomérase individu avait été résolu 10,11. Une percée majeure dans ce domaine est venu de la détermination de la structure cristalline des 12 actifs, sous-unité catalytique de T. télomérase castaneum, Tc TERT 1. Ici, nous présentons les méthodes pour produire de grandes quantités de l&#39;actif, Tc TERT solubles pour des études structurales et biochimiques, et la reconstitution du complexe RNP télomérase in vitro pour les tests d&#39;activité télomérase. Un aperçu des méthodes expérimentales utilisées est montré dans la figure 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

Les efforts déployés pour isoler la sous-unité catalytique de la télomérase, TERT, en quantité suffisante pour des études structurales, ont été atteints avec un succès limité pendant plus d&#39;une décennie. Ici, nous présentons les méthodes pour l&#39;isolement de la protéine recombinante TERT Tribolium castaneum (<em&gt; Tc</em&gt; TERT) et la reconstitution de la population active<em&gt; T. castaneum</emRibonucléoprotéine télomérase&gt; (RNP) complexes<em&gt; In vitro</em&gt;.

La reconstitution in vitro de l&#39;actif T. La télomérase castaneum

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 vues.  University of Pennsylvania. Les efforts déployés pour isoler la sous-unité catalytique de la télomérase, TERT, en quantité suffisante pour des études structurales, ont été atteints avec un succès limité pendant plus d'une décennie. Ici, nous présentons les méthodes pour l'isolement de la protéine recombinante TERT Tribolium castaneum ( Tc TERT) et la reconstitution de la population active T. castaneum (RNP) complex...

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Biologie

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.

Quantification des γH2AX Foci en réponse aux radiations ionisantes

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.

Évaluation de la distribution spatiale des rayonnements ionisants suivantes γH2AX

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.

Analyse de Schwann-astrocytes interactions à l&#39;aide In Vitro Tests

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 longueur des télomères et de la télomérase activité; Une Yin et Yang de la sénescence cellulaire

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.

Analyse des réactions de transformation de l&#39;ARN utilisant des cellules des systèmes gratuits: 3 &#39;Fin de clivage de pré-ARNm substrats<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.

Reconstitution de β-caténine dégradation dans<em&gt; Xenopus</em&gt; Extrait des oeufs

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.

Production de haploïdes embryons de poisson zèbre par<em&gt; In Vitro</em&gt; Engrais

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.

Reconstitution in vitro de complexes collecteurs de lumière de plantes et d&#39;algues vertes

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