La ricerca qui presentata è stata supportata dal Dipartimento della Salute della Pennsylvania, Il Medical Ellison, V e I fondamenti Smeraldo.

1. Espressione di ricombinante TERT Tc <ol><li> Inoculare 6 L di 2YT multimediali (6 x 2 L Perplesso beute contenenti 1 l di brodo 2YT in ciascuno) da sei piatti freschi di cellule trasformate pLysS Rosetta (DE3) contenente la sintesi Tc TERT plasmide con un TEV cleavable N-terminale hexahistidine- tag. </li><li> Crescere le cellule a 37 ° C sotto agitazione a 220 rpm in un OD 600 di 0,5-0,6. </li><li> Indurre l&#39;espressione delle proteine ​​con l&#39;aggiunta di 1 mM IPTG. </li><li> Ridurre la temperatura a 30 ° C, lenta agitazione a 150 rpm, e consentono alle cellule di esprimere la proteina per 4-5 h. </li><li> Celle di raccolta per centrifugazione a 4 ° C e 3500 rpm per 30 min. </li><li> Risospendere ogni L 1 pellet di cellule in 20 ml di tampone contenente 25 mM Tris, pH 7,5, 10% glicerolo, 0,5 M KCl, 15 mM imidazolo, 5 mM β-mercaptoetanolo, e 0,1 mM fenilmetil sulfonil fluoruro (PMSF) e benzamidine. </li><li> Flash congelare risospeso miscela di cellule dal lento stillicidio delle cellule in un tubo di plastica da 50 ml conica riempita con azoto liquido in modo che ogni goccia si blocca al momento dell&#39;impatto nel liquido N 2. Conservare cellule congelate a -80 ° C fino al momento di purificazione delle proteine. </li></ol> 2. Purificazione del Tc TERT <ol><li> Scongelare le cellule a temperatura ambiente fino a consistenza liquida viene raggiunta e poi passare a bicchieri di metallo in ghiaccio per sonicazione. </li><li> Sonicare cellule in ghiaccio per 2 minuti a intervalli di 30 secondi con 30 secondi si ferma, a circa 51 W di potenza. </li><li> Spin down cellule lisate a 18.000 rpm per 20 min per separare le frazioni proteiche solubili e insolubili e rimuovere con attenzione il sopranatante per cromatografia Nickel affinità. </li><li> Equilibrare la Ni-NTA (nichel-nitrilotriacetico acido) colonna con tampone contenente 25 mM Tris, pH 7,5, 10% glicerolo, 0,5 M KCl, 15 mM imidazolo, 5 mM β-mercaptoetanolo, e 0,1 mM PMSF e benzamidine. </li><li> Carico surnatante sopra la resina e raccogliere il flusso attraverso. </li><li> Lavare la colonna con tampone contenente 25 mM Tris, pH 7,5, 10% glicerolo, 500 mM KCl, 15 mM imidazolo, 5 mM β-mercaptoetanolo, e 0,1 mM PMSF per rimuovere tutte le proteine ​​residue e non specificamente legato. </li><li> Eluire Tc TERT del Ni-NTA resina utilizzando un gradiente di imidazolo di 15 - 300 mm e raccogliere 3 frazioni ml per l&#39;analisi SDS PAGE. </li><li> Utilizzare l&#39;analisi PAGE per determinare quali contengono frazioni proteiche Tc TERT e concentrare queste frazioni in un Amicon 30 kDa (Millipore) filtro. </li><li> Concentrato Tc frazioni TERT ad un volume inferiore a 10 ml per un&#39;ulteriore purificazione. </li><li> Il TERT Tc è ulteriormente purificato nel corso degli colonna a scambio cationico (HS poros, Applied Biosystems) pre-equilibrata con tampone contenente 25 mM Tris, pH 7,5, 10% glicerolo, 500 mM KCl, 1 mM DTT. </li><li> Caricare il mix di proteine ​​sulla colonna HS poros e lavarlo per rimuovere tutte le proteine ​​e acidi nucleici contaminanti. </li><li> Lavare la colonna con tampone contenente 500 mM KCl seguita da 700 mM KCl per rimuovere eventuali contaminanti rimanenti. </li><li> Eluire la proteina Tc TERT fuori la colonna HS poros con buffer di 1 M KCl. A questo punto la proteina deve essere superiore al 99% puro. </li><li> Rimuovere eventuali aggregati proteici passando la proteina in un Superdex-200 colonne dimensionamento (dimensione esclusione cromatografia - GE Healthcare), pre-equilibrata con tampone contenente 25 mM Tris, pH 7,5, 10% glicerolo, 500 mM KCl, 1 mM TCEP. </li></ol> 3. Tribolium castaneum scarabeo cultura <ol><li> Il T. castaneum coleotteri dove inizialmente come un dono dal Dott. S. Brown, Divisione di Biologia, Kansas State University. </li><li> Inizio T. cultura castaneum con l&#39;aggiunta di 100 coleotteri adulti a un contenitore con 28,5 g di farina e 1,5 g di lievito secco panettiere. </li><li> Conservare culture scarabeo a temperatura ambiente e in un ambiente buio e umido per tre o quattro settimane per consentire la riproduzione e la crescita. Posizionare un bicchiere contenente acqua nella zona di stoccaggio coleottero per assicurare un ambiente umido. </li><li> Raccogliere le larve scarabeo come necessario per l&#39;isolamento di RNA. Lasciare le larve ancora a maturare agli adulti, mantenendoli nella stessa cultura. </li><li> Ogni mese, trasferire i coleotteri più attivi (non più di un centinaio) in un pallone nuovo contenente farina e lievito fresco per ricostituire il loro approvvigionamento di cibo. </li></ol> 4. T. castaneum RNA totale isolamento <ol><li> Vendemmia 20 larve di scarabeo dalla cultura (ogni cultura dovrebbe contenere da 500-1000 larve), separandoli da coleotteri adulti e la farina / lievito madre. Eliminare le particelle di farina o lievito attaccato al scarabei prima della macinazione di loro. </li><li> Sterilizzare l&#39;area panchina, la malta e il pestello (si pensi trattare i guanti pure) utilizzato per questa procedura con RNaseZap (Ambion, Inc.) per rimuovere tutte le tracce di ribonucleasi prima della macinatura i coleotteri. </li><li> Grind larve in N 2 liquido in una polvere fine con un mortaio e pestello e transfer la polvere in una provetta da centrifuga da 50 ml, mantenendo il liquido N 2 per garantire che la polvere rimane congelato fino a quando il tampone di estrazione è aggiunto. </li><li> Lasciare che il liquido in eccesso N 2 a evaporare dalla polvere. Dopo che il liquido è evaporato N 2, omogeneizzare la polvere coleottero in 200 ml di tampone di estrazione (per 20 larve di scarabeo) contenente 25 mM Tris-HCl, 5 mM β-mercaptoetanolo, 1 mM EGTA, 0,1 benzamidine mm, 200 mm KCl, 10 % glicerolo, 10 mM imidazolo e 20U di RNasin, pH 7,5 e incubare in ghiaccio per 30 minuti. </li><li> Centrifugare l&#39;omogeneizzato a 15.000 rpm per 20 minuti a 4 ° C, raccogliere il surnatante e flash congelare il campione in un liquido N 2. Se necessario, i campioni possono essere conservati a -80 ° C durante la notte. </li><li> Estrarre l&#39;RNA totale secondo il RNeasy Mini Kit (Qiagen). </li></ol> 5. Ricostituzione in vitro della telomerasi castaneum Tribolium <ol><li> Mix 20 mg di ricombinante His-tag Tc TERT con 50 ml di T. castaneum RNA totale nel buffer vincolante contenente 25 mM Tris-HCl, 200 mM KCl, 10% glicerolo, 5 mM β-mercaptoetanolo, e 10 mM imidazolo, pH 7,5. </li><li> Incubare la TERT Tc - miscela di RNA totale per due ore a 22 ° C in T. lisato castaneum in presenza di inibitore RNasin per evitare la degradazione dell&#39;RNA. </li><li> Purificare il complesso nel corso di un Ni-NTA colonna per rimuovere le impurità lisato e RNA in eccesso. </li></ol> 6. Ripetere il protocollo di amplificazione telomerasi (TRAP) test <ol><li> Mix 4 mg di Ni-NTA purificata T. telomerasi castaneum in 50 microlitri allungamento tampone contenente 20 mM Tris-HCl (pH - 8,3), 7,5 mM MgCl 2, 63 mM KCl, 0,05% Tween 20, 1 mM EGTA, 0,01% BSA, 0,5 mM dNTPs, 1 mM TCAS-TS DNA primer (5&#39;-AAGCCGTCGAGCAGAGTC-3 &#39;), e incubare la miscela a 30 ° C per 60 minuti per consentire l&#39;allungamento degli ACT-TS fondo DNA dal ricostituito T. castaneum telomerasi. </li><li> Estrarre il allungata DNA a singolo filamento (ssDNA) con l&#39;aggiunta di un uguale volume di fenolo cloroformio alla miscela di reazione. Agitare la miscela delicatamente fino a quando l&#39;emulsione è omogenea. </li><li> Centrifugare il campione a 12.000 giri per cinque minuti a 4 ° C per separare il ssDNA da sostanze contaminanti. </li><li> Trasferire lo strato superiore acquoso del campione in una provetta eppendorf da 1,5 ml, aggiungere una concentrazione finale di 10 mM NaOAc (pH 5,0), 1 mM di MgCl 2 e 66,4% EtOH e quindi consentire il campione di precipitare durante la notte a -20 ° C. </li><li> Spin il campione a 15.000 rpm per 30 minuti a 4 ° C per far sedimentare il ssDNA precipitato. Decantare la soluzione con una pipetta e poi aggiungere 200 ml di EtOH 70% al campione di lavare rimanenti NaOAc e MgCl 2 dal campione. Centrifugare il campione per cinque minuti alla stessa velocità e la temperatura di ri-pellet del campione. </li><li> Decantare la soluzione EtOH utilizzando una pipetta e rimuovere ogni traccia residua di etanolo, consentendo il tubo eppendorf di incubare aperto a temperatura ambiente per tre minuti. Avendo presente nel campione di etanolo interferisce con la fase di amplificazione PCR. </li><li> Risospendere il pellet ssDNA (contenente il prodotto allungata telomerasi e il TCAS-TS primer) in 50 ml di tampone di reazione di PCR contenente 10 mM Tris-HCl (pH 8), 50 mM KCl, 2 mM MgCl 2, 100 mM dNTPs (dATP, dGTP e dTTP), 10 mM [32 P] dCTP, 1 mM TCAS-CX primer (5&#39;-GTGTGACCTGACCTGACC-3 &#39;) e 1,25 U di Taq DNA polimerasi. </li><li> PCR amplifica i prodotti telomerasi allungamento (29 cicli di 94 ° C per 30 secondi, 50 ° C per 30 secondi e 72 ° C per 1 minuto), in modo che siano visibili su un gel di poliacrilamide al 12%. </li><li> Pre-eseguire il gel al 12% con Tris-Borato-EDTA (TBE) in esecuzione tampone contenente Tris 44,5 mm, 44,5 mM di acido borico, 1 mM EDTA, pH 8 per trenta minuti prima di eseguire i campioni PCR. Dopo la pre-esecuzione e caricamento del gel con le reazioni di PCR, eseguire il gel a 185 volt per 1,5-2 ore a 4 ° C o su ghiaccio. </li><li> Asciugare il gel e utilizzare la piastra ai fosfori di imaging per visualizzare l&#39;attività. </li></ol> 7. Rappresentante dei risultati: Un esempio di Tc purificato TERT dopo cromatografia dimensione esclusione è mostrato nella Figura 2. La proteina concentrata dopo la depurazione viene eseguito su un 12% di SDS-poliacrilammide gel ed è dimostrato di essere più puro al 99% (Figura 2A). L&#39;esclusione dimensioni (S200) cromatogramma è prevista anche (Figura 2B) per dimostrare la purezza e la mancanza di inerti dal campione proteico purificato. Una resa di 5 mg di proteina dopo tutti i passaggi di purificazione sono stati completati e &#39;solitamente ottenuto anche se la resa finale può variare. UNA TRAPPOLA saggio rappresentativo per il ricostituito T. telomerasi castaneum è mostrato in figura 3 come precedentemente riportato da Mitchell et al. 12. Il graduale scala di prodotti telomerasi può essere visto in corsia 1, tuttaviasono assenti in entrambe le telomerasi RNasi trattati e il sito attivo TERT mutante (D251A) campioni in corsie 2 e 3 rispettivamente. <img src="/files/ftp_upload/2799/2799fig1.jpg" alt="Figura 1"/> Figura 1. Diagramma di flusso delle fasi coinvolte nella ricostituzione della ricombinante Tc TERT. In primo luogo, il ricombinante Tc TERT è over-espressa in E. coli. La proteina viene poi purificato da nichel, di scambio cationico, e di esclusione cromatografia dimensioni. Il T. coleotteri castaneum sono coltivati ​​in casa e le loro larve sono utilizzati per isolare l&#39;RNA totale. Il Tc TERT purificato e l&#39;RNA totale sono poi mescolati a ricostituire il complesso RNP telomerasi, e un saggio TRAP successivo è utilizzato per confermare il complesso RNP è un telomerasi attive. <img src="/files/ftp_upload/2799/2799fig2.jpg" alt="Figura 2"/> Figura 2. Dimensioni cromatogramma esclusione e analisi SDS PAGE di proteine ​​Tc TERT. (A) 12% SDS-poliacrilammide gel della proteina Tc TERT dopo cromatografia dimensione esclusione. La proteina Tc TERT (70 kDa) migra più velocemente sul gel SDS PAGE perché ha un elevato punto isoelettrico (pI - 9,8). (B) cromatogramma esclusione Dimensioni (Superdex S200) della proteina Tc TERT. <img src="/files/ftp_upload/2799/2799fig3.jpg" alt="Figura 3"/> Figura 3. Saggi TRAP del ricostituito in vitro Tribolium telomerasi castaneum adattato da Mitchell et al. 12. Corsia 1: wild type Tc TERT e RNA totale isolato da larve di scarabeo. Corsia 2: wild type Tc TERT e RNasi trattati RNA totale larve e estratti cellulari. Corsia 3: attivo il sito mutante Tc TERT (D251A) e larve di scarabeo larve RNA totale.

<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>

Nessun conflitto di interessi dichiarati.

Il metodo presentato qui permette la produzione di grandi quantità della subunità catalitica di T. castaneum TERT telomerasi nelle solubile, forma attiva per studi strutturali e biochimici. Il metodo di Tc TERT sovra-espressione è sensibile ai cambiamenti sottili di temperatura o la densità delle cellule da quelle di cui sopra e possono influenzare notevolmente i livelli di espressione della proteina. In particolare, abbiamo trovato che inducono le cellule per l&#39;espressione delle proteine ​?...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Rosetta (DE3) plysS Cellule </td><td> Novagen </td><td> 70956 </td></tr><tr><td> 2YT brodo </td><td> Teknova </td><td> Y0215 </td></tr><tr><td> IPTG </td><td> Oro Biotecnologie </td><td> I2481C </td></tr><tr><td> Misonix Sonicator 3000 </td><td> Qsonica, LLC. </td><td></td></tr><tr><td> Akta Purificatore FPLC </td><td> GE Life Sciences </td><td></td></tr><tr><td> Ni-NTA Superflow Resina </td><td> Qiagen </td><td> 30410 </td></tr><tr><td> Amicon Ultra-15 dispositivo centrifuga Filtro </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> POROS 50 HS scambio cationico forte Imballaggio </td><td> Applied Biosystems </td><td> 1-3359-06 </td></tr><tr><td> POROS 50 HQ scambio cationico forte Imballaggio </td><td> Applied Biosystems </td><td> 1-2559-06 </td></tr><tr><td> Superdex 200 10/300 Dimensione-esclusione Colonna </td><td> GE Life Sciences </td><td> 17-5175-01 </td></tr><tr><td> Fenolo: cloroformio: Isoamyl 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> Ricombinante l&#39;inibitore della ribonucleasi Rnasin </td><td> Promega </td><td> N251B </td></tr><tr><td> RNeasy Mini Kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> DNA oligonucleotidi </td><td> Integrated DNA Technologies </td><td></td></tr></tbody></table>

in vitro reconstitution of the active t. castaneum telomerase

Gli sforzi per isolare la subunità catalitica della telomerasi, TERT, in quantità sufficienti per studi strutturali, sono stati accolti con scarso successo da oltre un decennio. Qui, vi presentiamo i metodi per l&#39;isolamento dei ricombinanti Tribolium castaneum TERT (Tc TERT) e la ricostituzione del T. attivo castaneum telomerasi ribonucleoproteina (RNP) complessi in vitro. La telomerasi è una trascrittasi inversa specialisti 1 che aggiunge ripete DNA brevi, chiamate telomeri, porre fine al 3 &#39;dei cromosomi lineari 2 che servono per proteggerli da end-to-end di fusione e di degrado. A seguito di replicazione del DNA, un breve segmento è perso alla fine del cromosoma 3 e senza telomerasi, le cellule continuare a dividere fino alla fine raggiungendo il limite Hayflick 4. Inoltre, la telomerasi è latente nella maggior parte delle cellule somatiche 5 in adulti, ma è attiva nelle cellule tumorali 6, dove si promuove l&#39;immortalità delle cellule 7. L&#39;enzima telomerasi minimo è costituito da due componenti principali: la subunità proteiche (TER), che comprende la subunità catalitica dell&#39;enzima e parte integrante di RNA (TER), che contiene il TERT modello utilizza per sintetizzare i telomeri 8,9. Fino al 2008, solo le strutture per i domini telomerasi individuo era stato risolto 10,11. Un importante passo avanti in questo campo è venuto dalla determinazione della struttura cristallina dei 12 attivi, subunità catalitica di T. castaneum telomerasi, Tc TERT 1. Qui, vi presentiamo i metodi per la produzione di grandi quantità del principio attivo, TERT solubile Tc per studi strutturali e biochimici, e la ricostituzione del complesso RNP telomerasi in vitro per analisi attività della telomerasi. Una panoramica dei metodi sperimentali utilizzati è mostrata in 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

Gli sforzi per isolare la subunità catalitica della telomerasi, TERT, in quantità sufficienti per studi strutturali, sono stati accolti con scarso successo da oltre un decennio. Qui, vi presentiamo i metodi per l&#39;isolamento dei ricombinanti Tribolium castaneum TERT (<em&gt; Tc</em&gt; TERT) e la ricostituzione del principio attivo<em&gt; T. castaneum</em&gt; Ribonucleoproteina telomerasi (RNP) complesso<em&gt; In vitro</em&gt;.

Ricostituzione in vitro di Active T. castaneum telomerasi

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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Biology

In vitro Reconstitution of the Active T. castaneum Telomerase

11.6K Views.  University of Pennsylvania. Gli sforzi per isolare la subunità catalitica della telomerasi, TERT, in quantità sufficienti per studi strutturali, sono stati accolti con scarso successo da oltre un decennio. Qui, vi presentiamo i metodi per l'isolamento dei ricombinanti Tribolium castaneum TERT ( Tc TERT) e la ricostituzione del principio attivo T. castaneum Ribonucleoproteina telomerasi (RNP) complesso 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-linfociti Capture utilizzando un chip monouso Microfluidic per l&#39;HIV

Ricerca

Biologia

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.

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

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.

Valutazione della distribuzione spaziale delle γH2AX seguenti radiazioni ionizzanti

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.

Analisi di Schwann-astrociti Interazioni utilizzo In Vitro Saggi

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.

Lunghezza dei telomeri e telomerasi attività; Un Yin e Yang di senescenza cellulare

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.

Analisi delle reazioni RNA di trasformazione che utilizzano cellulari Sistemi gratis: 3 &#39;Fine Decolleté di pre-mRNA Substrati<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.

Ricostituzione della β-catenina degradazione nel<em&gt; Xenopus</em&gt; Estrai Egg

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.

Produzione di Haploid Zebrafish embrioni da<em&gt; In Vitro</em&gt; Concimazione

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.

In Vitro Ricostituzione di Complessi Light-raccolta di piante e alghe verdi

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

Methods Article

Ricostituzione in vitro di Active T. castaneum telomerasi