Die Forschung hier vorgestellte wurde von der Pennsylvania Department of Health, der Ellison Medical, Der V und The Emerald Stiftungen unterstützt.

1. Expression von rekombinanten Tc TERT <ol><li> Impfen 6 L von 2YT Medien (6 x 2 L Baffled Erlenmeyerkolben mit 1 l Brühe 2YT in jeder) aus sechs frische Platten von Rosetta (DE3) transformiert pLysS Zellen mit den synthetischen Tc TERT Plasmid mit einem TEV spaltbaren N-terminalen Hexa-Histidin- -Tag. </li><li> Wachsen Zellen bei 37 ° C unter Schütteln bei 220 Upm bis zu einer OD 600 von 0,5-0,6. </li><li> Induce Proteinexpression durch Zugabe von 1 mM IPTG. </li><li> Temperatur reduzieren und 30 ° C, langsam schüttelnd zu 150 Umdrehungen pro Minute und lassen Zellen auf Protein für 4-5 h ausdrücken </li><li> Ernten Sie die Zellen durch Zentrifugation bei 4 ° C und 3500 rpm für 30 min. </li><li> Resuspendieren je 1 L Zellpellet in 20 ml Puffer mit 25 mM Tris, pH 7,5, 10% Glycerin, 0,5 M KCl, 15 mM Imidazol, 5 mM β-Mercaptoethanol und 0,1 mM phenylmethyl Sulfonyl (PMSF) und Benzamidin. </li><li> Flash-Freeze resuspendiert Zellgemisch durch langsames Eintropfen der Zellen in eine 50 ml Kunststoff-konischen Rohr mit flüssigem Stickstoff gefüllt, so dass jeder Tropfen beim Aufprall gefriert in den flüssigen N 2. Shop gefrorenen Zellen bei -80 ° C bis bereit für die Proteinreinigung. </li></ol> 2. Reinigung von Tc TERT <ol><li> Thaw Zellen bei Raumtemperatur, bis eine flüssige Konsistenz erreicht ist und dann Metallbecher bewegen auf dem Eis für die Beschallung. </li><li> Mit Ultraschall-Zellen auf Eis für 2 min in Intervallen von 30 Sekunden mit 30 Sekunden Pause, bei etwa 51 W Leistung. </li><li> Spin down lysierten Zellen bei 18.000 rpm für 20 min auf die löslichen und unlöslichen Proteinfraktionen trennen und entfernen Sie vorsichtig den Überstand für Nickel-Affinitätschromatographie. </li><li> Äquilibrieren der Ni-NTA (Nickel-Nitrilotriessigsäure)-Säule mit Puffer mit 25 mM Tris, pH 7,5, 10% Glycerin, 0,5 M KCl, 15 mM Imidazol, 5 mM β-Mercaptoethanol und 0,1 mM PMSF und Benzamidin. </li><li> Laden Überstand über dem Harz und sammeln Sie die Durchströmung. </li><li> Die Säule wird mit Puffer mit 25 mM Tris, pH 7,5, 10% Glycerin, 500 mM KCl, 15 mM Imidazol, 5 mM β-Mercaptoethanol und 0,1 mM PMSF, um alle verbleibenden und nicht-spezifisch gebundene Proteine ​​zu entfernen. </li><li> Eluieren Tc TERT der Ni-NTA-Harz mit einem Imidazolgradienten von 15 bis 300 mM und sammeln 3 ml Fraktionen für SDS-PAGE-Analyse. </li><li> Verwenden Sie PAGE-Analyse, um festzustellen, welche Fraktionen enthalten Tc TERT Protein und konzentrieren diese Fraktionen in einer Amicon 30 kDa (Millipore-Filter). </li><li> Konzentrieren Sie sich Tc TERT Fraktionen auf ein Volumen von weniger als 10 ml zur weiteren Reinigung. </li><li> Die Tc TERT wird weiter über die KAT-Säule (HS poros, Applied Biosystems) voräquilibriert mit Puffer mit 25 mM Tris, pH 7,5, 10% Glycerin, 500 mM KCl und 1 mM DTT gereinigt. </li><li> Laden Sie die Protein-Mix auf der HS poros Spalte und waschen Sie es für alle Protein-und Nukleinsäure-Verunreinigungen zu entfernen. </li><li> Die Säule wird mit Puffer mit 500 mM KCl und 700 mM KCl, um alle verbleibenden Verunreinigungen zu entfernen. </li><li> Eluieren der Tc TERT Protein aus dem HS poros Säule mit 1 M KCl-Puffer. An diesem Punkt des Proteins sollte mehr als 99% rein. </li><li> Entfernen Sie alle Protein-Aggregate, indem das Protein über eine Superdex-200 Schlichte Spalte (Size Exclusion Chromatography - GE Healthcare), pre-äquilibriert mit Puffer mit 25 mM Tris, pH 7,5, 10% Glycerin, 500 mM KCl und 1 mM TCEP. </li></ol> 3. Tribolium castaneum Käfer Kultur <ol><li> Die T. castaneum Käfer, wo zunächst als ein Geschenk von Dr. S. Brown, Division of Biology, Kansas State University zur Verfügung gestellt. </li><li> Starten Sie T. castaneum Kultur, indem hundert erwachsenen Käfer in einen Behälter mit 28,5 g Mehl und 1,5 g getrocknete Bäckerhefe. </li><li> Shop Käfer Kulturen bei Raumtemperatur und in einer dunklen, feuchten Umgebung für drei Minuten vor vier Wochen, um für die Reproduktion und das Wachstum zu ermöglichen. Legen Sie ein Becherglas mit Wasser in der Käfer Lagerfläche auf einer feuchten Umgebung zu gewährleisten. </li><li> Ernten Sie die Käferlarven wie für RNA-Isolierung benötigt. Lassen Sie die restlichen Larven zu Erwachsenen reifen, halten sie in der gleichen Kultur. </li><li> Jeden Monat, Transfer zu den aktivsten Käfer (nicht mehr als ein Hundert) in eine neue Flasche mit frischem Mehl und Hefe, um ihre Versorgung mit Lebensmitteln zu ergänzen. </li></ol> 4. T. castaneum Gesamt-RNA isoliert <ol><li> Ernte 20 Käferlarven aus der Kultur (jede Kultur sollte zwischen 500-1000 Larven enthalten), indem Sie sie aus adulten Käfer und das Mehl / Hefekultur. Beseitigen Sie Mehl oder Hefepartikel an die Käfer vor dem Mahlen sie. </li><li> Sterilisieren der Bank-Bereich, die Mörser und Pistill (man denke an die Behandlung die Handschuhe auch) für dieses Verfahren mit RNaseZap (Ambion, Inc.) verwendet, um alle Spuren von Ribonukleasen vor dem Mahlen die Käfer zu entfernen. </li><li> Grind Larven in flüssigem N 2 zu einem feinen Pulver mit einem Mörser und Stößel und transfer das Pulver in ein 50 ml Zentrifugenröhrchen unter Beibehaltung der flüssigen N 2, um sicherzustellen, dass das Pulver gefroren bleibt bis zum Extraktionspuffer zugegeben. </li><li> Lassen Sie überschüssige Flüssigkeit N 2 aus dem Pulver verdampfen. Nachdem die Flüssigkeit verdampft ist N 2, Homogenisierung der Käfer Pulver in 200 ul Extraktionspuffer (je 20 Larven) mit 25 mM Tris-HCl, 5 mM β-Mercaptoethanol, 1 mM EGTA, 0,1 mM Benzamidin, 200 mM KCl, 10 % Glycerin, 10 mM Imidazol und 20U der RNasin, pH 7,5 und Inkubation auf Eis für 30 Minuten. </li><li> Centrifuge das Homogenat bei 15.000 rpm für 20 Minuten bei 4 ° C, den Überstand und Flash-Freeze der Probe in flüssigem N 2. Falls erforderlich, können die Proben bei -80 ° C über Nacht aufbewahrt werden. </li><li> Entpacken Sie die Gesamt-RNA mit dem RNeasy Mini Kit (Qiagen). </li></ol> 5. In vitro Rekonstitution der Tribolium castaneum Telomerase <ol><li> Mix 20 ug rekombinante His-Tag Tc TERT mit 50 ul T. castaneum Gesamt-RNA in den Bindungspuffer mit 25 mM Tris-HCl, 200 mM KCl, 10% Glycerin, 5 mM β-Mercaptoethanol und 10 mM Imidazol, pH 7,5. </li><li> Inkubieren Sie die Tc TERT - Gesamt-RNA-Gemisch zwei Stunden bei 22 ° C in T. castaneum Lysat in Anwesenheit von RNasin Inhibitor RNA-Abbau zu verhindern. </li><li> Purify der Komplex über eine Ni-NTA-Säule zu Lysat Verunreinigungen und überschüssige RNA zu entfernen. </li></ol> 6. Telomerase wiederholen Amplifikationsprotokoll (TRAP)-Assays <ol><li> Mix 4 pg Ni-NTA gereinigt T. castaneum Telomerase in 50 ul Dehnung Puffer mit 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 uM TCAS-TS DNA-Primer (5&#39;-AAGCCGTCGAGCAGAGTC-3 &#39;), und inkubieren Sie die Lösung bei 30 ° C für 60 Minuten Verlängerung des TCAS-TS DNA-Primer durch die rekonstituierte T. ermöglichen castaneum Telomerase. </li><li> Entpacken Sie die längliche Einzelstrang-DNA (ssDNA) durch Zugabe eines gleichen Volumen Phenol Chloroform zur Reaktionsmischung. Schütteln Sie die Mischung leicht, bis die Emulsion homogen ist. </li><li> Zentrifugieren Sie die Probe bei 12.000 rpm für 5 Minuten bei 4 ° C, die ssDNA von Verunreinigungen zu trennen. </li><li> Übertragen Sie die obere wässrige Schicht der Probe auf ein 1,5 ml Eppendorf-Röhrchen, fügen Sie eine Endkonzentration von 10 mM NaOAc (pH 5,0), 1 mM MgCl 2, und 66,4% EtOH und danach lässt man die Probe auf Niederschlag über Nacht bei -20 ° C. </li><li> Drehen Sie die Probe bei 15.000 rpm für 30 Minuten bei 4 ° C bis Pellet das ausgefallene ssDNA. Dekantieren der Lösung mit einer Pipette und dann mit 200 ul 70% EtOH auf die Probe, um die verbleibenden NaOAc und MgCl 2 aus der Probe zu waschen. Zentrifugieren Sie die Probe für 5 Minuten bei der gleichen Geschwindigkeit und Temperatur wieder Pellet der Probe. </li><li> Dekantieren der EtOH-Lösung mit einer Pipette und entfernen die letzten Spuren von Ethanol, indem sie die Eppendorf-Röhrchen, um bei Raumtemperatur inkubieren offen für drei Minuten. Nachdem Ethanol in der Probe vorhanden wird mit der PCR-Amplifikation Schritt stören. </li><li> Resuspendieren ssDNA Pellet (enthält die Telomerase verlängert Produkt und das TCAS-TS-Primer) in 50 ul PCR-Reaktion Puffer mit 10 mM Tris-HCl (pH 8), 50 mM KCl, 2 mM MgCl 2, 100 uM dNTPs (dATP, dGTP und dTTP), 10 uM [32 P] dCTP, 1 uM TCAS-CX-Primer (5&#39;-GTGTGACCTGACCTGACC-3 &#39;) und 1,25 U Taq DNA Polymerase. </li><li> PCR verstärken die Telomerase Dehnung Produkte (29 Zyklen von 94 ° C für 30 Sekunden, 50 ° C für 30 Sekunden und 72 ° C für 1 Minute), so dass sie sichtbar auf einem 12% Polyacrylamidgel werden. </li><li> Pre-run 12% Gel mit Tris-Borat-EDTA (TBE) Laufpuffer mit 44,5 mm Tris, 44,5 mM Borsäure, 1 mM EDTA, pH 8 für 30 Minuten vor der Ausführung des PCR-Proben. Nach einer Vorauswahl läuft und das Laden des Gels mit den PCR-Reaktionen, führen Sie das Gel bei 185 Volt für 1,5-2 Stunden bei 4 ° Celsius oder auf Eis. </li><li> Trocknen Sie das Gel und die Phosphor-Speicherfolien, um die Aktivität zu visualisieren. </li></ol> 7. Repräsentative Ergebnisse: Ein Beispiel für das gereinigte Tc TERT nach Größenausschlusschromatographie ist in Abbildung 2 dargestellt. Das Protein nach der Reinigung konzentrierte sich auf einem 12% SDS-Polyacrylamidgel laufen und wird gezeigt, dass mehr als 99% rein (Abbildung 2a). Die Größe Ausgrenzung (S200) Chromatogramm steht ebenfalls zur Verfügung (Abbildung 2B), um die Reinheit und das Fehlen von Aggregaten aus dem gereinigten Protein Probe zu demonstrieren. Eine Ausbeute von 5 mg Protein, nachdem alle Reinigungsschritte sind abgeschlossen ist in der Regel erhalten, obwohl die endgültige Ausbeute variieren kann. Ein Vertreter TRAP-Assay für die rekonstituierte T. castaneum Telomerase ist in Abbildung 3 dargestellt, wie zuvor von Mitchell et al. 12. Die stufenweise Leiter der Telomerase-Produkte können in Spur 1 zu sehen ist, jedochsie fehlen in den beiden RNase behandelt Telomerase und die TERT aktiven Zentrum Mutante (D251A) Proben in den Spuren 2 und 3 jeweils. <img src="/files/ftp_upload/2799/2799fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Flussdiagramm der Schritte in der Rekonstitution des rekombinanten Tc TERT beteiligt. Zuerst wird das rekombinante Tc TERT in E. überexprimiert coli. Das Protein wird dann durch Nickel, Kationenaustauschkapazität und Size-Exclusion-Chromatographie gereinigt. Die T. castaneum Käfer sind im Haus kultiviert und ihre Larven werden verwendet, um die Gesamt-RNA zu isolieren. Das gereinigte Tc TERT und der Gesamt-RNA werden dann gemischt zur Wiederherstellung der Telomerase RNP-Komplex und einem anschließenden TRAP-Assay verwendet, um die RNP-Komplex zu bestätigen ist eine aktive Telomerase. <img src="/files/ftp_upload/2799/2799fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Größe Chromatogramm und SDS-PAGE-Analyse von Tc TERT Protein. (A) 12% SDS-Polyacrylamidgel der Tc TERT Protein nach Größenausschlusschromatographie. Die Tc TERT Protein (70 kDa) wandert schneller auf dem SDS-PAGE-Gel, weil es einen hohen isoelektrischen Punkt (pI - 9,8) hat. (B) Größe Chromatogramm (Superdex S200) des Tc TERT Protein. <img src="/files/ftp_upload/2799/2799fig3.jpg" alt="Abbildung 3"/> Abbildung 3. TRAP-Assays der in vitro rekonstituiert Tribolium castaneum Telomerase von Mitchell et al angepasst. 12. Spur 1: Wildtyp-Tc TERT und Gesamt-RNA aus Käferlarven isoliert. Spur 2: Wildtyp-Tc TERT und RNase behandelt Larven Gesamt-RNA und Zellextrakten. Lane 3: aktive Zentrum mutierte Tc TERT (D251A) und Käferlarven insgesamt Larven RNA.

<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>

Die hier vorgestellte Methode ermöglicht die Produktion von großen Mengen der katalytischen Untereinheit von T. castaneum Telomerase TERT in lösliche aktive Form für strukturelle und biochemische Untersuchungen. Die Methode der Tc TERT Überexpression empfindlich auf subtile Veränderungen in Temperatur oder Zelldichte von den oben dargestellten und können dramatisch beeinflussen die Höhe der Proteinexpression. Insbesondere haben wir festgestellt, dass Induktion der Zellen für die Proteinexpress...

<table border="1"><tbody><tr><th> Name des Reagenz </th><th> Firma </th><th> Catalog Number </th></tr><tr><td> Rosetta (DE3) pLysS Cells </td><td> Novagen </td><td> 70956 </td></tr><tr><td> 2YT Broth </td><td> Teknova </td><td> Y0215 </td></tr><tr><td> IPTG </td><td> Gold-Biotechnologie </td><td> I2481C </td></tr><tr><td> MISONIX Sonicator 3000 </td><td> Qsonica, LLC. </td><td></td></tr><tr><td> ÄKTA Purifier FPLC </td><td> GE Life Sciences </td><td></td></tr><tr><td> Ni-NTA Superflow Resin </td><td> Qiagen </td><td> 30410 </td></tr><tr><td> Amicon Ultra-15 Zentrifugalfilter Geräte </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> POROS 50 HS starken Kationenaustausch Verpackung </td><td> Applied Biosystems </td><td> 1-3359-06 </td></tr><tr><td> POROS 50 HQ starken Kationenaustausch Verpackung </td><td> Applied Biosystems </td><td> 1-2559-06 </td></tr><tr><td> Superdex 200 10/300 Size-Exclusion Spalte </td><td> GE Life Sciences </td><td> 17-5175-01 </td></tr><tr><td> Phenol: Chloroform: Isoamylalkohol 25:24:1 mit 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> Rekombinante RNasin Ribonucleaseinhibitor </td><td> Promega </td><td> N251B </td></tr><tr><td> RNeasy Mini Kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> DNA-Oligonukleotide </td><td> Integrated DNA Technologies </td><td></td></tr></tbody></table>

in vitro reconstitution of the active t. castaneum telomerase

Die Bemühungen um die katalytische Untereinheit der Telomerase zu isolieren, TERT, in ausreichenden Mengen für strukturelle Studien haben mit begrenztem Erfolg seit mehr als einem Jahrzehnt getroffen. Hier präsentieren wir Methoden zur Isolierung des rekombinanten Tribolium castaneum TERT (Tc TERT) und die Wiederherstellung der aktiven T. castaneum Telomerase ribonucleoprotein (RNP)-Komplex in vitro. Telomerase ist ein spezialisierter Reverse-Transkriptase-1, die kurze DNA-Wiederholungen, die Telomere, die 3 &#39;Ende der linearen Chromosomen 2, die ihnen von End-to-End-Fusion und Abbau zu schützen dienen hinzu. Nach DNA-Replikation, ein kurzes Segment am Ende des Chromosoms 3 und ohne Telomerase ist verloren, weiterhin Zellen, bis schließlich der Erreichung ihrer Hayflick Limit 4 geteilt. Darüber hinaus ist die Telomerase ruhend in den meisten somatischen Zellen 5 bei Erwachsenen, sondern ist aktiv in Krebszellen 6, wo es fördert Zelle Unsterblichkeit 7. Die minimale Telomerase-Enzym besteht aus zwei Kernkomponenten: die Protein-Untereinheit (TERT), welche die katalytische Untereinheit des Enzyms und ein integraler RNA-Komponente (TER), der die Vorlage TERT verwendet, um Telomere 8,9 synthetisieren enthält umfasst. Vor 2008 hatten nur Strukturen für einzelne Telomerase-Domänen 10,11 gelöst. Ein wichtiger Durchbruch auf diesem Gebiet kamen aus der Bestimmung der Kristallstruktur der aktiven 12, katalytische Untereinheit von T. castaneum Telomerase, Tc TERT 1. Hier stellen wir Verfahren zur Herstellung von großen Mengen des aktiven, löslichen Tc TERT für strukturelle und biochemische Untersuchungen, und die Wiederherstellung der Telomerase RNP-Komplex in vitro für Telomerase-Aktivität Assays. Eine Übersicht über die experimentellen Methoden ist in Abbildung 1 dargestellt.

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

Die Bemühungen um die katalytische Untereinheit der Telomerase zu isolieren, TERT, in ausreichenden Mengen für strukturelle Studien haben mit begrenztem Erfolg seit mehr als einem Jahrzehnt getroffen. Hier präsentieren wir Methoden zur Isolierung des rekombinanten Tribolium castaneum TERT (<em&gt; Tc</em&gt; TERT) und die Wiederherstellung der aktiven<em&gt; T. castaneum</em&gt; Telomerase ribonucleoprotein (RNP)-Komplex<em&gt; In-vitro-</em&gt;.

In vitro Rekonstitution der Active T. castaneum Telomerase

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 Aufrufe.  University of Pennsylvania. Die Bemühungen um die katalytische Untereinheit der Telomerase zu isolieren, TERT, in ausreichenden Mengen für strukturelle Studien haben mit begrenztem Erfolg seit mehr als einem Jahrzehnt getroffen. Hier präsentieren wir Methoden zur Isolierung des rekombinanten Tribolium castaneum TERT ( Tc TERT) und die Wiederherstellung der aktiven T. castaneum Telomerase ribonucleoprotein (RNP)-Komplex In-vitro-.

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

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 &#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.

Die Quantifizierung der γH2AX Foci in Response to ionisierender Strahlung

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

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Biologie

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 &#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.

Auswertung der räumlichen Verteilung der γH2AX folgenden ionisierender Strahlung

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 von Schwann-Astrozyten Wechselwirkungen mit 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 ...

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.

Telomere und Telomerase-Aktivität Länge; Ein Yin und Yang der Zellalterung

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' 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.

Die Analyse der RNA-Prozessierung Reaktionen mit Handy Free Systems: 3 &#39;Ende Spaltung von prä-mRNA-Substrate<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 &#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.

Rekonstitution von β-Catenin-Abbau in<em&gt; Xenopus</em&gt; Egg Extract

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.

Die Produktion von Haploid Zebrafischembryonen durch<em&gt; In-vitro-</em&gt; Düngung

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-Rekonstitution der Lichtsammelkomplexe der Pflanzen und Grünalgen

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

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.