Pennsylvania Sağlık Bölümü, Ellison Tıp, V ve The Emerald Vakıflar tarafından desteklenen araştırma sundu.

1. Rekombinant Tc TERT Açıklanması <ol><li> TEV cleavable N-terminali ile sentetik Tc TERT plazmid içeren (de3) Rosetta dönüştürülmüş pLysS hücreleri altı taze plakalardan 2YT medya 6 L (6 x 2 L 1 L 2YT suyu içeren her Erlenmeyer şişeler şaşkın) İnokülasyon hexahistidine etiket. </li><li> 37 ° hücrelerin büyümesine ° C ise 0.5-0.6, OD 600 220 rpm sallayarak. </li><li> 1 mM IPTG ekleyerek protein ifadesi neden. </li><li> 30 sıcaklığını azaltın ° C, 150 rpm sallayarak yavaş ve hücreleri 4-5 saat protein ifade izin </li><li> Santrifüj yoluyla hasat hücreler 4 ° C ve 30 dakika 3500 rpm. </li><li> 20 ml 25 mM Tris, pH 7.5,% 10 gliserol, 0.5 M KCl, 15 mM imidazol, 5 mM β-mercaptoethanol ve 0.1 mM phenylmethyl Sülfonil florür (PMSF) ve benzamidine içeren tampon her 1 L hücre pelletini tekrar </li><li> Flaş dondurma hücrelerin yavaş yavaş her damla sıvı N 2 darbe donuyor böylece sıvı nitrojen ile dolu bir 50 ml plastik konik tüp içine damlayan yeniden süspanse hücre karışımı . , -80 Donmuş hücreleri Mağaza ° C protein saflaştırılması için hazır olana kadar. </li></ol> 2. Tc TERT saflaştırılması <ol><li> Çözülme hücreleri oda sıcaklığında sıvı bir kıvama kadar ulaştı ve ardından sonication buz metal bardak taşımaktır. </li><li> 30 saniyede yaklaşık 51 W güç, duraklar, 30 saniyelik aralıklarla 2 dakika süreyle buz hücrelerinin sonikasyon. </li><li> Çözünür ve çözünmez protein fraksiyonları ayrı ve dikkatle Nikel afinite kromatografisi süpernatant kaldırmak için 20 dakika süreyle 18.000 rpm parçalanmış hücreleri aşağı spin. </li><li> Ni-NTA (nikel-nitrilotriacetic asit) 25 mM Tris, pH 7.5,% 10 gliserol, 0.5 M KCl, 15 mM imidazol, 5 mM β-mercaptoethanol ve 0.1 mM PMSF ve benzamidine içeren tamponu ile sütun dengelenmesi. </li><li> Yük ve reçine üzerinde süpernatant üzerinden akışını toplamak. </li><li> Sütun bütün kalıntı ve non-spesifik bağlı proteinler kaldırmak için 25 mM Tris, pH 7.5,% 10 gliserol, 500 mM KCl, 15 mM imidazol, 5 mM β-mercaptoethanol ve 0.1 mM PMSF içeren tamponu ile yıkayın. </li><li> Zehir Tc, 15 imidazol degrade kullanarak Ni-NTA reçine TERT - 300 mM ve SDS PAGE analizi için 3 ml kesirler toplamak . </li><li> Hangi kesirler Tc TERT protein içerir belirlemek ve bu kesirler bir Amicon 30 kDa (Millipore) filtresi konsantre SAYFA analiz kullanın. </li><li> Tc TERT kesirler bir ses daha ileri saflaştırma işlemi için en az 10 ml konsantre ol. </li><li> Tc TERT katyon değişim sütun (HS Poros, Applied Biosystems) 25 mM Tris, pH 7.5,% 10 gliserol, 500 mM KCI, 1 mM DTT içeren tampon ile ön dengelenmiş üzerinde daha arındırılır. </li><li> HS Poros sütun üzerinde protein karışımı yerleştirin ve tüm protein ve nükleik asit kirleticiler çıkarmak için yıkama. </li><li> 500 mM KCl kalan tüm kirleri çıkarmak için 700 mM KCl içeren tamponu ile sütun yıkayın. </li><li> 1 M KCI tamponu ile HS Poros sütun kapalı Tc TERT protein Zehir. Bu noktada daha fazla protein% 99 saflıkta olmalıdır. </li><li> Protein geçen bir Superdex-200 boyutlandırma sütun (boyut dışlama kromatografisi - GE Healthcare) üzerinde herhangi bir protein agrega çıkarın, 25 mM Tris, pH 7.5,% 10 gliserol, 500 mM KCI, 1 mM TCEP içeren tampon ile ön-dengelenmiş. </li></ol> 3. Tribolium castaneum böceği kültür <ol><li> T. castaneum böcekler başlangıçta Dr. S. Brown, Biyoloji Bölümü, Kansas Eyalet Üniversitesi tarafından bir hediye olarak verilmiştir. </li><li> Başlangıç ​​T. bir kapta un 28.5 g ve 1.5 g kuru ekmek mayası ile yüz yetişkin böcekler ekleyerek castaneum kültür. </li><li> Oda sıcaklığında ve 3-4 hafta boyunca karanlık, nemli ortamlarda saklayın böceği kültürlerde üreme ve büyüme için izin vermek. Böceği depolama alanında su içeren nemli bir ortam sağlamak için bir behere koyun. </li><li> RNA izolasyonu için gerekli böceği larvaları Hasat. Kalan larvalar aynı kültürü tutmak, yetişkin olgun izin verin. </li><li> Her ay en aktif böcekler (en fazla yüz daha), kendi gıda tedarik doldurmak için taze un ve maya içeren yeni bir balona transfer. </li></ol> 4. T. castaneum toplam RNA izolasyonu <ol><li> Hasat 20 yetişkin böcekler ve un / maya kültürü ayırarak kültür böceği larvaları (her kültür, 500-1000 larvaları arasında içermelidir). Un veya taşlama önce böcekler bağlı maya parçacıkları ortadan kaldırır. </li><li> Tezgah alanı, harç ve böcekler taşlama önce ribonucleases tüm izlerini kaldırmak için RNaseZap (Ambion A.Ş.) ile bu işlem için kullanılan havaneli (eldiven gibi tedavi düşünün) sterilize edin. </li><li> Sıvı N 2, bir havanda ve t kullanarak ince bir toz haline larvaları Grindtoz ekstraksiyon tamponu eklenir kadar toz donmuş kalmasını sağlamak için sıvı N 2 korurken, 50 ml santrifüj tüpüne ransfer. </li><li> Aşırı sıvı N 2 toz buharlaştırmak için izin verin. Sıvı N 2 buharlaştı sonra, 25 mM Tris-HCl, 5 mM β-mercaptoethanol, 1 mM EGTA, 0,1 mM benzamidine, 200 mM KCl, 10 içeren ekstraksiyon tamponu 200 ul böceği tozu (20 böceği larvaları başına) homojenize gliserol,% 10 mM, 30 dakika boyunca buz üzerinde RNasin, pH 7.5 ve inkübe imidazol ve 20U. </li><li> 4 20 dakika 15.000 rpm&#39;de Homojenat Santrifüj ° C, supernatant ve flash toplamak sıvı N 2 örnek dondurmak. Gerekirse, numuneler -80 ° C gece boyunca saklanabilir. </li><li> RNeasy Mini Kiti (Qiagen) kullanılarak total RNA ayıklayın. </li></ol> 5. Tribolium castaneum telomeraz in vitro sulandırıldıktan <ol><li> T. 50 ul ile 20 mg rekombinant Onun etiketli Tc TERT karıştırın castaneum toplam RNA bağlayıcı tampon içeren 25 mM Tris-HCl, 200 mM KCl,% 10 gliserol, 5 mM β-mercaptoethanol ve 10 mM imidazol, pH 7.5. </li><li> Toplam RNA karışımı az iki saat için 22 ° C T. - Tc TERT inkübe RNA bozulmasını önlemek için RNasin inhibitörü varlığı castaneum lizat. </li><li> Ni-NTA sütun üzerinde lizat kirleri ve aşırı RNA kaldırmak için karmaşık arındırın. </li></ol> 6. Telomeraz tekrar amplifikasyon protokolü (TRAP) deneyleri <ol><li> Ni-NTA saflaştırılmış T. 4 mikrogram Mix 50 ul uzama tampon castaneum telomeraz içeren 20 mM Tris-HCl (pH 8.3), 7.5 mM MgCl 2, 63 mM KCl,% 0.05 &#39;Tween 20, 1 mM EGTA,% 0.01 BSA, 0.5 mM dNTP, 1 mcM TCAS-TS DNA astar (5&#39;-AAGCCGTCGAGCAGAGTC-3 &#39;), ve 30 ° C 60 dakika, sulandırılmış T. TCAS-TS DNA astar uzama izin karışımı inkübe castaneum telomeraz. </li><li> Reaksiyon karışımı eşit miktarda fenol kloroform eklenerek uzatılmış tek iplikli DNA (ssDNA) ayıklayın. Emülsiyon homojen karışımı hafifçe kadar çalkalayınız. </li><li> Örnek Santrifüj beş dakika 12.000 rpm&#39;de 4 ° C kirleticilerden uzak ssDNA ayrı. </li><li> 1,5 ml Eppendorf tüp örnek üst sulu tabaka transfer nihai konsantrasyonu 10 mM NaOAc (pH 5.0), 1 mM MgCl 2 ve% 66.4 EtOH ve sonra örnek -20 geceleme çökelmesine izin ° C. </li><li> 4 otuz dakika 15.000 rpm&#39;de örnek Spin ° C pelet çöktürülmüş ssDNA. Bir pipet kullanarak çözüm Durusu ve sonra, kalan NaOAc ve MgCl 2 örnek yıkamak için örnek% 70 EtOH 200 ul ekleyin . Aynı hız ve sıcaklık örnek yeniden pelet az beş dakika için örnek santrifüjleyin. </li><li> Bir pipet kullanarak EtOH çözüm Durusu ve Eppendorf tüp, üç dakika boyunca oda sıcaklığında açık inkübe izin vererek etanol kalan izlerini ortadan kaldırmak. Örnek etanol mevcut olan PCR adım ile müdahale edecektir. </li><li> PCR reaksiyon tamponu içeren 10 mM Tris-HCl (pH 8), 50 mM KCl, 2 mM MgCl 2, 100 mcM dNTP (dATP, 50 ul ssDNA pelet (telomeraz uzatılmış ürün ve TCAS-TS astar içeren) yeniden süspanse dGTP ve dTTP), 10 mcM [32 P] dCTP, 1 mcM TCAS-CX astar (5&#39;-GTGTGACCTGACCTGACC-3 &#39;) ve 1.25 U Taq DNA polimeraz. </li><li> PCR% 12 poliakrilamid jel üzerinde görünür, böylece telomeraz uzama ürünleri (94 ° C, 30 saniye boyunca 50 ° C 30 saniye ve 72 ° C için 1 dakika 29 siklus) yükseltmek. </li><li> Tris-borat-EDTA (TBE) 44.5 mM Tris, 44.5 mM Borik Asit, 1 mM EDTA, otuz dakika, pH 8 çalıştırmadan önce PCR örnekleri içeren tampon çalışan% 12 jel Öncesi çalıştırın. Ön çalışan ve PCR reaksiyonları ile jel yükledikten sonra 1.5-2 saat boyunca, 4 ° Celsius veya buz üzerinde 185 Volt jel çalıştırın. </li><li> Jel Kuru ve aktivite görselleştirmek için fosfor görüntüleme plaka kullanın. </li></ol> 7. Temsilcisi Sonuçlar: Boyut dışlama kromatografisi sonra saflaştırılmış Tc TERT bir örneği Şekil 2&#39;de gösterilmiştir. Arıtma sonra konsantre protein,% 12 SDS-poliakrilamid jel üzerinde çalışan ve saf fazla% 99 (Şekil 2A) olduğu gösterilmiştir. Boyutu hariç tutma (S200) kromatogram ve aynı zamanda saflık ve saflaştırılmış protein örnek agrega eksikliği göstermek için (Şekil 2B). Son verim değişebilir rağmen tamamlanmıştır tüm arıtma adımlardan sonra 5 mg protein verimi elde edilir. Sulandırılmış T. için bir temsilci TRAP yönteminin Mitchell ve arkadaşları tarafından rapor olarak castaneum telomeraz Şekil 3&#39;te gösterilmiştir. 12. Ancak, telomeraz ürünleri basamaklı merdiveni şeritli 1 görülebilirşeritleri 2 ve 3 sırasıyla RNaz tedavi telomeraz ve TERT aktif sitesi mutant (D251A) örnekleri mevcut değildir. <img src="/files/ftp_upload/2799/2799fig1.jpg" alt="Şekil 1"/> Şekil 1. Rekombinant Tc TERT sulandırıldıktan ilgili adımları akış şeması. İlk olarak, rekombinant Tc TERT E. fazla ifade edilir coli. Protein sonra nikel, katyon değişimi ve boyut dışlama kromatografisi arındırılır. T. castaneum böcekler evde kültür ve larvaların total RNA izole etmek için kullanılır. Aktif telomeraz saflaştırılmış Tc TERT ve toplam RNA sulandırmak telomeraz RNP kompleksi sonra karıştırılır ve bir sonraki TRAP yönteminin RNP kompleks onaylamak için kullanılır. <img src="/files/ftp_upload/2799/2799fig2.jpg" alt="Şekil 2"/> Şekil 2. Tc TERT protein Boyut dışlama kromatogram ve SDS PAGE analizi. Tc TERT protein boyut dışlama kromatografisi sonra (A)% 12 SDS-poliakrilamid jel. Yüksek bir izoelektrik noktası (pI 9.8), çünkü Tc TERT protein (70 kDa) SDS PAGE jel üzerinde daha hızlı geçirir. Tc TERT protein (B) Ölçü dışlama kromatogram (Superdex S200) . <img src="/files/ftp_upload/2799/2799fig3.jpg" alt="Şekil 3"/> Şekil 3. In vitro TRAP deneyleri Tribolium castaneum telomeraz Mitchell ve ark adapte sulandırılmış 12. Lane 1: vahşi tip Tc TERT ve toplam RNA izole böceği larvaları. Lane 2: vahşi tip Tc TERT ve RNaz larvaları toplam RNA ve hücre özleri tedavi. Lane 3: aktif sitesi mutant Tc TERT (D251A) ve böcek larvaları toplam larva 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>

Burada sunulan yöntemi büyük miktarlarda T. katalitik subunit üretim için izin verir çözünür, yapısal ve biyokimyasal çalışmalar için aktif olarak castaneum telomeraz TERT. Tc TERT üzerinden ifade yöntemi yukarıda belirtilen sıcaklık ya da hücre yoğunluğu ince değişikliklere duyarlı ve protein ekspresyon seviyeleri önemli ölçüde etkileyebilir . Özellikle, biz optik yoğunluğu 600 nm&#39;de 0.5 ulaşmadan önce protein ekspresyonu hücreleri indükleyen ya da sıc...

<table border="1"><tbody><tr><th> Reaktif Adı </th><th> Şirket </th><th> Katalog Numarası </th></tr><tr><td> Rosetta (de3) plysS Hücreler </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> Altın Biyoteknoloji </td><td> I2481C </td></tr><tr><td> MISONIX sonikatör 3000 </td><td> Qsonica, LLC. </td><td></td></tr><tr><td> Akta Arıtma FPLC </td><td> GE Life Sciences </td><td></td></tr><tr><td> Ni-NTA Superflow Reçine </td><td> Qiagen </td><td> 30410 </td></tr><tr><td> Amicon Ultra-15 Santrifüj Filtre Cihaz </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> Ambalaj Poros 50 HS Güçlü Katyon Değişim </td><td> Applied Biosystems </td><td> 1-3359-06 </td></tr><tr><td> Ambalaj Poros 50 HQ Güçlü Katyon Değişim </td><td> Applied Biosystems </td><td> 1-2559-06 </td></tr><tr><td> Superdex 200 10/300 Boyut Dışlama Sütun </td><td> GE Life Sciences </td><td> 17-5175-01 </td></tr><tr><td> Fenol: Kloroform: 10mM Tris, pH: 8, 1mm EDTA ile izoamil 25:24:1 </td><td> Sigma </td><td> P3803-100 ml </td></tr><tr><td> RNaseZap </td><td> Ambion </td><td> AM9780 </td></tr><tr><td> Rekombinant Rnasin Ribonükleaz İnhibitörü </td><td> Promega </td><td> N251B </td></tr><tr><td> RNeasy Mini Kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> DNA oligonükleotidler </td><td> Integrated DNA Technologies </td><td></td></tr></tbody></table>

in vitro reconstitution of the active t. castaneum telomerase

Telomeraz katalitik altbirim izole etmek için çabalar, TERT, yapısal çalışmalar için yeterli miktarda, bir on yıl daha sınırlı bir başarı ile yerine getirildiğini var. Burada, rekombinant Tribolium castaneum TERT (Tc TERT) izolasyonu ve aktif T. sulandırıldıktan yöntemleri mevcut in vitro castaneum telomeraz ribonükleoprotein (RNP) karmaşık. Telomeraz, bir uçtan-uca füzyon ve bozulmaya karşı korumak için hizmet doğrusal kromozomların 2 3 &#39;sonuna kadar, telomer denilen kısa DNA tekrarlar, ekleyen özel bir ters transkriptaz 1. DNA replikasyonu takiben, kısa segment 3 kromozom sonunda kaybolur ve telomeraz olmadan sonunda kendi Hayflick Sınırı 4 ulaşana kadar, hücre bölünmesi devam eder. Ayrıca, telomeraz yetişkinlerde en somatik hücre 5 içinde uyuyan, ancak kanser hücreleri, hücre ölümsüzlük 7 teşvik 6 aktif . Enzimin katalitik alt birim ve şablon TERT telomer 8,9 sentezlemek için kullandığı içeren ayrılmaz bir RNA bileşeni (TER), oluşan protein altbirim (TERT), minimal telomeraz enzimi iki temel bileşenden oluşmaktadır. 2008&#39;den önce, ayrı ayrı telomeraz etki alanları için sadece yapıları 10,11 çözüldü. T. katalitik altbirim aktif 12 kristal yapının belirlenmesi, bu alanda büyük bir atılım geldi castaneum telomeraz, Tc TERT 1. Burada, yapısal ve biyokimyasal çalışmalar için aktif, çözünür Tc TERT ve telomeraz aktivitesi testleri için in vitro telomeraz RNP kompleks sulandırıldıktan büyük miktarlarda üretmek için yöntemler sunuyoruz. Kullanılan deneysel yöntemler hakkında genel bir bakış Şekil 1&#39;de gösterilmiştir.

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

Telomeraz katalitik altbirim izole etmek için çabalar, TERT, yapısal çalışmalar için yeterli miktarda, bir on yıl daha sınırlı bir başarı ile yerine getirildiğini var. Burada, rekombinant Tribolium castaneum TERT izolasyonu (yöntemleri mevcut<em&gt; Tc</em&gt; TERT) ve aktif sulandırıldıktan<em&gt; T. castaneum</em&gt; Telomeraz ribonükleoprotein (RNP) karmaşık<em&gt; In vitro</em&gt;.

Active T. in vitro Sulandırma castaneum Telomeraz

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 Görüntüleme.  University of Pennsylvania. Telomeraz katalitik altbirim izole etmek için çabalar, TERT, yapısal çalışmalar için yeterli miktarda, bir on yıl daha sınırlı bir başarı ile yerine getirildiğini var. Burada, rekombinant Tribolium castaneum TERT izolasyonu (yöntemleri mevcut Tc TERT) ve aktif sulandırıldıktan T. castaneum Telomeraz ribonükleoprotein (RNP) karmaşık In vitro.

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

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Biyoloji

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.

İyonlaştırıcı Radyasyon Cevabı γH2AX Foci Kantitasyonu

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.

Radyasyondan aşağıdaki γH2AX Mekansal Dağılımı Değerlendirilmesi

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.

Schwann-astrosit Etkileşimleri Analizi In Vitro Tahliller

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.

Telomer uzunluğu ve Telomeraz Aktivitesi, Hücre Yaşlılık bir Yin ve Yang

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.

Pre-mRNA Substratlarm 3 &#39;End Bölünme: Hücre Ücretsiz Sistemlerini Kullanma RNA İşleme Reaksiyonları Analizi<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.

Β-katenin Bozulması Of Sulandırma<em&gt; Xenopus</em&gt; Yumurta Özü

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.

Haploit Zebra balığı Embriyolar üretimi ile<em&gt; In Vitro</em&gt; Gübreleme

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.

Bitkiler ve Yeşil Alg Işık hasat Kompleksleri İn Vitro Sulandırma içinde

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