A pesquisa apresentada aqui foi apoiado pelo departamento de Pensilvânia da saúde, o Medical Ellison, The V e As Fundações Esmeralda.

1. Expressão de recombinante TERT Tc <ol><li> Inocular 6 L de 2YT media (6 x 2 L Intrigado Frascos Erlenmeyer contendo 1 L de caldo de 2YT em cada) de seis pratos frescos de células transformadas pLysS Rosetta (DE3) contendo o sintético Tc TERT plasmídeo com um TEV N-terminal clivável hexahistidine- tag. </li><li> Cultivar células a 37 ° C agitando a 220 rpm para um OD 600 de 0,5-0,6. </li><li> Induzir a expressão da proteína pela adição de 1 mM IPTG. </li><li> Reduzir a temperatura a 30 ° C, lenta agitação a 150 rpm, e permitir que as células expressam a proteína por 4-5 h. </li><li> Células colheita por centrifugação a 4 ° C e 3500 rpm por 30 min. </li><li> Ressuspender cada 1 L pellet celular em 20 ml de tampão contendo 25 mM Tris, pH 7,5, glicerol 10%, 0,5 M KCl, 15 mM imidazol, 5 mM β-mercaptoetanol e 0,1 mM de fluoreto phenylmethyl sulfonil (PMSF) e benzamidine. </li><li> Flash congelar ressuspenso mistura células por gotejamento lentamente as células em um tubo de plástico 50 ml cônico cheio de nitrogênio líquido para que cada gota congela no impacto no N 2 líquido. Armazenar células congeladas a -80 ° C até que esteja pronto para a purificação de proteínas. </li></ol> 2. Purificação do Tc TERT <ol><li> Células descongelar à temperatura ambiente até que uma consistência líquida é atingido e depois passar para copos de metal em gelo por sonicação. </li><li> Sonicate células no gelo por 2 min, em intervalos de 30 segundos com 30 segundo faz uma pausa, em cerca de 51 W de potência. </li><li> Spin down células lisadas a 18.000 rpm por 20 min para separar as frações de proteínas solúveis e insolúveis e remova cuidadosamente o sobrenadante para cromatografia de afinidade de níquel. </li><li> Equilibrar a Ni-NTA (níquel-nitrilotriacético ácido) coluna com tampão contendo 25 mM Tris, pH 7,5, glicerol 10%, 0,5 M KCl, 15 mM imidazol, 5 mM β-mercaptoetanol e 0,1 mM PMSF e benzamidine. </li><li> Sobrenadante de carga sobre a resina e recolher a fluir. </li><li> Lavar a coluna com tampão contendo 25 mM Tris, pH 7,5, glicerol 10%, 500 mM KCl, 15 mM imidazol, 5 mM β-mercaptoetanol e 0,1 mM PMSF para remover todas as proteínas residual e não especificamente ligados. </li><li> Eluir Tc TERT da resina Ni-NTA usando um gradiente de imidazol de 15 - 300 mm e coletar 3 ml de frações SDS PAGE análise. </li><li> Usar a análise de PAGE para determinar quais frações de proteínas contêm Tc TERT e concentrar essas frações em um Amicon 30 kDa (Millipore) filtro. </li><li> Concentre-se frações Tc TERT a um volume inferior a 10 ml para posterior purificação. </li><li> A TERT Tc é mais purificada sobre a coluna de troca catiônica (HS poros, a Applied Biosystems) pré-equilibrada com tampão contendo 25 mM Tris, pH 7,5, glicerol 10%, 500 mM KCl, e 1 mM DTT. </li><li> Carregar o mix de proteínas na coluna poros HS e lavá-lo para remover todos os contaminantes de proteína e ácido nucléico. </li><li> Lavar a coluna com tampão contendo 500 mM KCl seguido por 700 mM KCl para remover quaisquer contaminantes restantes. </li><li> Eluir a Tc proteína TERT fora da coluna poros HS com um tampão KCl M. Neste ponto, a proteína deve ser mais de 99% pura. </li><li> Remova todos os agregados de proteínas, passando a proteína mais de uma coluna de dimensionamento Superdex-200 (tamanho da cromatografia de exclusão - GE Healthcare), pré-equilibrada com tampão contendo 25 mM Tris, pH 7,5, glicerol 10%, 500 mM KCl, e 1 TCEP mM. </li></ol> 3. Tribolium castaneum cultura besouro <ol><li> O T. castaneum besouros onde inicialmente fornecido como um presente pelo Dr. S. Brown, Divisão de Biologia, Kansas State University. </li><li> Iniciar T. cultura castaneum adicionando cem besouros adultos para um recipiente com 28,5 g de farinha e 1,5 g de fermento de padeiro seco é. </li><li> Culturas Loja besouro em temperatura ambiente e em um ambiente escuro, úmido por três a quatro semanas para permitir a reprodução e crescimento. Coloque um copo contendo água na área de armazenamento besouro para garantir um ambiente úmido. </li><li> Colher as larvas do besouro, conforme necessário para o isolamento do RNA. Permitir que as larvas restantes para amadurecer a adultos, mantendo-os na mesma cultura. </li><li> Cada mês, transferir os besouros mais ativos (não mais de uma centena) para um balão novo contendo farinha eo fermento fresco para reabastecer seu suprimento de alimentos. </li></ol> 4. T. castaneum RNA total isolamento <ol><li> Colheita larvas do besouro 20 da cultura (cada cultura deve conter entre 500-1000 larvas), separando-os dos besouros adultos e da cultura da farinha / fermento. Eliminar a farinha ou partículas de fermento anexado ao besouros antes de triturar-los. </li><li> Esterilizar a área do banco, a argamassa eo pilão (considerar tratar as luvas bem) usado para este procedimento com RNaseZap (Ambion, Inc.) para remover todos os vestígios de ribonucleases antes de triturar os besouros. </li><li> Moer larvas em N 2 líquido em um pó fino com um pilão e tRANSFERÊNCIA o pó para um tubo de centrífuga de 50 ml, mantendo o N 2 líquido para garantir que o pó fica congelada até que o tampão de extração é adicionado. </li><li> Permitir que o excesso de líquido evaporar N 2 a partir do pó. Depois que o N 2 líquido tenha evaporado, homogeneizar o pó em 200 mL besouro de tampão de extração (por 20 larvas do besouro), contendo 25 mM Tris-HCl, 5 mM β-mercaptoetanol, 1 mM EGTA, 0,1 benzamidine mM, 200 mM KCl, 10 glicerol%, 10 mM imidazol e 20U de RNasin, pH 7,5 e incubar no gelo por 30 minutos. </li><li> Centrifugar o homogeneizado a 15.000 rpm por 20 minutos a 4 ° C, recolher o sobrenadante e flash congelar a amostra em N 2 líquido. Se necessário, as amostras podem ser armazenadas a -80 ° C durante a noite. </li><li> Extrair o RNA total usando o RNeasy Mini Kit (Qiagen). </li></ol> 5. Reconstituição in vitro de Tribolium castaneum telomerase <ol><li> Mix 20 mcg de recombinante His-tag Tc TERT com 50 ul de T. RNA castaneum total no buffer de ligação contendo 25 mM Tris-HCl, KCl 200 mM, glicerol 10%, 5 mM β-mercaptoetanol, e 10 mM imidazol, pH 7.5. </li><li> Incubar a TERT Tc - mistura de RNA total por duas horas a 22 ° C em T. castaneum lisado na presença de RNasin inibidor para prevenir a degradação do RNA. </li><li> Purificar o complexo mais de uma coluna Ni-NTA para remover as impurezas lisado e RNA em excesso. </li></ol> 6. Telomerase protocolo de amplificação de repetição (TRAP) ensaios <ol><li> Misture 4 mg de T. purificada Ni-NTA telomerase castaneum em 50 de buffer alongamento l contendo 20 mM Tris-HCl (pH - 8,3), 7,5 mM MgCl 2, 63 mM KCl, 0,05% Tween 20, 1 mM EGTA, BSA 0,01%, 0,5 mM dNTPs, 1 mM TCAS-TS Primer DNA (5&#39;-AAGCCGTCGAGCAGAGTC-3 &#39;), e incubar a mistura a 30 ° C por 60 minutos para permitir o alongamento do primer DNA TCAS-TS pelo T. reconstituído telomerase castaneum. </li><li> Extrair o DNA de fita simples alongada (ssDNA), adicionando um volume igual de clorofórmio fenol à mistura de reação. Agitar a mistura suavemente até que a emulsão é homogênea. </li><li> Centrifugar a amostra a 12.000 rpm por cinco minutos a 4 ° C para separar o ssDNA de contaminantes. </li><li> Transferir a camada superior aquosa da amostra a um tubo eppendorf 1,5 ml, adicionar uma concentração final de 10 mM NaOAc (pH 5,0), 1 mM de MgCl 2, e 66,4% EtOH e depois deixar a amostra para precipitar durante a noite a -20 ° C. </li><li> Girar a amostra a 15.000 rpm para 30 minutos a 4 ° C para agregar as ssDNA precipitado. Decantar a solução com uma pipeta e, em seguida, adicionar 200 mL de EtOH 70% para a amostra de lavar NaOAc restantes e MgCl 2 a partir da amostra. Centrifugar a amostra por cinco minutos com a mesma velocidade e temperatura de re-pellet a amostra. </li><li> Decantar a solução EtOH usando uma pipeta e remover vestígios de etanol, permitindo que o tubo eppendorf para incubar aberta à temperatura ambiente por três minutos. Tendo etanol presente na amostra irá interferir com a etapa de amplificação por PCR. </li><li> Ressuspender o sedimento ssDNA (contendo o produto telomerase alongado eo primer TCAS-TS) em 50 ul de tampão de PCR reação contendo 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 de primer TCAS-CX (5&#39;-GTGTGACCTGACCTGACC-3 &#39;) e 1,25 U Taq DNA polimerase. </li><li> PCR amplificar os produtos telomerase alongamento (29 ciclos de 94 ° C por 30 segundos, 50 ° C por 30 segundos e 72 ° C por 1 minuto), de modo que sejam visíveis em gel de poliacrilamida 12%. </li><li> Pré-executar o gel 12% com Tris-Borato-EDTA (TBE) rodando tampão contendo 44,5 mM Tris, 44,5 mM de ácido bórico, 1 mM EDTA, pH 8 para 30 minutos antes de executar as amostras do PCR. Após a pré-execução e carregamento do gel com as reações de PCR, execute o gel de 185 Volts para 1,5-2 horas a 4 ° C ou em gelo. </li><li> Secar o gel e use o fósforo placa de imagem para visualizar a atividade. </li></ol> 7. Resultados representativos: Um exemplo do Tc purificada TERT após cromatografia de exclusão de tamanho é mostrado na Figura 2. A proteína concentrada após purificação é executado em um 12% de gel de poliacrilamida SDS-e mostra-se mais de 99% puro (Figura 2A). O tamanho da exclusão cromatograma (S200) também é fornecida (Figura 2B) para demonstrar a pureza ea falta de agregados a partir da amostra de proteína purificada. A produção de 5 mg de proteína após todas as etapas de purificação foram concluídos geralmente é obtido apesar de o rendimento final pode variar. Um ensaio de TRAP representante para o T. reconstituído telomerase castaneum é mostrado na Figura 3 como já foi relatado por Mitchell et al. 12. A escada passo a passo de produtos telomerase pode ser visto na pista 1, no entantoeles estão ausentes tanto na telomerase RNase tratados e os ativos do site TERT mutante (D251A) amostras em faixas 2 e 3, respectivamente. <img src="/files/ftp_upload/2799/2799fig1.jpg" alt="Figura 1"/> Figura 1. Fluxograma das etapas envolvidas na reconstituição do recombinante Tc TERT. Primeiro, o recombinante Tc TERT é sobre-expressa em E. coli. A proteína é então purificado por níquel, de troca catiônica, e cromatografia por exclusão de tamanho. O T. castaneum besouros são cultivadas em casa e suas larvas são usadas para isolar o RNA total. A purificada Tc TERT eo RNA total é então misturado para reconstituir o complexo RNP telomerase, e um ensaio TRAP subseqüente é usado para confirmar o complexo RNP é um telomerase ativa. <img src="/files/ftp_upload/2799/2799fig2.jpg" alt="Figura 2"/> Figura 2. Cromatograma tamanho exclusão e SDS PAGE análise de proteína TERT Tc. (A) 12% gel de poliacrilamida SDS da proteína TERT Tc após cromatografia de exclusão de tamanho. O Tc proteína TERT (70 kDa) migra mais rápido no gel SDS PAGE porque tem um ponto alto isoelétrico (pI - 9,8). (B) cromatograma exclusão de tamanho (Superdex S200) da proteína TERT Tc. <img src="/files/ftp_upload/2799/2799fig3.jpg" alt="Figura 3"/> Figura 3. Ensaios de TRAP do in vitro reconstituído Tribolium castaneum telomerase adaptada de Mitchell et al. 12. Pista 1: tipo selvagem Tc TERT e RNA total isolado de larvas de besouro. Faixa 2: tipo selvagem Tc TERT e RNase tratados larvas RNA total e extratos de células. Pista 3: ativa local mutante Tc TERT (D251A) e larvas de besouro larvas RNA total.

<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>

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O método aqui apresentado permite a produção de grandes quantidades da subunidade catalítica de T. castaneum TERT telomerase na forma solúvel ativo para estudos estruturais e bioquímicos. O método de Tc TERT sobre-expressão é sensível a mudanças sutis na temperatura ou densidade celular daqueles acima mencionados e pode afetar drasticamente os níveis de expressão da proteína. Especificamente, nós descobrimos que as células para induzir a expressão da proteína antes de a densidade ópt...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Rosetta (DE3) Células plysS </td><td> Novagen </td><td> 70956 </td></tr><tr><td> 2YT Caldo </td><td> Teknova </td><td> Y0215 </td></tr><tr><td> IPTG </td><td> Biotecnologia ouro </td><td> I2481C </td></tr><tr><td> MISONIX sonicador 3000 </td><td> Qsonica, LLC. </td><td></td></tr><tr><td> AKTA Purifier FPLC </td><td> GE Life Sciences </td><td></td></tr><tr><td> Ni-NTA Resina Superflow </td><td> Qiagen </td><td> 30410 </td></tr><tr><td> Amicon Ultra-15 Dispositivo Centrífuga Filtro </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> POROS 50 HS de troca catiônica forte Embalagem </td><td> Applied Biosystems </td><td> 1-3359-06 </td></tr><tr><td> POROS 50 HQ de troca catiônica forte Embalagem </td><td> Applied Biosystems </td><td> 1-2559-06 </td></tr><tr><td> Superdex 200 10/300 coluna por exclusão de tamanho- </td><td> GE Life Sciences </td><td> 17-5175-01 </td></tr><tr><td> Fenol: Clorofórmio: 25:24:1 isoamilo com 10mM Tris, pH 8, EDTA 1mM </td><td> Sigma </td><td> P3803-100mL </td></tr><tr><td> RNaseZap </td><td> Ambion </td><td> AM9780 </td></tr><tr><td> Recombinante inibidor da ribonuclease Rnasin </td><td> Promega </td><td> N251B </td></tr><tr><td> RNeasy Mini Kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> Oligonucleotides DNA </td><td> Integrada DNA Technologies </td><td></td></tr></tbody></table>

in vitro reconstitution of the active t. castaneum telomerase

Esforços para isolar a subunidade catalítica da telomerase, TERT, em quantidades suficientes para estudos estruturais, foram cumpridos com sucesso limitado por mais de uma década. Aqui, apresentamos métodos para o isolamento do recombinante Tribolium castaneum TERT (Tc TERT) ea reconstituição do T. ativos telomerase castaneum ribonucleoproteína complexos (RNP) in vitro. A telomerase é uma transcriptase reversa que adiciona uma especializados repete DNA curtas, chamadas telômeros, a extremidade 3 &#39;dos cromossomas lineares duas que servem para protegê-los de ponta a ponta-de fusão e degradação. Após a replicação do DNA, um pequeno segmento é perdido no fim do cromossomo 3, e sem telomerase, as células continuam dividindo até que finalmente atingir o seu limite Hayflick 4. Além disso, a telomerase está adormecida na maioria das células somáticas 5 em adultos, mas é ativo em células de câncer 6, onde promove a imortalidade celular 7. A enzima telomerase mínima consiste em dois componentes principais: a subunidade de proteína (TERT), que compreende a subunidade catalítica da enzima e um componente integrante RNA (TER), que contém o TERT modelo usa para sintetizar telômeros 8,9. Até 2008, apenas as estruturas de domínios telomerase indivíduo tinha sido resolvido 10,11. Um grande avanço neste campo veio a determinação da estrutura cristalina dos 12 ativos, subunidade catalítica de T. castaneum telomerase, Tc TERT 1. Aqui, apresentamos métodos para produzir grandes quantidades do ativo TERT Tc, solúvel para estudos estruturais e bioquímicos, e da reconstituição do complexo RNP telomerase in vitro para ensaios de atividade telomerase. Uma visão geral dos métodos experimental utilizado é mostrado na 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

Esforços para isolar a subunidade catalítica da telomerase, TERT, em quantidades suficientes para estudos estruturais, foram cumpridos com sucesso limitado por mais de uma década. Aqui, apresentamos métodos para o isolamento do recombinante Tribolium castaneum TERT (<em&gt; Tc</em&gt; TERT) ea reconstituição do ativo<em&gt; T. castaneum</em&gt; Ribonucleoproteína telomerase (RNP) complexo<em&gt; In vitro</em&gt;.

Na reconstituição in vitro da atividade T. Telomerase castaneum

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

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Biology

In vitro Reconstitution of the Active T. castaneum Telomerase

11.6K visualizações.  University of Pennsylvania. Esforços para isolar a subunidade catalítica da telomerase, TERT, em quantidades suficientes para estudos estruturais, foram cumpridos com sucesso limitado por mais de uma década. Aqui, apresentamos métodos para o isolamento do recombinante Tribolium castaneum TERT ( Tc TERT) ea reconstituição do ativo T. castaneum Ribonucleoproteína telomerase (RNP) complexo In vitro.

Vídeo: 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.

Quantificação de γH2AX Focos em resposta à radiação ionizante

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

Biologia

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.

Avaliação da Distribuição Espacial da γH2AX seguintes Radiação Ionizante

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.

Análise de Schwann-astrócito Interações Usando In Vitro Ensaios

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.

Comprimento dos telômeros e da telomerase atividade; Um Yin e Yang da senescência celular

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

Rapid Generation of Amyloid from Native Proteins In vitro

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

Rapid Generation of Amyloid from Native Proteins In vitro

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

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

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

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

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' 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.

Análise das reacções de processamento de RNA utilizando células Sistemas gratuitas: 3 &#39;Fim de clivagem de pré-mRNA Substratos<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.

Reconstituição de β-catenina degradação na<em&gt; Xenopus</em&gt; Extrato de ovo

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.

Produção de Haplóide embriões Zebrafish por<em&gt; In Vitro</em&gt; Fertilização

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 Reconstituição de Complexos de Light-colheita de plantas e algas verdes

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.