Este trabalho é apoiado em parte por fundos fornecidos pela Universidade de Carolina do Norte em Charlotte, fundo Wachovia base para a excelência do corpo docente, e uma bolsa de NIGMS (R15GM101571).

1. Preparação LSE <ol><li> Rãs do sexo feminino (Xenopus laevis) são injetadas duas vezes para coleta de ovos. A primeira injecção (ferragem) é de 100 U PMSG (gonadotrofina de égua prenhe) por sapo. Rãs deve ser preparada pelo menos dois dias antes de ovo indução assentamento e rãs condicionadas são utilizáveis ​​por até duas semanas. Para rãs primos, injetar por via subcutânea PMSG nos sacos linfáticos dorsal usando uma seringa de 3 ml e 27 agulha G. </li><li> Para induzir a postura de ovos, injetar 500 U hCG (gonadotrofina coriônica humana) por sapo preparado por via subcutânea nos sacos linfáticos dorsal usando uma agulha de 27 G. Incubar rãs injetados em baldes separados contendo 2 litros de solução modificado 1x Marc Ringer (MMR). Permitir rãs 14-20 horas para colocar ovos antes da coleta. </li><li> Retire as rãs dos baldes e despeje a solução MMR até cerca de 100 ml está à esquerda. Obter ovos dos baldes, transferindo os ovos para uma proveta de 250 ml. </li><li> Ovos Dejelly por adição de 100 ml de 2% de cisteína (ajustarpH a 7,8 com KOH 10 M). Agite suavemente os ovos com uma pipeta de vidro invertida Pasteur (0,7 cm de diâmetro), aproximadamente a cada 30 seg. Decantar e substituir com cisteína fresco duas vezes durante a incubação. O processo está completo dejellying em cerca de 5-15 min quando os ovos formar uma camada mais condensada no fundo da proveta. </li><li> Rejeitar a solução cisteína e lavar os ovos três vezes com MMR 0,25 x. Redemoinho os ovos na solução por uma pipeta de vidro. &quot;Bad&quot; ovos são determinadas por inspeção visual simples e são brancos &quot;inchado&quot; na aparência. Os &quot;maus&quot; ovos irá acumular-se no centro do copo, após agitação. Remover &quot;maus&quot; ovos por uma pipeta de Pasteur. </li><li> Lavar os ovos com Lise Egg Buffer (ELB) três vezes. Remover quaisquer adicionais &quot;maus&quot; ovos por uma pipeta de Pasteur. Despeje os ovos em um tubo Falcon 14 ml. </li><li> Gire o tubo de Falcon por 55 segundos a 188 xg (1.100 rpm) com o CL2 IEC centrífuga de mesa clínica com um rotor basculante para compactar os ovos. Remover o excesso de tampãotopo da camada de ovo. Adicione 0,5 ul da Aprotinina estoque Leupeptin / e 0,5 ul de estoque citocalasina B por ml de ovos compactadas. </li><li> Centrifugar ovos em 16.500 xg (10.000 rpm) durante 15 min a 4 ° C utilizando o RC6 Sorvall mais centrifugadora superspeed com HB6 rotor basculante. Após centrifugação, os ovos são fraccionados em três camadas do tubo: extracto de lípidos, e / gema de pigmento a partir de cima para baixo, respectivamente. Perfurar a parede do tubo Falcon na porção inferior da camada de meio de extracto com uma agulha G 21. Remova cuidadosamente a agulha como a agulha de perfuração pode ser obstruída por plástico. Insira uma nova agulha 21 G ligada a uma seringa de 1 ml para o local de punção para recolher o extracto. Lentamente, aspirar o extracto para dentro da seringa, para evitar as bolhas de ar e contaminação com o lípido e / gema de pigmento camadas. </li><li> Colocar-se os extractos num tubo refrigerado microcentrífuga de 1,5 ml. Por cada mililitro de extracto de ovo, adicionar as seguintes soluções de produtos químicos para o emconcentração final dicated (mostrada entre parêntesis): (1) 10 ul de cicloheximida (100 ug / ml), (2) 1 ul de aprotinina / leupeptina (10 | ig / ml de cada), (3) 1 ul de citocalasina B (5 ug / ml ), (4) 1 ul de ditiotreitol (1 mM), e (5) Nocodazole 0,33 ul (3 ug / ml). Inverter o ovo extrair pelo menos 10 vezes, com cuidado. Este é o LSE, a qual deve ser feita e utilizada dentro de 4 hr. A qualidade da LSE é comprometido após 4 h ou congelação-descongelação. </li></ol> 2. Tratamento da cromatina de espermatozóides com abordagens danificar o DNA <ol><li> Prepare cromatina espermática normal de acordo com o método descrito previamente 13. </li><li> Prepare MMS-tratado cromatina espermática <ol><li> Ressuspender cromatina espermática normal em 0,5 ml de tampão de X. </li><li> Adicionar ~ pi 5,5 de MMS (9,1 M em stock) a 500 ul de cromatina ressuspendidas a uma concentração final de 100 mM. </li><li> Incubar cromatina espermática em tubo de microcentrífuga à temperatura ambiente durante 30 min, com rotação. </li><li> Gire a mtubo icrocentrifuge a 686 xg (2100 rpm) durante 10 min à temperatura ambiente usando a centrífuga IEC CL2 clínico com rotor basculante e adaptadores de tubos. </li><li> Descartar o sobrenadante e ressuspender o sedimento em 0,5 ml de tampão X mais BSA (3%), DTT (0,1 mM) e aprotinina / leupeptina (10 | ig / ml de cada). </li><li> Repetir os passos (4) e (5) duas vezes para lavar a cromatina de esperma. </li><li> Determinar a concentração de cromatina espermática com um hemocitómetro e diluir a 100.000 esperma / ul. Guardar aliquotas de 5 uL a -80 ° C congelador para posterior utilização. </li></ol></li><li> Prepare UV-tratado cromatina espermática <ol><li> Adicionar a quantidade desejada (por exemplo, 10 ul) da cromatina espermática normal sobre a superfície de uma peça de Parafilm. </li><li> Coloque o Parafilm num reticulador de UV. Ajuste o parâmetro de energia desejado, dependendo da sua experiência, e começar a danificar a cromatina de espermatozóides através da luz UV. Por exemplo, demora aproximadamente 21 segundos para atingir 1000 J / m 2. </li><li> Depois de irradiação UVdiação, cromatina espermática UV-tratados devem ser adicionadas a extractos de ovos imediatamente. </li></ol></li></ol> 3. Preparação de um DNA Damage Estrutura mimetizando-(AT70) <ol><li> Dissolver os dois oligonucleótidos sintéticos de poli-(dA) 70 (designada como A70) e poli-(dT) 70 (designado como T70) em água até uma concentração de 2 ug / uL, respectivamente. </li><li> Adicionar 100 ul de A70 e 100 ul de T70 para um tubo de microcentrífuga de 1,5 ml. Ferver solução mista oligo sintético durante 5 min a 95 ° C em um heatblock. </li><li> Leve o bloco de banho seco para fora (com o tubo contendo EM mistura no mesmo), e deixe esfriar até a temperatura ambiente em um banco de laboratório. Este ajustamento de temperatura leva cerca de 45-60 min. </li><li> A mistura arrefecida é AT70, com uma concentração final de 2 ug / uL. Ul da loja 10 da AT70 alíquotas em congelador -20 ° C para posterior utilização. </li></ol> 4. Provocando o Checkpoint danos no DNA em LSE com cromatina esperma danificado ou um dano de DNAImitando-Estrutura <ol><li> Induzir checkpoint danos no DNA em LSE com cromatina de esperma danificado <ol><li> Adicionar 50 ul de LSE para um tubo de microcentrífuga de 1,5 ml e complementar com 1 ul de mistura de energia e 2 ul de cromatina espermática danificado (concentração final ~ 4000 esperma / ul de reacção). </li><li> Incubar o tubo de reacção à temperatura ambiente durante 90 min e apertar o tubo a cada 10 min. </li><li> Pipetar 1 ul de mistura de reacção sobre uma lâmina de microscópio e suplementado com 1 ul de solução de corante nuclear após 30 min de incubação. Coloque uma lamela sobre a mistura de reacção e verificar se a formação de núcleos de via microscópio de fluorescência. Tipicamente, os núcleos redondos irá formar após 30 min de incubação, indicando que a replicação do ADN tenha iniciado. </li><li> Tomar 10 ul da mistura de reacção em 90 ul de tampão de amostra. As amostras são analisadas através de imunotransf erência utilizando anticorpos anti-P-S344 Chk1 ou anti-anticorpos de Chk1. </li></ol></li><li> Checkpoint induzir danos no DNA em LSE com aAT70 <ol><li> Adicionar 50 ul de LSE para um tubo de microcentrífuga de 1,5 ml suplementado com 1 ul de mistura de energia e de 1,6 ul de estoque Tautomycin. </li><li> Adicionar 1,25 ul de pré-fabricados AT70 (2 ug / uL) e água (como controlo negativo) a cada reacção. Incubar as reacções à temperatura ambiente durante 90 min. Flick os tubos de reacção a cada 10 minutos. </li><li> Adicionam-se 10 ul da mistura de reacção em 90 ul de tampão de amostra. As amostras são analisadas por meio de imunoblot utilizando anti-P-S344 Chk1 ou anti-anticorpos de Chk1. </li></ol></li></ol> 5. Resultados representativos A cromatina espermática danificadas ou estrutura danificam o DNA imita pode desencadear o checkpoint danos ATR/Chk1-mediated ADN no sistema de extracto de ovo de Xenopus. Figura 2a mostra que o MMS induz fosforilação Chk1 a Ser344 (Chk1 P-S344), o qual é um indicador da ATR activação da cinase. Figura 2B mostra que AT70, como um dano do ADN que imita-structure, também aciona Chk1 fosforilação. Amostras totais Chk1 são usados ​​como controlos de carga em ambos os exemplos. <img alt="Figura 1" src="/files/ftp_upload/4449/4449fig1.jpg" /> Figura 1. Um diagrama de ponto de verificação da sinalização de danos ao DNA. <img alt="Figura 2" src="/files/ftp_upload/4449/4449fig2.jpg" /> Figura 2. Chk1 fosforilação é induzida por uma ou MMS AT70 tratamentos em extractos de Xenopus ovo. (A) MMS-danificada cromatina espermática (MMS) ou cromatina espermática normal (Con) são incubados em extractos de ovos durante 90 min. Chk1 fosforilação em Ser344 (Chk1 P-S344) e Chk1 total em extractos de ovos são examinadas através de imunotransf erência. (B) ou de água AT70 (Con) são adicionados em extractos de ovos, respectivamente. As amostras são também analisados ​​através de imunotransf erência como em (A).

<ol>
	<li>Lohka, M. J., Masui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+in+vitro+of+sperm+pronuclei+and+mitotic+chromosomes+induced+by+amphibian+ooplasmic+components.">Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.</a> Science. 220 (4598), 719-721 (1983).</li><li>Cimprich, K. A., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR:+an+essential+regulator+of+genome+integrity.">ATR: an essential regulator of genome integrity.</a> Nat. Rev. Mol. Cell Biol. 9 (8), 616-627 (2008).</li><li>Lupardus, P. J., Van, C., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+the+ATR-mediated+checkpoint+using+Xenopus+egg+extracts.">Analyzing the ATR-mediated checkpoint using Xenopus egg extracts.</a> Methods. 41 (2), 222-231 (2007).</li><li>Lupardus, P. J., Byun, T., Yee, M. C., Hekmat-Nejad, M., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+requirement+for+replication+in+activation+of+the+ATR-dependent+DNA+damage+checkpoint.">A requirement for replication in activation of the ATR-dependent DNA damage checkpoint.</a> Genes Dev. 16 (18), 2327-2332 (2002).</li><li>Stokes, M. P., Van Hatten, R., Lindsay, H. D., Michael, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+is+required+for+the+checkpoint+response+to+damaged+DNA+in+Xenopus+egg+extracts.">DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts.</a> J. Cell Biol. 158 (5), 863-872 (2002).</li><li>Kato, K., Strauss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+an+intermediate+in+DNA+synthesis+by+HEp.2+cells+treated+with+methyl+methanesulfonate.">Accumulation of an intermediate in DNA synthesis by HEp.2 cells treated with methyl methanesulfonate.</a> Proc. Natl. Acad. Sci. U.S.A. 71 (5), 1969-1973 (1974).</li><li>Paulovich, A. G., Hartwell, L. 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+checkpoint+regulates+the+rate+of+progression+through+S+phase+in+S.+cerevisiae+in+response+to+DNA+damage.">A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage.</a> Cell. 82 (5), 841-847 (1995).</li><li>Guo, Z., Kumagai, A., Wang, S. X., Dunphy, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+Atr+in+phosphorylation+of+Chk1+and+cell+cycle+regulation+in+response+to+DNA+replication+blocks+and+UV-damaged+DNA+in+Xenopus+egg+extracts.">Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts.</a> Genes Dev. 14 (21), 2745-2756 (2000).</li><li>Jazayeri, A., Balestrini, A., Garner, E., Haber, J. E., Costanzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mre11-Rad50-Nbs1-dependent+processing+of+DNA+breaks+generates+oligonucleotides+that+stimulate+ATM+activity.">Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity.</a> EMBO. J. 27 (14), 1953-1962 (2008).</li><li>Kumagai, A., Dunphy, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Claspin,+a+novel+protein+required+for+the+activation+of+Chk1+during+a+DNA+replication+checkpoint+response+in+Xenopus+egg+extracts.">Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts.</a> Mol. Cell. 6 (4), 839-849 (2000).</li><li>Yan, S., Lindsay, H. D., Michael, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+requirement+for+Xmus101+in+ATR-mediated+phosphorylation+of+Claspin+bound+Chk1+during+checkpoint+signaling.">Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling.</a> J. Cell Biol. 173 (2), 181-186 (2006).</li><li>Shiotani, B., Zou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-stranded+DNA+orchestrates+an+ATM-to-ATR+switch+at+DNA+breaks.">Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks.</a> Mol. Cell. 33 (5), 547-558 (2009).</li><li>Tutter, A. V., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+DNA+replication+in+a+soluble+cell-free+system+derived+from+Xenopus+eggs.">Chromosomal DNA replication in a soluble cell-free system derived from Xenopus eggs.</a> Methods Mol. Biol. 322, 121-137 (2006).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Fractionation+of+Xenopus+laevis+Egg+Extracts.">Preparation and Fractionation of Xenopus laevis Egg Extracts.</a> J. Vis. Exp. (18), e891 (2008).</li></ol>

Não há conflitos de interesse declarados.

Existem várias vantagens em estudar o posto danos no DNA utilizando extratos de Xenopus ovo. A utilização de extractos de ovo fornece uma grande quantidade de extractos livres de células sincronizadas em interfase do ciclo celular. Os extractos de ovos pode ser feita fácil e barata. É relativamente fácil para danificar o DNA ou cromatina e revelar um defeito no ponto de verificação, após danos no ADN immunodepleting uma proteína alvo a partir de extracto de ovo. Subsequentemente, um defeito da funç?...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reagentes </td></tr><tr><td> Anticorpo anti-P-Chk1 S344 </td><td> Sinalização celular </td><td> 2348L </td><td></td></tr><tr><td> Anticorpo anti-Chk1 </td><td> Santa Cruz </td><td> SC7898 </td><td></td></tr><tr><td> Aprotinina </td><td> Biomedicals MP </td><td> 0219115880 </td><td></td></tr><tr><td> Cicloheximida </td><td> Sigma </td><td> C7698-5G </td><td></td></tr><tr><td> Citocalasina B </td><td> EMD </td><td> 250233 </td><td></td></tr><tr><td> Ditiotreitol (DTT) </td><td> VWR </td><td> JTF780-2 </td><td></td></tr><tr><td> hCG </td><td> Sigma </td><td> CG10-10VL </td><td></td></tr><tr><td> L-Cisteína </td><td> Sigma </td><td> C7352-1KG </td><td></td></tr><tr><td> Leupeptina </td><td> VWR </td><td>97063-922 </td><td></td></tr><tr><td> Metanossulfonato de metilo (MMS) </td><td> Sigma </td><td> 129.925-5G </td><td></td></tr><tr><td> Nocodazol </td><td> Sigma </td><td> M1404-2mg </td><td></td></tr><tr><td> PMSG </td><td> Calbiochem </td><td> 367222 </td><td></td></tr><tr><td> Tampão de amostra </td><td> Sigma </td><td> S3401 </td><td></td></tr><tr><td> Tautomycin </td><td> Wako Chemicals EUA </td><td> 209-12041 </td><td></td></tr><tr><td></td><td></td><td></td><td> Equipamento </td></tr><tr><td> Balde para a postura de ovos </td><td> Produtos Rubbermaid comerciais </td><td> 6308 </td><td></td></tr><tr><td> CL2 centrífuga IEC com rotor basculante </td><td> Thermo Scientific </td><td> 004260F </td><td></td></tr><tr><td> HB6 rotor de caçamba móvel </td><td> Thermo Scientific </td><td> 11860 </td><td></td></tr><tr><td> Sorvall RC6 mais centrífuga superspeed </td><td> Thermo Scientific </td><td> 46910 </td><td></td></tr><tr><td> Reticulador UV </td><td> UVP </td><td> 95-0174-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Soluções </td></tr><tr><td> 1x MMR </td><td></td><td></td><td> 100 mM de NaCl, 2 mM de KCl, 0,5 mM de MgSO4, 2,5 mM de CaCl 2, 5 mM de HEPES, pH ajustado a 7,8 com NaOH 10 M </td></tr><tr><td> Aprotinina / estoque Leupeptin </td><td></td><td></td><td> 10 mg / ml de cada uma em água. Armazenar 20 ul de aliquotas a -80 ° C. </td></tr><tr><td> Tampão X </td><td></td><td></td><td> 0,2 M de sacarose, 80 mM de KCl, 15 mM NaCl, 5 mM de MgCl2, 1 mM de EDTA, 10 mM de HEPES, pH ajustado a 7,5 por HCl </td></tr><tr><td> Estoque cicloheximida </td><td></td><td></td><td> 10 mg / ml em água. Loja de aliquotas de 1 ml a -20 ° C. </td></tr><tr><td> Citocalasina B estoque </td><td></td><td></td><td> 5 mg / ml em DMSO. Armazenar 20 ul de aliquotas a -20 ° C. </td></tr><tr><td> Ditiotreitol estoque (TDT) </td><td></td><td></td><td> 1 M em água. Loja de aliquotas de 1 ml a -20 ° C. </td></tr><tr><td> ELB </td><td></td><td></td><td> Sacarose 0,25 M, 1 mM de DTT, 50 ug / ml de cicloheximida, 2,5 mM MgCl2, 50 mM de KCl, 10 mM de HEPES, pH7.7 </td></tr><tr><td> Nocodazol estoque </td><td></td><td></td><td> 10 mg / ml em DMSO. Armazenar aliquotas de 5 uL a -80 ° C. </td></tr><tr><td> Mistura de energia </td><td></td><td></td><td> 375 mM de fosfato de creatina, ATP 50 mM e 25 mM de MgCl 2. Alíquotas são guardadas a -80 ° C. </td></tr><tr><td> Solução de corante nuclear </td><td></td><td></td><td> 0,4 ug / ml de Hoechst 33258 de glicerol, 25%(V / v), em PBS 1x </td></tr><tr><td> Estoque Tautomycin </td><td></td><td></td><td> 100 uM em DMSO. Armazenar aliquotas de 10 uL a -80 ° C. </td></tr></tbody></table>

study of the dna damage checkpoint using xenopus egg extracts

Numa base diária, as células são sujeitas a uma variedade de insultos endógenos e ambientais. Para combater esses insultos, as células desenvolveram posto de controle de danos ao DNA sinalização como um mecanismo de vigilância para detectar danos no DNA e diretos respostas celulares a danos no DNA. Há diversos grupos de proteínas chamados sensores, transdutores e efetores envolvidos na sinalização de DNA checkpoint de dano (Figura 1). Neste caminho complexo de sinalização, ATR (ATM e Rad3-relacionados) é uma das quinases importantes que podem responder a danos no ADN e stress replicação. Activated ATR pode fosforilar os seus substratos a jusante, tais como o Chk1 (Ponto de verificação quinase 1). Consequentemente, fosforiladas e activadas Chk1 conduz a muitos efeitos a jusante do ponto de verificação, incluindo danos no DNA paragem do ciclo celular, a activação da transcrição, reparação de danos do ADN, e a apoptose ou senescência (Figura 1). Quando o DNA é danificado, não para ativar o DNA resultados checkpoint danos em unrepdanos ao ar e, posteriormente, instabilidade genômica. O estudo dos danos de ADN do ponto de verificação será elucidar como as células a manter a integridade do genoma e proporcionar um melhor entendimento de como as doenças humanas, tais como o cancro, se desenvolver. Extratos de ovos de Xenopus laevis estão surgindo como um poderoso modelo de sistema livre de células extrato no DNA pesquisa checkpoint danos. De baixa velocidade de extracto (LSE) foi inicialmente descrita por grupo Masui 1. A adição de cromatina espermática demembranated para os resultados na formação de núcleos de LSE, onde o DNA é replicado em uma forma semiconservativa uma vez por ciclo celular. A via de sinalização ATR/Chk1-mediated checkpoint é acionado por dano de DNA ou estresse replicação 2. Dois métodos são usados ​​atualmente para induzir o posto danos ao DNA: abordagens de DNA e DNA danos prejudiciais imitando-estruturas 3. Dano do ADN pode ser induzida por irradiação com ultravioleta (UV), γ-irradiação, metil metanesulfonate (MMS), mitomicina C (MMC), 4-nitroquinolina-1-óxido de (4-NQO), ou afidicolina 3, 4. O MMS é um agente alquilante que inibe a replicação do DNA e ativa o ponto de verificação danos ATR/Chk1-mediated DNA 4-7. Irradiação UV também provoca a ATR/Chk1-dependent DNA 8 checkpoint danos. A estrutura de ADN que imita dano-AT70 é um complexo de recozido de dois oligonucleótidos de poli-(dA) 70 e poli-(dT) 70. O sistema AT70 foi desenvolvido no laboratório Bill Dunphy e é amplamente utilizada para induzir a sinalização de checkpoint ATR/Chk1 9-12. Aqui, nós descrevemos protocolos (1) para preparar extractos isentos de células de ovos (LSE), (2) para o tratamento de Xenopus com cromatina de esperma dois ADN diferentes abordagens danificar (MMS e UV), (3) para preparar o DNA de danos estrutura imita AT70, e (4) para accionar o ponto de verificação ATR/Chk1-mediated danos no DNA LSE com cromatina espermática danificado ou uma estrutura de ADN que imita danos.

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Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extracto é um sistema modelo útil para investigar o checkpoint danos no DNA. Este protocolo é para a preparação de extractos de ovos de Xenopus e os danos do ADN reagentes indutores de ponto de verificação. Estas técnicas são adaptáveis ​​a uma variedade de abordagens que danificam o ADN no estudo da sinalização checkpoint danificam o DNA.

Estudo da Checkpoint Dano ao DNA usando<em&gt; Xenopus</em&gt; Os extratos de ovos

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

study-of-the-dna-damage-checkpoint-using-xenopus-egg-extracts

Universidad de Cartagena UDEC

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JoVE Journal

Biology

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

16.6K visualizações.  University of North Carolina at Charlotte. Xenopus egg extracto é um sistema modelo útil para investigar o checkpoint danos no DNA. Este protocolo é para a preparação de extractos de ovos de Xenopus e os danos do ADN reagentes indutores de ponto de verificação. Estas técnicas são adaptáveis ​​a uma variedade de abordagens que danificam o ADN no estudo da sinalização checkpoint danificam o DNA.

Vídeo: Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biologia

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Gerados por computador Modelo Estímulos animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Preparação e fracionamento de Xenopus laevis Egg Extractos

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

In Vitro Assembléia Nuclear Usando Extractos Egg fracionado Xenopus

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Fertilização de Oócitos Xenopus Utilizando o método de transferência do host

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Duas e três dimensões imagens de células vivas de Proteínas de DNA resposta a danos

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Produção de<em&gt; Xenopus tropicalis</em&gt; Egg Extrai identificar RNAs associada aos microtúbulos

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

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 ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Detecção e análise de danos no DNA em Rato Músculo Esquelético<em&gt; In Situ</em&gt; Usando o método TUNEL

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificação, sequenciamento de próxima geração, e Genomic DNA Mapeamento das retrovirais Integração Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Estudo da Checkpoint Dano ao DNA usando

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