Name: study of the dna damage checkpoint using xenopus egg extracts
Uploaded: 2012-11-05T13:00:00
Description: Watch this Scientific Journal Video about Study of the DNA Damage Checkpoint using Xenopus Egg Extracts at JoVE.com

这项工作是支持的，部分由北卡罗莱纳大学夏洛特，美联银行基金优秀教师，并授予NIGMS（R15GM101571）提供资金。

1。 LSE准备<ol><li>集蛋，雌蛙（ 非洲爪蟾 ）注射两次。第一次注射（启动）为100 U PMSG（孕马血清促性腺激素）每青蛙。青蛙必须催芽前至少两天诱导产卵和引物的青蛙可用于长达两个星期。黄金青蛙，用3毫升注射器和27 G针在背淋巴囊皮下注射PMSG。 </li><li>诱导产卵，注入500 U HCG（人绒毛膜促性腺激素）每底漆青蛙在使用27 G针背淋巴囊皮下。孵育注入青蛙在不同的桶含2升1个马克的改性?...

<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. 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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. 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在研究非洲爪蟾卵提取物的DNA损伤检验点有以下几个优点。卵提取物的使用提供了一个大的数量的无细胞提取物在相间的细胞周期同步。卵提取物，可以容易且廉价地作出。这是比较容易损坏的DNA或染色质，并揭示后从卵提取物immunodepleting目标蛋白在DNA损伤检验点缺陷。随后，一个潜在的功能缺损可以“救出”addback的野生型或突变型重组蛋白质。因此， 非洲爪蟾卵提取物是一种功?...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td>反Chk1的P-S344抗体</td><td>细胞信号转导</td><td> 2348L </td><td></td></tr><tr><td>抗-Chk1的抗体</td><td>圣克鲁斯</td><td> SC7898 </td><td></td></tr><tr><td>抑肽酶</td><td> MP Biomedicals </td><td> 0219115880 </td><td></td></tr><tr><td>放线菌酮</td><td>西格玛</td><td> C7698-5G </td><td></td></tr><tr><td>细胞松弛素B </td><td> EMD </td><td> 250233 </td><td></td></tr><tr><td>二硫苏糖醇（DTT）的</td><td> VWR </td><td> JTF780-2 </td><td></td></tr><tr><td>人绒毛膜促性腺激素</td><td>西格玛</td><td> CG10-10VL </td><td></td></tr><tr><td> L-半胱氨酸</td><td>西格玛</td><td> C7352-1KG </td><td></td></tr><tr><td>亮肽素</td><td> VWR </td><td>97063-922 </td><td></td></tr><tr><td>甲基磺酸甲酯（MMS） </td><td>西格玛</td><td> 129925-5G </td><td></td></tr><tr><td>诺考达唑</td><td>西格玛</td><td> M1404-2MG </td><td></td></tr><tr><td> PMSG </td><td> Calbiochem公司</td><td> 367222 </td><td></td></tr><tr><td>上样缓冲液</td><td>西格玛</td><td> S3401 </td><td></td></tr><tr><td>互变</td><td>光化工（美国） </td><td> 209-12041 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>产蛋桶</td><td> Rubbermaid的商业产品</td><td> 6308 </td><td></td></tr><tr><td> CL2 IEC离心机转子的摆动</td><td> Thermo Scientific的</td><td> 004260F </td><td></td></tr><tr><td> HB6摆动转子</td><td> Thermo Scientific的</td><td> 11860 </td><td></td></tr><tr><td> SORVALL RC6加超高速离心机</td><td> Thermo Scientific的</td><td> 46910 </td><td></td></tr><tr><td> UV交联</td><td> UVP </td><td> 95-0174-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> 解决方案 </td></tr><tr><td> 1个MMR </td><td></td><td></td><td> 100mM的NaCl，2mM的氯化钾，0.5mM的MgSO 4干燥 ，2.5mM的CaCl 2的 ，5mM的HEPES，pH值调节至7.8，用10 M NaOH </td></tr><tr><td>抑肽酶/亮肽素股票</td><td></td><td></td><td> 10毫克/毫升在水中。将20μL分装-80°C </td></tr><tr><td> x的缓冲区</td><td></td><td></td><td> 15mM的NaCl的80 mM KCl中，0.2M蔗糖，5毫摩尔MgCl 2，1mM的EDTA，10mM的HEPES，pH值调节至7.5，用HCl </td></tr><tr><td>放线菌酮股票</td><td></td><td></td><td> 10毫克/毫升在水中。存储1毫升的等分试样在-20℃。 </td></tr><tr><td>细胞松弛素B股</td><td></td><td></td><td> 5毫克/毫升在DMSO中。 20μL分装-20℃保存</td></tr><tr><td>二硫苏糖醇（DTT）的股票</td><td></td><td></td><td> 1 M中水。存储1毫升的等分试样在-20℃。 </td></tr><tr><td> ELB </td><td></td><td></td><td> 0.25M蔗糖，1mM的DTT，50微克/毫升放线菌酮，2.5毫摩尔MgCl 2，50 mM KCl中，10mM的HEPES，pH7.7 </td></tr><tr><td>诺考达唑股票</td><td></td><td></td><td>在DMSO中的10毫克/毫升。将5微升等分在-80°C </td></tr><tr><td>能量合剂</td><td></td><td></td><td> 375 mM的磷酸肌酸，50mM的ATP，和25mM的MgCl 2。等分试样在-80℃下保存</td></tr><tr><td>核染料溶液</td><td></td><td></td><td> 0.4μg/ ml的Hoechst 33258染色，25％甘油（体积/体积），在1x PBS </td></tr><tr><td>变构股票</td><td></td><td></td><td>在DMSO中的100μM的。在-80℃下存储10微升等份</td></tr></tbody></table>

study of the dna damage checkpoint using xenopus egg extracts

在每天的基础上，将细胞进行的各种内源性和环境的侮辱。为了解决这些侮辱，细胞进化作为一个监督机制，检测DNA损伤和直接的细胞反应对DNA损伤的DNA损伤检验点的信号。有几组的蛋白质称为DNA损伤检查点的信号（ 图1）中所涉及的传感器，传感器和效应器。在这种复杂的信号转导通路，ATR（ATM和RAD3相关）是主要的激酶，可以响应DNA损伤和复制压力之一。激活ATR磷酸化其下游底物，如检查点激酶1（Chk1的）。因此，磷酸化和激活Chk1的导致许多下游的影响，在DNA损伤检验点包括细胞周期阻滞，转录激活，DNA损伤修复，细胞凋亡或衰老（ 图1）。当DNA被破坏，无法激活DNA损伤检验点在UNREP结果播出损伤，且随后，基因组不稳定。细胞DNA损伤检验点的研究阐明了如何保持基因组的完整性，并提供了一​​个更好地了解人类疾病，如癌症，如何发展。 非洲爪蟾卵提取物正在成为一个强大的无细胞提取DNA损伤检验点的研究模型系统，。低速提取物（LSE）最初是由升井组1。去膜精子染色质核形成DNA的半保留复制的方式一次细胞周期LSE结果。 ATR/Chk1-mediated检查点的信号转导通路所引发的DNA损伤或复制压力2。目前使用的两种方法诱导的DNA损伤检查点：DNA损伤的方法和DNA损伤模仿结构体3。 DNA损伤可诱导的紫外线（UV）照射，γ-照射，甲基甲烷esulfonate（MMS），丝裂霉素C（MMC），4 -硝基喹啉-1 -氧化物（4-NQO），或aphidicolin的3，4。 MMS是一种烷化剂，抑制DNA的复制和激活ATR/Chk1-mediated DNA损伤检验点4-7。紫外线照射触发的ATR/Chk1-dependent的的DNA损伤检验点8。的DNA损伤模仿结构AT70退火是一个复杂的两个寡核苷酸的聚（dA的）70和聚-（dT）的70。 AT70比尔·邓菲的实验室系统的开发和广泛使用9-12，诱导ATR/Chk1检查点的信号。 在这里，我们描述协议（1）准备细胞的卵提取物（LSE），（2）治疗爪蟾精子染色质与两种不同的DNA损伤的方法（MMS和UV），（3）准备的DNA损伤模仿的结构AT70，和（4）来触发的ATR/Chk1-mediated DNA损伤检查点LSE相损坏的精子染色质或DNA损伤模仿结构。

Watch this Scientific Journal Video about Study of the DNA Damage Checkpoint using Xenopus Egg Extracts at JoVE.com

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

非洲爪蟾卵提取物是一个有用的模型系统，研究DNA损伤检验点。该协议是非洲爪蟾卵提取物和DNA损伤检验点的诱导试剂的准备。这些技术是适应各种DNA损伤的方法，在研究的DNA损伤检查点的信号。

研究DNA损伤检验点<em&gt;爪蟾</em&gt;卵提取物

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

Research

JoVE Journal

Biology

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

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

测试的速度和运动方向的视觉灵敏度在蜥蜴

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

科研

生物学

Computer-Generated Animal Model Stimuli

计算机生成的动物模型的刺激

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

制备及分馏爪蟾卵提取

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

在使用分馏爪蟾卵提取物的体外核大会

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

Fertilization of Xenopus oocytes using the Host Transfer Method

施肥爪蟾卵母细胞使用主机传输方法

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

二维和三维活细胞成像的DNA损伤反应蛋白

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

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

生产<em&gt;热带爪蟾</em&gt;卵提取物识别微管相关的RNA

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

Reconstitution Of ÃƒÅ½Ã‚Â²-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 ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

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

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

检测和DNA损伤分析小鼠骨骼肌<em&gt;原位</em&gt;使用TUNEL法

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