这项工作是由来自美国国立健康和希望之城贝克曼研究所研究院的资金支持。我们要感谢他们建设性的意见，清晰的手稿的评论。

1。何刺激的易位频率<ol><li> 10-20独立YPGly / LAC培养基（1％酵母提取物，2％蛋白胨，3％甘油和3％乳酸）1毫升的文化所需的基因型的单菌落接种。足够的时间达到了约5X10 7 - 1 × 10 8细胞/ ml的细胞密度，在30 ° C的肩，或轻轻摇动孵育过夜，或文化。 </li><li>添加半乳糖的文化终浓度为2％，以诱使何内切酶的定向双链断裂的HIS3 -Δ3“（第三号染色体） 和HIS3Δ5”（第十五号染色体）易位基板。 </li><li>孵育30 ° C的肩，轻轻摇动或4小时。 </li><li> 4 h后，板适当稀释到YPD（酵母提取物1％，2％蛋白胨，2％葡萄糖）的文化产生每盘约100至200个菌落，并到中型组氨酸缺乏足够数量的细胞产生可观察到的他的重组菌落数。孵育板在30 ° C为两到三天。 </li></ol> 注：1.4步介绍易位频率选择性的条件下镀上的文化中缺乏组氨酸的决心。此法可非选择性的条件下也可进行电镀到YPD文化，然后复制电镀到他板两到三天后出现的殖民地。这些方法产生类似的易位频率（ 图2C）。 <ol start="5"><li>易位的频率来确定的组氨酸prototrophic菌落数除以镀活菌总数（上YPD电镀稀释确定）。确定中位数的易位频率和95％的置信区间 15 。 </li></ol> 2。电镀效率<ol><li>每个过夜培养的细胞中取出等份，并确定由hemacytometer计数（百特医疗用品公司Catolog＃：B3178 - 1）的细胞数。 </li><li>约100至200个细胞，用适当稀释到YPD板。孵育两到三天，在30 ° C（如用1 × 10 8细胞/ ml的细胞密度的文化，应板100-200 10-5稀释每板液）。 </li><li>添加20％的半乳糖终浓度为2％的文化。 </li><li> 4小时后，取出细胞从每一种文化等分，确定hemacytometer计数细胞数，板约100至200个细胞，用适当稀释到YPD。两到三天的孵育30 ° C。 </li><li>确定出现细胞镀上YPD的殖民地，这个商数乘以100除以电镀效率。确定95％的置信区间中位数的百分比。 </li></ol> 3。基因组南方杂交分析<ol><li>选择一个单一的他+每个独立审判的重组殖民地和准备的基因组DNA 16 。 </li><li>用BamHI限制性内切酶消化约4微克的DNA。 </li><li>独立的酶BamHI酶切片段在0.7％琼脂糖凝胶，并转移到一个带正电荷的尼龙膜（Hybond N +，通用电气保健产品代码：RPN303B）17。 </li><li>一个P - 32标记的探针与随机引物（Amersham Biosciences公司产品编号：RPN1604）获得一个1.8 kb的酶BamHI / BamHI酶切基因组含有HIS3基因的克隆与杂交。 </li><li>可视化放射自显影或磷光的DNA片段。 </li></ol> 4。染色体杂交分析使用分离的染色体轮廓钳位均匀电场（厨师）： <ol><li>准备从选中他的琼脂糖插头18 +重组体的染色体： <ul><li>液体培养长出了他的 +候选人TXV：III染色体含有重组在5毫升YPD，约1-2 x 10 8细胞/毫升。 </li><li>降速和50毫米EDTA洗细胞2次。 </li><li>悬浮细胞，在约200μL50 mM的EDTA，和温暖至50 ° C。 </li><li>新增的熔融等体积的2％（W ​​/ V），低熔点琼脂糖带来至50℃，调匀。 </li><li>免除到插头模具约80μL分装，并让他们冷却30分钟，在4 ° C。 </li><li>拉伸从模具中插入一个12孔的菜。每口井可放入多达五个插头。 </li><li>加入3毫升新鲜配制的spheroplasting解决方案（2 -β-巯基乙醇14毫米，20毫米EDTA，0.5毫克/毫升Zymolyase 20T，10毫米的Tris - HCl，pH值7.5，1米山梨醇），每孔。在37 ° C孵育4小时，轻轻摇动。 </li><li>删除spheroplasting的解决方案，并取代LDS的解决方案（3毫升10毫米的Tris - HCl，pH值8.0，100毫米EDTA，1％（W / V）十二烷基硫酸钠锂，adjus吨pH值至8.0）。在37 ° C下15分钟，轻轻摇动。 </li><li>删除LDS的解决方案，并取代另一个LDS 3毫升分装。在37 ° C过夜，轻轻摇动。 </li><li>删除LDS和与NDS（5克的N -月桂酰肌氨酸，93克二钠EDTA二水，0.6克Tris碱，调节pH值至8.0，与卫生署2 500毫升）0.2 × 3毫升取代。在室温下孵育30分钟，轻轻摇动。删除NDS和重复2次。 </li><li>删除NDS和替换3毫升TE。在室温下30分钟洗净，轻轻摇动。重复4次。 </li><li>商店插头在4 ° C在2毫升的TE。插头可以保留最多的一年。 </li></ul></li><li>独立的染色体上的1％琼脂糖凝胶，用一个Bio - Rad公司的厨师DRII仪器在14 ° C（产品编号：170-3612）。 </li></ol> 参数： 第一座：70年代的开关时间，6V/cm 15H 。 第二块：120S开关时间，在6V/cm 11H <ol start="3"><li>可视化染色与1μg/ml溴化乙锭为30分钟，60兆焦耳的紫外线照射在紫外线Stratalinker（Stratagene公司）的染色体，染色30分钟DH 2 O辐照溴化乙锭染色染色体缺口的DNA，以便有效地转移到细胞膜。 </li><li>染色体转移到一个带正电的膜（Hybond N + GE医疗产品编号：RPN303B。）在变性条件下的毛细作用（0.4N的NaOH，1.5M氯化钠）。 </li><li>杂交与一个32 P标记探针与随机引物（Amersham Biosciences公司产品编号：RPN1604）获得了1.8 kb的酶BamHI / BamHI酶切基因组含有 HIS3 基因 17的克隆。 </li><li>可视化放射自显影或磷光染色体。 </li></ol> 5。代表性的成果： 描绘在染色体水平（ 图1）易位检测的一个图形表示。实验过程的示意图还显示（ 图2A） 。前和上岗后的电镀效率是可行的，集落形成细胞总数除以总数由胞体在hemacytometer计数（图4B）所确定的文化决定的。前和上岗后电镀效率没有显着不同的野生型细胞（p值= 0.1400）（ 表1）。 <img src="/files/ftp_upload/3150/3150table1.jpg" alt="表1" /> 表1。前和在野生型细胞诱导后的电镀效率 。 hemacytometer计数的每毫升细胞机构的数量确定。 b的每毫升的活菌数是由非选择性培养基上镀适当稀释，产生约100-200殖民地。 C的前和诱导后的电镀效率除以总数的决定可行的，集落形成，细胞总数hemacytometer数确定，胞体在文化。 这表明，两种易位染色体的存在或不存在不影响生存的能力DSB的形成。染色体易位的频率可以计算出电镀到YPD（ 图2B）确定的活菌总数除以组氨酸prototrophic殖民地。此法可使用不同基因型的菌株，以确定蛋白质的功能丧失如何影响SSA（即rad51Δ - / - ）进行。然后，可以绘制在不同菌株中确定的重组频率比较这些菌株的公共福利金（图2C）HO -内切酶诱导双链断裂的修复能力的差异。下选择性的野生型二倍体株（2.2x10 -2）和非选择性（2.17x10 -2）的条件下获得的易位频率在统计学上没有不同，从对方（p值= 0.9131），而易位的频率选择性地获得（6.0x10 -2）和非选择性（11.9x10 -2）与rad51Δ - / -纯合子相似，但统计学差异（p值= 0.0089） 。获得的频率选择性（p值= 0.0001）和非选择性（p值= 0.0002）与RAD51 ° - / -纯合子有统计学使用相应的条件，与野生型菌株获得的不同。 假定易位轴承克隆可进一步研究基因组Southern杂交和染色体杂交分析（ 图3）。南部分析，基因组DNA琼脂糖凝胶电泳前用BamHI内切酶消化，杂交和杂交32P标记1.8kb HIS3探头可视化诊断0.8 KBhis3Δ200，1.7 kb的HIS3 -Δ3“，4 KB tIII：第十五5 KB TXV：第三，和8 KB HIS3 -Δ5”片段（ 图3B.1） 。可以编制完整的染色体，由厨师（ 图3B.2）分隔，抹杀尼龙和32个杂交标记的P - 1.8 KB HIS3探头可视化1.1 MB第十五完整的染色体，0.8 MB TXV：三易位染色体，0.6 MB tIII：第十五易位染色体，0.3 MB，完整的染色体三（图 3B.3） 。一个图形化的地图描绘预计酶BamHI内切酶消化基因组DNA片段，以及家长和重组的染色体，是描绘（ 图3A） 。 <img src="/files/ftp_upload/3150/3150fig1.jpg" alt="图1" /> 图1。易位染色体形成单链退火（SSA），1）双链断裂的HIS3Δ3和HIS3 -Δ5“基板何内切酶（染色体十五和第三，分别）创建下面的另外半乳糖文化。 2）DSB的处理，生成破碎的染色体末端的3&#39;单链。 3）SSA机械退火互补311或60核苷酸的单链 HIS3重组基板形成序列。经退火形成的非同源的尾巴是由内切酶消化中删除。互补四个BP出挑何酶切形成的，其余的染色体片段也可退火。 4）结扎总结创造一个完整的基因HIS3和TXV：SSA三，易位染色体。携带此染色体的细胞，可以选择为他们的成长中缺乏组氨酸的能力。结扎也可能会产生互惠tIII：第十五易位染色体由一个NHEJ类似机制。 <img src="/files/ftp_upload/3150/3150fig2.jpg" alt="图2" /> 图2。确定SSA易位的频率含量 。）毫升YPGly / LAC文化是一个选择基因型的细胞单菌落接种，并成长为一个适当的密度约5X10 7 - 1 × 10 8细胞/毫升。半乳糖添加到终浓度2％，第三和第十五号染色体上的重组基板，以创建双链断裂。要选择性的条件下进行检测，适当稀释液等，约100到200个细胞镀到YPD和足够数量的细胞中缺乏组氨酸镀上产生一个观察到他的重组菌落数。无选择的进行检测，大约100到200个细胞接种到YPD，种植两到三天，以单菌落，然后到中期缺乏组氨酸的副本镀。二）易位频率可确定的殖民地，在他的板增长的比例增长YPD数量除以。 C）株不同基因型（ 即野生型和rad51Δ易位频率- / - ）可以绘制比较这些菌株的公共福利金的双链断裂的修复能力的差异。 <img src="/files/ftp_upload/3150/3150fig3.jpg" alt="图3" /> 图3。确定电镀效率 （一）等分是从过夜培养前和后DSB诱导的细胞，适当稀释，由hemacytometer计数，以确定细胞每毫升文化机构的总数。镀到非选择性培养基，以确定适当的稀释总数每毫升的活菌计数菌落出现在YPD。 （二）电镀效率，然后由细胞体的总数除以活菌总数，并乘以100这个商数确定。 <img src="/files/ftp_upload/3150/3150fig4.jpg" alt="图4" /> 图4。检测基因组Southern杂交和染色体杂交分析染色体易位的事件 。 一）有关染色体的图形表示前（左）后（右）易位形成 。 家长和重组染色体的大小列出megabase对（MB）。包含从父和重组菌株的基因组DNA经BamHI消化所产生的相关序列的限制片段的大小，以及一个1.8 kb的BamHI位HIS3基因组克隆杂交印迹显示，在碱基对（KB）上市。染色体是不是按比例绘制。 二）物理分析公认的易位轴承克隆。 （1）基因组Southern杂交分析 - 收集和基因组DNA用BamHI限制性内切酶消化，电泳，BLO分馏拟合尼龙，和一个P -标记32 1.8 kb的HIS3探针杂交，以可视化下面的片段 ：his3Δ200KB 0.8，1.7 kb的HIS3Δ3“，4 KB tIII：第十五5 KB TXV：：三， 8 KB HIS3 -Δ5“。车道：）的母公司二倍体，B） 他+非相互易位重组，C） 他 +相互易位重组。 （2）厨师凝胶 - 完整的染色体制备琼脂糖插头，由厨师分离，溴化乙锭染色，紫外光下可视化。车道：如上。 （3）染色体印迹-分离染色体涂抹尼龙和标记的P - 32 1.8 kb的HIS3探针杂交可视染色体：1.1 MB，0.8 MB TXV完整的染色体十五：三易位染色体，0.6 MB tIII：第十五易位染色体，0.3 MB完整的染色体三。车道：如上。

<ol>
	<li>Sen, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+genomic+deletions+mediated+by+recombination+between+Alu+elements.">Human genomic deletions mediated by recombination between Alu elements.</a> The American Journal of Human Genetics. 79, 41-53 (2006).</li><li>Han, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alu+recombination-mediated+structural+deletions+in+the+chimpanzee+genome.">Alu recombination-mediated structural deletions in the chimpanzee genome.</a> Plos Genetics. 3, 1939-1949 (2007).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copy+number+variation+in+human+health,+disease,+and+evolution.">Copy number variation in human health, disease, and evolution.</a> Annual Review of Genomics and Human Genetics. 10, 451-481 (2009).</li><li>Goodier, J. L., Kazazian, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrotransposons+revisited:+The+restraint+and+rehabilitation+of+parasites.">Retrotransposons revisited: The restraint and rehabilitation of parasites.</a> Cell. 135, 23-35 (2008).</li><li>Lanktree, M., Hegele, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copy+number+variation+in+metabolic+phenotypes.">Copy number variation in metabolic phenotypes.</a> Cytogenet Genome Res. 123, 169-175 (2008).</li><li>Mullighan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+genetic+alterations+in+acute+lymphoblastic+leukaemia.">Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia.</a> Nature. 446, 758-764 (2007).</li><li>Mattarucchi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microhomologies+and+interspersed+repeat+elements+at+genomic+breakpoints+in+chronic+myeloid+leukemia.">Microhomologies and interspersed repeat elements at genomic breakpoints in chronic myeloid leukemia.</a> Genes Chromosomes Cancer. 47, 625-632 (2008).</li><li>Stenger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biased+distribution+of+inverted+and+direct+Alus+in+the+human+genome:+implications+for+insertion,+exclusion,+and+genome+stability.">Biased distribution of inverted and direct Alus in the human genome: implications for insertion, exclusion, and genome stability.</a> Genome Res. 11, 12-27 (2001).</li><li>Hedges, D., Deininger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inviting+instability:+Transposable+elements,+double-strand+breaks,+and+the+maintenance+of+genome+integrity.">Inviting instability: Transposable elements, double-strand breaks, and the maintenance of genome integrity.</a> Mutat Res. 616, 46-59 (2007).</li><li>Symington, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+RAD52+epistasis+group+genes+in+homologous+recombination+and+double-strand+break+repair.">Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair.</a> Microbiol Mol Biol Rev. 66, 630-670 (2002).</li><li>Lewis, L., Resnick, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tying+up+loose+ends:+nonhomologous+end-joining+in+Saccharomyces+cerevisiae.">Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae.</a> Mutat Res. 451, 71-89 (2000).</li><li>Pannunzio, N. R., Manthey, G. M., Bailis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAD59+is+required+for+efficient+repair+of+simultaneous+double-strand+breaks+resulting+in+translocations+in+Saccharomyces+cerevisiae.">RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae.</a> DNA Repair (Amst). 7, 788-800 (2008).</li><li>Nicholas, R., Pannunzio, G. M. M., Bailis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAD59+and+RAD1+cooperate+in+translocation+formation+by+single-strand+annealing+in+Saccharomyces+cerevisiae.">RAD59 and RAD1 cooperate in translocation formation by single-strand annealing in Saccharomyces cerevisiae.</a> Curr Genet. 56, 87-100 (2010).</li><li>Manthey, G. M., Bailis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad51+Inhibits+Translocation+Formation+by+Non-Conservative+Homologous+Recombination+in+Saccharyomyces+cerevisiae.">Rad51 Inhibits Translocation Formation by Non-Conservative Homologous Recombination in Saccharyomyces cerevisiae.</a> Plos One. 5, (2010).</li><li>Hoffman, C. S., Winston, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ten-minute+DNA+preparation+from+yeast+efficiently+releases+autonomous+plasmids+for+transformation+of+Escherichia+coli.">A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli.</a> Gene. 57, 267-272 (1987).</li><li>Sambrook, J., MacCallum, P., Russell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , (2001).</li><li>Iadonato, S. P., Gnirke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RARE-cleavage+analysis+of+YACs.">RARE-cleavage analysis of YACs.</a> Methods Mol. Biol. , 75-85 (1999).</li><li>Argueso, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+breaks+associated+with+repetitive+DNA+can+reshape+the+genome.">Double-strand breaks associated with repetitive DNA can reshape the genome.</a> Proc. Natl. Acad. Sci. U. S. A. , 11845-11850 (2008).</li><li>Batzer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alu+repeats+and+human+disease.">Alu repeats and human disease.</a> Molecular Genetic Metabolism. 67, 183-193 (1999).</li><li>Fasullo, M., Dave, P., Rothstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-damaging+agents+stimulate+the+formation+of+directed+reciprocal+translocations+in+Saccharomyces+cerevisiae.">DNA-damaging agents stimulate the formation of directed reciprocal translocations in Saccharomyces cerevisiae.</a> Mutat. Res. 314, 121-133 (1994).</li><li>Richardson, C., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+chromosomal+translocations+induced+by+DNA+double-strand+breaks.">Frequent chromosomal translocations induced by DNA double-strand breaks.</a> Nature. 405, 697-700 (2000).</li><li>Look, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+altered+by+chromosomal+translocations+in+leukemias+and+lymphomas.">Genes altered by chromosomal translocations in leukemias and lymphomas.</a> The Genetic Basis of Human Cancer. , (2002).</li><li>Fasullo, M. T., Davis, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direction+of+chromosome+rearrangements+in+Saccharomyces+cerevisiae+by+use+of+his3+recombinational+substrates.">Direction of chromosome rearrangements in Saccharomyces cerevisiae by use of his3 recombinational substrates.</a> Mol. Cell. Biol. 8, 4370-4380 (1988).</li><li>Manthey, G. M., Naik, N., Bailis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Msh2+Blocks+an+Alternative+Mechanism+for+Non-Homologous+Tail+Removal+during+Single-Strand+Annealing+in+Saccharomyces+cerevisiae.">Msh2 Blocks an Alternative Mechanism for Non-Homologous Tail Removal during Single-Strand Annealing in Saccharomyces cerevisiae.</a> PLoS One. 4, e7488-e7488 (2009).</li><li>Muller, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-course+of+radiation-induced+chromosomal+aberrations+in+tumor+patients+after+radiotherapy.">Time-course of radiation-induced chromosomal aberrations in tumor patients after radiotherapy.</a> Int. J. Radiation Oncology Biol. Phys. 63, 1214-1220 (2005).</li><li>Lin, F. L., Sperle, K., Sternberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+for+homologous+recombination+during+transfer+of+DNA+into+mouse+L+cells:+role+of+DNA+ends+in+the+recombination+process.">Model for homologous recombination during transfer of DNA into mouse L cells: role of DNA ends in the recombination process.</a> Mol. Cell. Biol. 4, 1020-1034 (1984).</li><li>Ivanov, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Requirements+for+the+single-strand+annealing+pathway+of+double-strand+break+repair+in+Saccharyomyces+cerevisiae.">Genetic Requirements for the single-strand annealing pathway of double-strand break repair in Saccharyomyces cerevisiae.</a> Genetics. 142, 693-704 (1996).</li></ol>

没有利益冲突的声明。

高剂量的电离辐射，目前通过大量的DSB 的 19代的基因组不稳定的固有风险。真核基因组充斥着重复序列，产生易位和其他基因组重排 20,21的优秀基板。经常观察12,21,22的重复序列之间的介绍时，DSB的人力资源染色体易位。大量证据表明，大部分可以归结为染色体易位的白血病和淋巴瘤相关的基因组不稳定，突出重要性的认识这个机制是如何发生在真核生物中 22,23 。我们已经开发出在芽殖酵母为研究上的大小相似，分散在整个酵母和人类基因组的重复元素不同的染色体易位染色体之间的同源性短地区形成DSB诱导的HR系统。 在试验中，HIS3Δ3“易位基板位于之一第十五号染色体的复制上。其他HIS3基因（HIS3Δ200）的HIS3启动子和编码序列，以防止这个序列被用来作为模板修复24〜1KB删除。 HIS3Δ5“基板位于LEU2轨迹上的第三号染色体的一个副本，其他含有一个不变的LEU2等位基因（ 图 1）与第三号染色体的的复制。一个半乳糖诱导HO的内切酶表达盒标有侃 MX插入到第四号染色体（TRP1：GAL中，何侃MX）TRP1的位点。每个易位基板两侧是由HO -内切酶识别序列，除了中期通过半乳糖诱导的何基因的表达，可用于裂解针对性。何内切酶诱导HIS3 -Δ3和HIS3Δ5“基板切割后，细胞可以有效地使用共享短HIS3序列同源性呼吸道（311 bp或60 BP）的人力资源来修复破碎的染色体，产生易位染色体一个完整的HIS3基因12-14,25。 由于母细胞缺乏HIS3基因的完整的副本，他们是无法生长，他的媒介。只有有发生易位事件中缺乏组氨酸的细胞可以选择。因此，染色体易位的频率可以计算活菌镀镀上YPD确定，总数除以组氨酸prototrophic殖民地。基因组DNA和完整的染色体，然后，可以分离出代表他的 +克隆和基因组南方和染色体杂交分析证实易位染色体的存在。 仔细分析，使我们能够收集更多的有关重要信息检测12。基因组Southern杂交分析提供了证据表明，有十五和第三号染色体的切割，HO -内切酶诱导后30分钟几乎是完整的，因此，在人口没有完整无缺的染色体基板显著的背景（G. Manthey＆A。Bailis未发表的结果）。他的染色体基因组南方杂交分析-幸存者表示，细胞经常失去一个，其他的，或切割染色体，并保持活力（L.利德尔＆A. Bailis，未发表的结果） 。更重要的是，几乎相当于非选择性培养基上的电镀效率表明，修复损坏的染色体既没有失败，也未能挽留易位染色体影响生存的能力DSB的形成前后诱导HO -内切酶的表达。与此相一致，TXV：三，易位染色体已被证明在没有选择的情况下，在有丝分裂细胞不稳定。这体现在越来越多TXV：三载有他的 +重组子过夜非选择性，非选择性板电镀到单菌落，和副本电镀到缺乏组氨酸的选择性培养基。 10 70％的殖民地这些板块所产生的已经失去的TXV：三易位染色体（N. Pannunzio＆A. Bailis，未公布结果）。 由暴露在人体的红外产生的易位染色体表现出了类似的不稳定26。这表明，易位的形成可能有助于促进杂合性缺失在肿瘤发生的早期事件。二，广泛的遗传和分子生物学分析表明，SSA，人力资源的效率和强制性非保守的机制，是以下两条染色体12,27,28 DSB的同时创造易位形成人力资源的主要机制。这是合作nsistent的发现，在创建多个重复序列在酵母基因组中相邻的休息了双链断裂足够的密度，并通过人力资源的易位形成的高频率的红外的结果大剂量。总之，这些观察表明的红外暴露在人类可能致癌的影响，部分从人力资源的有效机制，产生易位，通过其内在的不稳定性，促进推出肿瘤发生的遗传变化的双链断裂的修复结果。因为辐射是经常被用来治疗癌症，基因组重排造成的辐射诱导的DNA双链断裂的修复可能会导致患者经常出现的继发性肿瘤的产生。因此，这种模式可以帮助我们更好地了解获得的遗传和分子基础的一个重要的临床反应IR的治疗。

quantitation and analysis of the formation of ho-endonuclease stimulated chromosomal translocations by single-strand annealing in saccharomyces cerevisiae

遗传变异通常是介导的基因组重排，通过互动之间产生分散在每一个真核基因组中存在的重复元素。这个过程是一个用于生成之间以及生物体中的1-3的多样性的重要机制。人类基因组包含大约40％的重复序列，反转录转座子的起源，包括各种线和正弦 4 。这些重复的元素之间的交流活动可以导致基因组重排，包括易位，能够破坏基因的剂量和表达，可能导致自身免疫性疾病和心血管疾病，以及在人类癌症 6-9 。 多种方式重复元素之间的交流发生在。共享完美（或近乎完美的）的同源性的序列之间的交流出现这个过程被称为同源重组（HR）。相比之下，非同源末端连接（NHEJ）用于交换10,11很少或没有序列同源性。 ，在有丝分裂细胞，人力资源的主要目的是修复双链断裂（DSB的）所产生的异常DNA的复制和氧化损伤的内源性，或暴露于电离辐射（IR）和其他外源DNA损伤剂。 在这里描述的检测，DSB的同时创建两个不同的染色体二倍体细胞中的基因位点，由半乳糖诱导HO -内切酶（图1）接壤重组基板。修复破碎的染色体染色体易位产生单链退火（SSA）的，其中同源序列的染色体末端附近的一个过程共价键加入后退火。其中的基板，HIS3 -Δ3“，包含了3个” 截断 HIS3基因位于染色体第十五的复制在本地HIS3轨迹。第二基板，HIS3Δ5“，位于LEU2轨迹上的第三号染色体的一个副本，并包含一个5&#39; 截断 HIS3基因。这两种基板两侧由HO HO -内切酶的切口，可用于有针对性的内切酶识别位点。何内切酶的识别位点的原生的 MAT轨迹，在第三号染色体的的两个副本，已在所有菌株中删除。这可防止复合基板和其他干扰检测破碎染色体末端之间的互动。 侃MX -标记的半乳糖诱导HO内切酶表达盒插入TRP1第四号染色体上的轨迹。基板份额311基点或60 BP HIS3编码序列，由福利金，可用于人力资源机械修理。使用这些基板的人力资源来修复破碎染色体的细胞形成一个完整的 HIS3等位基因和一个TXV：III染色体易位可以选择的能力，成长中缺乏组氨酸（ 图2A） 。通过人力资源的易位的频率计算除以组氨酸prototrophic殖民地，在选择性培养基上出现的非选择性培养基（ 图 2B）镀上适当稀释后产生的活菌总数。已被用于多种DNA修复突变体研究易位形成的遗传控制，使用本系统12-14 SSA。

Watch this Scientific Journal Video about Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae at 

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

何刺激的易位检测监控以下二倍体多个位点的DNA双链断裂创造单链退火<em&gt;酿酒酵母</em&gt;。这种机制可能模式在高等真核生物体细胞暴露于高剂量的电离辐射的基因组重排。

定量分析和HO -核酸内切酶的形成，刺激单链退火染色体易位酿酒酵母</em

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

quantitation-analysis-formation-ho-endonuclease-stimulated

Research

JoVE Journal

Biology

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

14.6K 次观看.  Irell & Manella Graduate School of Biological Sciences. 何刺激的易位检测监控以下二倍体多个位点的DNA双链断裂创造单链退火酿酒酵母。这种机制可能模式在高等真核生物体细胞暴露于高剂量的电离辐射的基因组重排。

视频: Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

剖析酿酒酵母 ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

科研

生物学

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 &#181;M. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

EPR监控的辅助因子氧化还原滴定<em&gt;酿酒酵母</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA的衰变率的测量<em&gt;酿酒酵母</em&gt;使用<em&gt; rpb1-1</em&gt;株

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...