我们感谢提供质粒和酵母S. Jinks罗伯逊和E Heidenreich; P. Pham和中频古德曼帮助了与TLS的检测。这项工作是支持中的一部分，美国联邦和衰老研究补助金由NIH AG20642，AG025135。

两个寿命模型被用来研究在 S老化酵母 :RLS和CLS。复制（萌芽）寿命（RLS）的基础上观察，酵母母细胞进行有限数量的3-6部门。我们将重点讨论的时间寿命（CLS），模型的基础上，文化或板7-11非分裂酵母的时间生存。野生型酵母生长指数为10-12小时，在合成葡萄糖完成（SDC）中型。当血糖水平降低，酵母发酵呼吸的开关，这个过程称为diauxic转变。细胞继续分裂期间慢慢进入G 0逮捕前约48小时后diauxic阶段。细胞的代谢率仍然高至5-6天（0天接种日）。随着时间的推移细胞的生存能力可以访问按时间顺序老化细胞的百分比抽样，每两天，可以退出 G 0逮捕和形式丰富YPED板殖民地。 1。酵母顺序寿命（CLS）的液体培养<ol><li>单个菌落接种到1毫升合成完整的葡萄糖培养基（SDC，表1）孵育过夜振荡（220转）30 ° C。三个独立的殖民地接种应提供生物副本的每个应变准备。 </li><li>食入SDC的介质（通常为10毫升）稀释过夜培养外径= 0.05-0.1（〜1:100稀释）和孵化用颤抖的（220转）30 ° C。这个时间点被认为是0天的时间老化。保持一个文化/烧瓶的体积（10毫升50毫升的文化，或长/大实验在125毫升25毫升文化）1:5的比例，以确保适当的曝气。 </li><li>从第3天开始，取出10μL从烧瓶等分，YEPD培养基高压灭菌的水，10μL稀释的文化（即10月4 -10）（1％酵母提取物，细菌用胰蛋白胨2％，2％葡萄糖稀释10000倍2％琼脂）板，并在30℃孵育48-72小时前殖民地形成团结（CFU）的计算。 </li><li> 3天CFU被认为是100％生存。老化在随后的日子里文化的稀释因素，应作相应调整，以确保〜20-100的CFU检测菌落（例如，10月4 -10，10月4 -30 10 3 -10等）。对于野生型细胞，在DBY746背景的华彩达到1％，生存11天左右。 </li></ol> 在原位的可行性实验的变化包括: <ul><li>热量限制（CR）葡萄糖限制。稀释葡萄糖有限的SC培养基培养过夜（如0.5或0.05％，而不是标准的2％葡萄糖），一般会导致低人口密度在第3天的饱和度，但很多扩展的平均值和最大值（10％ 的生存）的寿命12。 </li><li>极端CR /饥饿，灭菌水冲洗3天的固定相的文化（通常10毫升，如在上述第3步，）（悬浮细胞，在同等体积的水和自旋向下在2500转5分钟，重复3次）。 Incubationof细胞在水中模仿饥饿的条件下，酵母可能会遇到在野外。每两天后，老龄化的文化应与灭菌水清洗，并在同等体积的水悬浮删除裂解死细胞释放任何营养素。执行采样老化文化如上所述的可行性实验。这种饥饿状况将导致几乎DBY746在野生型菌株12平均华彩翻番。 </li></ul> 在原位的可行性实验2。 在液体培养基中，存活细胞的一小部分可能会重新进入细胞周期和成长，利用剩下的营养素或这些发布的形式溶解在介质中的死细胞， 称为表型再生/喘气13。我们开发了一个可行性上一个直接制版系统，利用DBY746应变（TRP1）的营养缺陷型来规避再生/喘气问题，也让不断曝光，剥夺或外部的各种营养素或酵母刺激的效果测试CLS 11。 在原位可行性实验，这也模仿复制寿命模型，细胞不断暴露寿命分析的时间，以丰富的营养物质。 <ol><li>单个菌落接种到1毫升合成完整的葡萄糖培养基（SDC）和孵育过夜振荡（220转）30 ° C。至少有三个独立的殖民地接种应提供生物副本每个应变准备。 </li><li>让文化成长〜10 8细胞/ ml（外径600 =〜10），淡化水culturewith蒸压100-200 cell/10μL（通常是10 的 4倍稀释）和1000 cells/10μL（10 3倍稀释）。 </li><li>板块上一个色氨酸辍学SDC板10-30μl稀释文化的两个等分。对于ř每一株，准备8日至20板的设置，根据电镀密度和标记（例如，10的4倍稀释，板10μL或10 4 -10 10 -30 10 3 -10 10 3 -30等），确保10-100殖民地可以算在预期下降的可行性，在衰老过程中。 </li><li>孵育寿命分析的时间设置在30 ° C板。 </li><li>电镀一天，每2天随后，取出一板和明智的下拉添加色氨酸（2毫克/毫升）0.5毫升，让可行的细胞生长。额外的48-72小时，在30 ° C孵育板。统计色氨酸除了一天的可行性CFU。电镀一天的CFU被认为是100％的存活率。 </li></ol> 在原位的可行性实验的变化包括: <ul><li>平板时代的细胞（极端饥饿状态）。加入1 ml 2X YPED色氨酸，而不是在随后的日子里，让经济增长和CFU 计数 14 。 </li><li>色氨酸辍学合成完全培养基不同碳源，如各种浓度的葡萄糖（葡萄糖限制热量的限制），各种碳源，如乙醇，甘油，醋酸14板的年龄细胞。加入1毫升的葡萄糖/色氨酸溶液（20％葡萄糖和1mg/ml的色氨酸），让经济增长和CFU计数。 </li><li>其他营养物质的操纵，如氮。 </li></ul> 3。在按时间顺序衰老的DNA损伤和突变频率 刀豆电阻（CAN R）和CAN1测序 自发突变频率可以通过测量刀豆电阻（CAN R）按时间顺序老化文化频率进行评估。 CAN1（YEL063）基因，它编码的血浆精氨酸通透，的突变使细胞抵抗的模拟L - canavanine.Can精氨酸ř在不同时间点收集的殖民地，也可用于基因组DNA 的提取和随后的CAN1测序保存基因，它可以提供突变谱数据（突变测量师，SoftGenetics）。 碱基（TRP +回归） TRP1 - 289菌株在TRP1编码序列中包含一个琥珀突变（C403T） 。测量频率色氨酸+回归15 TRP1 - 289允许在酵母顺序老化中的碱基替换率的估计。 移码突变赖氨酸-应变EH150（ 马塔，lys2ΔBglII，TRP1 -Δ，HIS3 -Δ200，URA3 - 52，ADE2 - 1O）港口通过插入4个核苷酸，以创建一个经 Bgl II限制在 LYS2基因的酶切位点，构建一个lys2ΔBglII突变。由此产生的4转向开放阅读赖氨酸营养缺陷型的框架，可以通过小的插入/ 缺失突变16-17逆转结果。 总值的染色体重排（GCRs） 为了检测染色体重排总值（GCRs），我们产生了突变株，其中HXT13（YEL069），编码一个高度冗余的己糖转运，是一个 URA3 卡带 18中断。HXT13位于第五号染色体上的基因突变是7.5 kb的端粒， 以 CAN1两个CAN1和URA3基因使细胞的耐药性分别为L -刀豆和5 -氟酸（5FOA），。考虑到，在这两个基因， 可以 5FOA ř频率分析发生点突变频率低，提供了一个估算的GCRs结果，在这两个基因的损失。 同源和homeologous重组要监视的同源水平（100％），在时间顺序老化homeologous重组（91％），我们产生了突变体的线性质粒（HIS3:内含子IR - URA3）携带"100％同源反向重复序列（IRS）（ pSR406 ）或homeologous IRS HIS3轨迹19（pSR407）的91％ 。国税局之间的重组，允许功能HIS3蛋白的表达。 <ol><li>在正常的可行性实验（如上所述）的同时，取出一个细胞老化文化适量， <ol><li> 可以突变，开始〜2 × 10 7细胞（200μL）3天文化; </li><li>色氨酸+回归，开始〜10 8个细胞（500-1000微升3天的文化）; </li><li>赖氨酸+帧移码突变，启动〜10 8个细胞（500-1000第3天文化液） ; </li><li> GCRs，开始2 - 3x10 8细胞（2-3毫升3天的文化）; </li><li>同源和homeologous重组，启动及波浪线5X10 7细胞（200-500微升3天的文化）; </li></ol>调整电镀量，减少总的可行性和突变频率的增加在时间顺序老化（ 见表 2 ）。株hypermutator表型（如DNA修复缺陷的）的情况下，相应地减少取样量。 </li><li>颗粒与台式离心机（6000转）5分钟的细胞。 </li><li>再次重悬在1毫升蒸压水，和颗粒细胞的细胞。 </li><li>悬浮细胞沉淀在100μL水。板细胞， <ol><li> ř突变，精氨酸辍学合成完整的中小板，（SDC -精氨酸，辅以60微克/毫升硫酸L -刀豆）; </li><li>色氨酸+回归，色氨酸辍学板（SDC -色氨酸）; </li><li>赖氨酸+帧移码突变，赖氨酸辍学板（SDC -赖氨酸）; </li><li>为GCRs，精氨酸辍学板（SDC -精氨酸）辅以5FOA（1毫克/毫升）和L -刀豆硫酸（60微克/毫升）; </li><li>同源和homeologous重组，补充组氨酸辍学板，半乳糖（2％）。 </li></ol></li><li>计数CFUs 3-4天的潜伏期后，30 ° C。突变频率是正常化的活菌数。 </li></ol>补充到原位的可行性实验，年龄依赖性色氨酸+回归，赖氨酸+移码突变，重组，或者可以ř也可以在铭牌上老化细胞研究。 <ol><li>板细胞（〜10 8细胞/板），色氨酸，赖氨酸，他辍学，或L -刀豆氨酸的合成完整的中板（如上所述）。 </li><li>每两天（或感兴趣的时间点），得分新兴的殖民地。 </li><li>突变频率估计正常化新兴殖民地特定的一天活菌总数。 </li></ol> 4。 Translesion合成性（TLS） 年龄依赖性的突变频率的增加可能涉及增加大分子的损伤，减少细胞保护/修复，并增加在衰老过程中的DNA修复错误（如Polζ依赖translesion合成，TLS）。例如，长寿命的sch9Δ突变体表现出SOD2升高表达，表达下降的容易出错的DNA修复酶 REV1 20 。在这里，我们描述了一个实验，受损的DNA模板和整个核提取物相结合 ，以评估在体外的TLS。 <ol><li>准备核提取物Wang 等人所述的 21个;量化BCA法测定（皮尔斯）的蛋白浓度。 </li><li> 0，5，10或20微克至50μL（终体积）反应，包含TLS的缓冲液（20毫米HEPES，MgCl 2的7毫米，1毫米的数码地面电视，25毫米氯化钠，pH值7.8），200μMdNTPs和100 nm的核提取物5&#39; - 32 P标记的12 - mer引物（5&#39; - CGA TGG TAC GGA - 3&#39;）退火48 - MER模板包含利益的损害DNA（5&#39; - TCG的ATA CTG的多伦多注册税务师ATG的AT＆T的AAC GAC中TXA AGC ACG TCC GTA CCA TCG的3&#39;，其中X是损坏的部位，例如，8 - 氧代- G碱基网站，等）。 </li><li>孵育的混合物，在30 ° C为30分钟，增加2.5μLEDTA（20毫米）和2μL蛋白酶K（10毫克/毫升）终止反应。 </li><li>苯酚抽提和乙醇沉淀分离纯化的DNA。 </li><li>悬浮在凝胶样缓冲液50μL的DNA（20毫米EDTA，98％甲酰胺和染料）。 </li><li>解决19％聚丙烯酰胺凝胶translesion的合成产品。 </li><li>量化的TLS使用磷光（ 图1）。 </li></ol> 5。代表性的成果我们通常使用为100％的标准液体华彩分析生存3天的CFU计数。生存，可以安装在随后几天的百分比计算的平均值（50％的生存）和最高（10％ 的生存）寿命12。寿命从原位可行性检测得到的结果通常是一致的，但与那些使用液体华彩检测与减少的平均寿命，这是由于部分细胞不断暴露营养素。葡萄糖存在抑制保护细胞的活性，减少应激反应转录因子，如MSN2 / 4和Gis1 12的反式显示。 年龄相关的基因突变频率很大程度上取决于应变的背景下，遗传操纵，文化条件和年龄而异。表2显示了在野生型菌株（DBY746）获得的典型结果。 <table border="1"><tbody><tr><th>组件</th><th> g / L的</th></tr><tr><td> D -葡萄糖</td><td> 20 </td></tr><tr><td>硫酸铵</td><td> 5 </td></tr><tr><td>氮基（-AS/-AA） </td><td> 1.8 </td></tr><tr><td>磷酸二氢钠</td><td> 1.4 </td></tr><tr><td></td><td> 毫克/升 </td></tr><tr><td>腺嘌呤</td><td> 80 </td></tr><tr><td> L -精氨酸</td><td> 40 </td></tr><tr><td> L -天冬氨酸</td><td> 100 </td></tr><tr><td> L -谷氨酸</td><td> 100 </td></tr><tr><td> L -组氨酸</td><td> 80 </td></tr><tr><td> L -异亮氨酸</td><td> 60 </td></tr><tr><td> L -亮氨酸</td><td> 120 </td></tr><tr><td> L -赖氨酸</td><td> 60 </td></tr><tr><td> L -蛋氨酸</td><td> 80 </td></tr><tr><td> L -苯丙氨酸</td><td> 60 </td></tr><tr><td> L -丝氨酸</td><td> 400 </td></tr><tr><td> L -苏氨酸</td><td> 200 </td></tr><tr><td> L -色氨酸</td><td> 80 </td></tr><tr><td> L -酪氨酸</td><td> 40 </td></tr><tr><td> L -缬氨酸</td><td> 150 </td></tr><tr><td>尿嘧啶</td><td> 80 </td></tr></tbody></table> 表1合成完整的葡萄糖培养基，SDC（用NaOH调整pH至6.0） 。超过4倍，组氨酸，亮氨酸，色氨酸和尿嘧啶包括以弥补营养缺陷型的DBY746毒株。 <table border="1"><tbody><tr><th></th><th>第3天（平均± SEM） </th><th> （在衰老过程中） </th></tr><tr><td> 可以ř </td><td> 1.76 ± 0.12 × 10 -6 </td><td> 6-8 X10 -6 </td></tr><tr><td> 色氨酸+的回归 </td><td> 6.60 ± 1.70 × 10 -8 </td><td> 1.60 × 10 -6 </td></tr><tr><td> 赖氨酸+帧移码突变 </td><td> 3.00 ± 0.78 × 10 -8 </td><td> 0.70 × 10 -6 </td></tr><tr><td> GCRs </td><td> 0.62 ± 0.10 × 10 -8 </td><td> 0.30 X10 -6 </td></tr><tr><td> 同源重组 </td><td> 5.55 ± 2.74 × 10 -6 </td><td> 27.00 X10 -6 </td></tr><tr><td> Homeologous重组 </td><td> 0.12 ± 0.04 × 10 -6 </td><td> 0.48 × 10 -6 </td></tr></tbody></table> 表2。典型的野生型细胞的突变频率，在时间顺序老化（DBY746 ）。 <img alt="图1" src="/files/ftp_upload/3030/3030fig1.jpg" /> 图1。translesion合成（TLS）20的结果。从3日龄固定相（DBY746）野生型和sch9Δ突变的细胞的核提取物与未受损害或碱基的网站，其中包含30分钟的DNA模板，在30 ° C孵育TLS产品是由固体（完好的模板）或点（损坏的模板）线。有没有translesion sch9Δ突变体合成核提取物中观察到。免费引物的开箭头表示。

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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+simple+model+system+for+age-dependent+DNA+damage+and+cancer.">A simple model system for age-dependent DNA damage and cancer.</a> Mech Ageing Dev. 128, 45-49 (2007).</li><li>Wei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+span+extension+by+calorie+restriction+depends+on+Rim15+and+transcription+factors+downstream+of+Ras/PKA,+Tor,+and+Sch9.">Life span extension by calorie restriction depends on Rim15 and transcription factors downstream of Ras/PKA, Tor, and Sch9.</a> PLoS Genet. 4, e13-e13 (2008).</li><li>Fabrizio, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+is+a+mediator+of+an+altruistic+aging+program+in+Saccharomyces+cerevisiae.">Superoxide is a mediator of an altruistic aging program in Saccharomyces cerevisiae.</a> J Cell Biol. 166, 1055-1067 (2004).</li><li>Wei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tor1/Sch9-regulated+carbon+source+substitution+is+as+effective+as+calorie+restriction+in+life+span+extension.">Tor1/Sch9-regulated carbon source substitution is as effective as calorie restriction in life span extension.</a> PLoS Genet. 5, e1000467-e1000467 (2009).</li><li>Capizzi, R. L., Jameson, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+table+for+the+estimation+of+the+spontaneous+mutation+rate+of+cells+in+culture.">A table for the estimation of the spontaneous mutation rate of cells in culture.</a> Mutat Res. 17, 147-148 (1973).</li><li>Heidenreich, E., Novotny, R., Kneidinger, B., Holzmann, V., Wintersberger, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-homologous+end+joining+as+an+important+mutagenic+process+in+cell+cycle-arrested+cells.">Non-homologous end joining as an important mutagenic process in cell cycle-arrested cells.</a> Embo J. 22, 2274-2283 (2003).</li><li>Heidenreich, E., Wintersberger, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-dependent+and+selection-induced+mutations+in+respiration-competent+and+respiration-deficient+strains+of+Saccharomyces+cerevisiae.">Replication-dependent and selection-induced mutations in respiration-competent and respiration-deficient strains of Saccharomyces cerevisiae.</a> Mol Gen Genet. 260, 395-400 (1998).</li><li>Chen, C., Kolodner, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gross+chromosomal+rearrangements+in+Saccharomyces+cerevisiae+replication+and+recombination+defective+mutants.">Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants.</a> Nat Genet. 23, 81-85 (1999).</li><li>Datta, A., Adjiri, A., New, L., Crouse, G. F., JinksRobertson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+crossovers+between+diverged+sequences+are+regulated+by+mismatch+repair+proteins+in+Saccaromyces+cerevisiae.">Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae.</a> Mol Cell Biol. 16, 1085-1093 (1996).</li><li>Madia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogene+homologue+Sch9+promotes+age-dependent+mutations+by+a+superoxide+and+Rev1/Polzeta-dependent+mechanism.">Oncogene homologue Sch9 promotes age-dependent mutations by a superoxide and Rev1/Polzeta-dependent mechanism.</a> J Cell Biol. 186, 509-523 (2009).</li><li>Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+expression+of+recombinant+genes+and+preparation+of+cell-free+extracts+in+yeast.">Controlled expression of recombinant genes and preparation of cell-free extracts in yeast.</a> Methods Mol Biol. 313, 317-331 (2006).</li><li>Fabrizio, P., Pletcher, S. D., Minois, N., Vaupel, J. W., Longo, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronological+aging-independent+replicative+life+span+regulation+by+Msn2/Msn4+and+Sod2+in+Saccharomyces+cerevisiae.">Chronological aging-independent replicative life span regulation by Msn2/Msn4 and Sod2 in Saccharomyces cerevisiae.</a> FEBS Lett. 557, 136-142 (2004).</li><li>Allen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+quiescent+and+nonquiescent+cells+from+yeast+stationary-phase+cultures.">Isolation of quiescent and nonquiescent cells from yeast stationary-phase cultures.</a> J Cell Biol. 174, 89-100 (2006).</li><li>Madia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+mutation+in+SCH9+prevents+recombination+errors+and+premature+genomic+instability+in+a+Werner/Bloom+model+system.">Longevity mutation in SCH9 prevents recombination errors and premature genomic instability in a Werner/Bloom model system.</a> J Cell Biol. 180, 67-81 (2008).</li></ol>

没有利益冲突的声明。

液体老化文化的某个时候表现出的再生/喘气表型的13，其中的华彩分析的复杂性。愈合，通常会出现超过90-99％的人口已经失去了可行性。这种表型往往与增加氧化应激和/或降低保护细胞。例如，这种表型的频率超过在缺乏胞质超氧化物歧化酶的细胞双打，并大大降低了在长寿的突变体 （例如，sch9Δ或ras2Δ）或22突变体的过度表达超氧化物歧化酶。在实践中，我们定义为增加可行性或稳定在3连续采样在时间顺序老化，死亡率高阶段的可行性再生。 年龄相关的不同的DNA突变频率差异很大，取决于应变的背景下，遗传操纵，文化条件，以及再生/喘气和极低的生存。应进行预实验，在时间点，如与突变检测的各种电镀密度平均和最大的生存前performingthe满刻度的寿命分析，以确定突变频率范围。野生型菌株应始终包含在一个寿命或突变频率的研究，平行任何治疗或基因突变等实验间的变化可占。多种生物副本应包括在研究，液体和在应进行原位可行性/突变分析证实的结果。 而是把重点放在一个特定类型的DNA突变分析，组合使用多种检测年龄相关的基因组不稳定，可能揭示出特定的DNA损伤和DNA损伤修复系统（S），年龄相关的基因组不稳定。例如，在毛的染色体重排（GCRs）显著增加，比其他DNA突变的，是观察野生型酵母提示elevationof双链断裂和/或减值的非同源末端加入酵母期间（NHEJ）按时间老化（见表2）。 CAN1突变谱（CAN1的基因测序）在野生型老化酵母建议时间老化和容易出错的DNA修复过程中的增加，而在长寿命sch9Δ突变体，少氧化DNA损伤和氧化损伤大大减少错误容易发生translesion合成观察20。 此处描述的方法可以进一步支出来研究年龄相关的基因组不稳定。例如，静态和非静态的细胞可以分离出酵母固定相使用密度梯度法Allen 等人所描述的文化23。这里所描述的基因突变检测相结合，我们曾报道，一大截的年龄相关的基因突变，从静态的细胞，而不是分割，损坏或细胞凋亡20,24，出现。

studying age-dependent genomic instability using the s. cerevisiae chronological lifespan model

使用酿酒酵母衰老模型的研究已发现的寿命是部分在高等真核生物 1-2中保守的调控途径。酵母衰老模型的简单性和力量，也可以探讨研究DNA损伤和基因组维修以及他们的贡献，在衰老过程中的疾病。在这里，我们描述了一个系统来研究年龄相关的DNA突变，包括碱基置换，移码突变，染色体重排总值，并同源/ homeologous重组，以及通过简单的DNA相结合的酵母顺序寿命核DNA修复活性损伤和突变检测。这里所描述的方法应便于识别基因/途径调节基因组的不稳定和机制的基础在哺乳动物中年龄依赖性的DNA突变与癌症。

Watch this Scientific Journal Video about Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model at JoVE.com

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

在这里，我们描述了一套与酵母顺序寿命模型来研究基因/通路调节，或在衰老过程中作出贡献基因组DNA不稳定，可结合DNA突变检测。

研究年龄相关的基因组不稳定性使用 S。酵母年代的寿命模型

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

studying-age-dependent-genomic-instability-using-s-cerevisiae

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

15.5K 次观看.  University of Southern California, Los Angeles. 在这里，我们描述了一套与酵母顺序寿命模型来研究基因/通路调节，或在衰老过程中作出贡献基因组DNA不稳定，可结合DNA突变检测。

视频: Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

化验粘附和酿酒酵母琼脂入侵

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

科研

生物学

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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 ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

斑马鱼肾功能评价采用荧光测定间隙

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

在单核级LINE-1逆转录转座子分析

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

研究年龄相关的基因组不稳定性使用 S。酵母年代的寿命模型

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