我们感谢所有当前和以前的安德鲁·库恩克尔实验室成员的工作和讨论与协议和重复使用的数据在这里提出。这项工作得到了 ES065070 的项目 Z01 的支持, T.A.K. 国家卫生研究院 (NIH) 的内部研究司 (NIEHS)。

1. 碱性水解和链特定的南方印迹 (图 1)
<ol><li>细胞生长与基因组 DNA 分离<ol>
<li>根据制造商的说明, 使用酵母 DNA 纯化试剂盒分离酵母基因组 dna。使用处于生长指数阶段的区域性 (介于0.5 和1之间的光密度之间)。并用重悬在35毫升的 1x TE 缓冲器 (10 毫米三盐酸, pH 7.5, 1 毫米乙二胺乙酸 (EDTA)) 的最终 DNA 颗粒。</li>
		<li>添加1毫升的5毫克/毫升 RNase A 到 DNA 和孵育30分钟在37摄氏度。</li>
		<li>用0.1 容量的3米醋酸钠 (pH 5.2) 和2.5 卷100% 乙醇在冰上沉淀 DNA。</li>
		<li>离心机在 1.6万 x g 20 分钟在4摄氏度。用吸管取出上清液。</li>
		<li>用500毫升70% 乙醇清洗颗粒。用吸管取出上清液。</li>
		<li>用35毫升的 1x TE 缓冲器将并用重悬上的颗粒干燥。</li>
		<li>定量使用 fluorimeter (材料表) 与 dsDNA BR 试剂盒进行 DNA 检测。</li>
		<li>5毫克的 DNA 从每个样品使用0.1 容量3米醋酸钠, pH 5.2 和2.5 容量100% 乙醇为总容量200毫升 (增加 H2O 如果必要, 需要另外的5毫克脱氧核糖核酸, 如果中性凝胶控制是必要的)。在冰上孵育20分钟。</li>
		<li>离心机在 1.6万 x g 20 分钟在4摄氏度。用吸管取出上清液。</li>
		<li>把球团干在板凳上。并用重悬的 DNA 在14毫升的 H2O。</li>
	</ol></li>
	<li>碱性水解与凝胶电泳<ol>
<li>添加6毫升1米的 KOH 和孵育55°c 2 小时。</li>
		<li>在冰上孵化样品5分钟。</li>
		<li>添加4毫升6x 碱性 DNA 加载缓冲器: 300 毫米 KOH, 6 毫米 EDTA, pH 8.0, 18% 聚蔗糖 (材料表), 0.15% 甲酚绿色, 0.25% 二甲苯蓝 FF。</li>
		<li>铸造碱性凝胶, 由1% 琼脂糖, 50 毫米氢氧化钠, 1 毫米 EDTA, pH 8.0。使用一个梳子, 允许24毫升的 DNA 样本, 每车道加载。</li>
		<li>在室温下运行凝胶1x 碱性电泳缓冲器:50 毫米氢氧化钠, 1 毫米 EDTA, pH 8.0。包括适当的 DNA 大小标记。</li>
		<li>简单地向下旋转管子, 将整个24毫升的样品装入碱性琼脂糖凝胶中。Electrophorese 为30分钟在 30 V, 其次是 18-20 h 在 10 v。</li>
		<li>2孵化的凝胶在室温下与温和的搅拌在中和缓冲 I: 1 米三盐酸, 1.5 米氯化钠。</li>
		<li>将中性凝胶浸泡在200毫升水中, 其中含有 1/1万稀释的 SYBR 金染色, 室温下以温和摇动的方法对 DNA 进行染色。</li>
		<li>使用紫外线 transilluminator 可视化 DNA。增加碱敏感性导致小 dna 片段的出现, 表明在 dna5中失修 ribonucleotides 的密度较高。</li>
	</ol></li>
	<li>南部转移<ol>
<li>用毛细管动作10将碱性凝胶中的 DNA 转移到正电荷的尼龙膜上。理查德·山姆布鲁克和罗素的手册10中提供了如何设置此转移的示例。</li>
		<li>碱性转移缓冲器室温下浸泡碱性凝胶15分钟: 0.4 米氢氧化钠, 1 米氯化钠。</li>
		<li>湿尼龙膜 (切到胶粒的大小) 与水简单, 然后浸泡5分钟碱性转移缓冲器。</li>
		<li>通过在玻璃烘烤盘中反转3玻璃烧杯 (100 毫升), 建立转移平台。这道菜应该比琼脂糖凝胶的尺寸稍大一些。把玻璃盘子放在上面。</li>
		<li>在玻璃板上悬垂2大块3毫米的色谱纸 (切到凝胶的宽度大小和可以在边缘上悬垂的更长的边)。</li>
		<li>将碱性转移缓冲液倒入色谱纸上, 半填充玻璃碟。用5毫升的玻璃吸管平滑气泡。</li>
		<li>将琼脂糖凝胶放在顶部, 再平滑气泡。用 parafilm 包围凝胶的所有边缘。将少量碱性转移缓冲液倒入凝胶上。</li>
		<li>将预浸泡过的尼龙膜放在凝胶的顶部。把气泡弄平。</li>
		<li>湿2张纸切到大小的琼脂糖凝胶。将它们放在膜的顶端, 并平滑出任何气泡。</li>
		<li>在转移三明治的顶端放置一大堆纸巾 (切到比琼脂糖凝胶稍大一点的尺寸)。小心地将玻璃盘子放在纸巾上。将加权对象添加到顶部 (一本质量为400克的书工作良好)。</li>
		<li>让转移在室温下通宵进行。</li>
		<li>小心地把转移栈分开, 在室温下将膜浸泡15分钟, 中和缓冲 II: 0.5 米三盐酸盐, pH 7.2, 1 米氯化钠。</li>
		<li>用紫外交联剂将 DNA 与带电的尼龙膜交交。将膜放在交叉链接器中, 并使用 autocrosslink 设置。</li>
	</ol></li>
	<li>南方探头准备 注意: 此处描述的用于检测 DNA 链中初生的前导与滞后链中碱敏感点的方法基于以前发布的协议11。在我们的实验中 , 探测是在URA3报告基因上进行的 , 它入到与ARS306复制起源 (图 3A) 相邻的两个方向 ( OR1 或 OR2 ) 之一。在酵母基因组 DNA 中存在碱敏感点是核苷酸的一个指标, 允许使用 ribonucleotides 作为 DNA 聚合酶活性12、13、14的生物标志物。这种方法, 使用的目标 DNA 序列相邻的有效ARS306起源的复制, 应服从其他基因组的位置和目标基因, 以及。<ol>
<li>PCR-放大URA3报告基因的520基对段使用以下引物对: URA3-F1 (5´ GCTACATATAAGGAACGTGCTGC) 和 URA3-R1 (5´ CTTTGTCGCTCTTCGCAATGTC)。</li>
		<li>用 10-20 ng 酵母基因组 DNA 作为模板进行 PCR 反应, 4 ul 每底漆 (10 微米股票), 10 ul 10x 前 Taq 缓冲器 (Mg2 +加), 8 ul dNTP 混合物 (2.5 毫米每), 0.5 ul 的前 Taq DNA 聚合酶 (5 U/ul), 并调整最终音量为 100 ul 与 H2O。</li>
		<li>运行以下 PCR 程序:95 °c 为5分钟;30周期95°c 为1分钟, 55 °c 为1分钟, 72 °c 为2分钟;72°c 为10分钟并且保持在4°c。</li>
		<li>用 pcr 纯化试剂盒提纯 pcr 产物, 用50毫升的 H2O 洗脱 DNA。现在可以将其用作 radiolabeling 反应中的模板。</li>
		<li>使用以下引物为链特定的 radiolabeling: 使用 URA3-A (5´ CTCATCCTAGTCCTGTTGCTGCC), 以可视化的 DNA, 退火探测 A。这对应于新生的领先链 dna, 当URA3是在 OR2 和 tnascent 滞后链 dna 时URA3在 OR1。使用 URA3-B (5´-CAGTACCCTTAGTATATTCTCCAG) 可视化新生 DNA, 退火探针 B。这相当于新生的领先链 dna 时, 当URA3是在 OR1 和新生的滞后链 dna 时, URA3在 OR2 (图 3)。<ol>
<li>使用 10-20 ng 放大URA3片段作为模板 DNA 进行 radiolabeling 反应, 2 底漆 ul (10 微米库存), 5 ul 10x 前 Taq 缓冲器 (Mg2 +加), 4 毫米 dNTPs (减去 dCTP), 2.5 ul (5 μCi) 的一个 50 P dCTP, 0.5 ul ExTaq 脱氧核糖核酸聚合酶 (5 U/ul) 和调整最后容量到 50 ul 与 H2o 运行以下程序为 radiolabeling:94 °c 为5分钟;25周期94°c 为1分钟, 55 °c 为三十年代, 72 °c 为1分钟;72°c 为5分钟并且保持在4°c。</li>
		</ol>
</li>
		<li>使用 G-25 旋转列删除未合并的放射性标签。在添加核素样品之前, 将该列离心1分钟, 在 720 x g 和 removethe 缓冲器吹打。负载核素反应产品和离心机2分钟在 720 x g 洗脱标记探针。</li>
		<li>在95摄氏度孵育5分钟, 在杂交前立即变性探针。在冰上短暂冷却。</li>
	</ol></li>
	<li>南方杂交<ol>
<li>将润湿膜滚入杂交管中, 加入25毫升新制备的杂交缓冲液: 0.5 米磷酸钠缓冲液、pH 7.2、7% 月桂酸钠 (SDS)、1% 牛血清白蛋白 (BSA)。孵育在65°c 1-2 h 与旋转在一个杂交烤箱。</li>
		<li>小心地倒出溶液, 加入25毫升的杂交缓冲器加上 radiolabelled 探针。65摄氏度孵育 16-18 小时, 旋转。</li>
		<li>将溶液倒入放射性废物容器中。在室温下用磷酸盐-SDS 洗涤液进行5洗涤5分钟:40 毫米磷酸钠缓冲液, pH 7.2, 5% SDS, 0.5% BSA, 1 毫米 EDTA, pH 8.0。</li>
		<li>执行2洗涤15分钟每65°c 与磷酸盐 SDS 洗涤解决方案 II:40 毫米磷酸钠缓冲液, ph 7.2, 1% SDS, 1 毫米 EDTA, ph 值8.0。</li>
		<li>使用镊子将膜从杂交管中取出, 并在打开的塑料套筒保护器中放置。覆盖并暴露在成像板上。尝试多种不同的曝光时间, 从4小时到4维不等。</li>
		<li>如有必要, 用不同探针对印迹进行剥离和重新探测, 大约 3-4 次。在25毫升的50% 甲酰胺, 2x SSPE 溶液中, 将膜培养为2小时65摄氏度。 注: 20x SSPE 库存解决方案: 3 米氯化钠, 0.2 米 NaH2PO4, 0.02 米 EDTA, pH 7.4。</li>
		<li>将溶液倒入放射性废物容器中。在磷酸盐-SDS 洗涤液中, 每65°c 执行2次洗涤15分钟。</li>
		<li>检查膜与盖格计数器, 并重复剥离协议, 如果信号仍然存在。</li>
		<li>执行前述杂交和杂交。</li>
	</ol></li>
</ol>2.酵母菌株的变异率和特异性测定
<ol><li>准备细胞培养<ol>
<li>接种一个单一的殖民地 (健康酵母细胞需要 2-3 天在30°c 形成适当大小的殖民地) 生长在 YPD 琼脂补充与100毫克/毫升腺嘌呤入5毫升 YPDA 汤补充与250毫克/毫升腺嘌呤 (补充腺嘌呤是仅必要的, 如果使用的菌株是腺嘌呤营养缺陷型)。生长在一个振动筛孵化器或在30°c 的转子, 直到文化达到饱和 (36-48 小时的菌株, 没有严重的生长缺陷)。</li>
		<li>将单元格向下旋转 2000 x g, 5 分钟。</li>
		<li>丢弃上清液, 用5毫升的无菌水冲洗细胞。</li>
		<li>并用重悬500毫升的无菌水中的细胞。</li>
	</ol>
</li>
	<li>细胞培养和电镀的稀释<ol>
<li>对于非选择控制, 将96井板中的细胞以160万的因子和每种培养基100毫升的系数稀释到完整的 (COM) 板上。</li>
		<li>为了选择ura3突变体, 将100毫升未稀释的细胞放在 5 FOA 板上, 用于野生型 (应变)。如果预期的菌株的突变率高于某一应变, 则应相应稀释细胞。</li>
		<li>为can1突变体选择, 稀释细胞5倍和板100毫升到一个 canavanine 的板 (可以) 的一个重量应变。稀释系数为0.5。</li>
		<li>孵育板材在30°c 直到酵母殖民地形成。对于没有生长缺陷的菌株, 2-3 天通常是足够的 COM 和罐板和 3-5 天为 5 FOA 板。为了比较不同菌株之间的突变率, 最好选择同一天的生长来计算菌落数。把盘子放在冷房间里 (摄氏4摄氏度)。</li>
	</ol>
</li>
	<li>自发突变率的计算<ol>
<li>计算每个板块上可见的菌落数并记下菌落数</li>
		<li>用德雷克公式计算突变率: ÷ ( μNt) 。f是根据最终培养的细胞数除以突变体数量计算出的突变频率。Nt 是最终文化中的细胞数, μ是突变率。该方程可以用牛顿-拉夫逊法等迭代法求解。还有其他估计方法, 包括李和库尔森测试, 以计算突变率15。</li>
		<li>计算假设对数正态分布的 utation 速率的置信区间。最常用的是95% 置信区间。</li>
	</ol>
</li>
	<li>变异谱分析<ol>
<li>选择一个单一的殖民地从每个 5 FOA 或可以板块和刷新 YPDA 板块。</li>
		<li>执行菌落 pcr 放大URA3或CAN1基因和序列, 以检测突变使用以下引物: URA3-F (5 '-CGCATATGTGGTGTTGAAGAA-3 ') 和 URA3-R (5 '-TGTGAGTTTAGTATACATGCA-3 ') 的 pcr 扩增和800 bp URA3基因的测序;CAN1-F (5 '-CTTCTACTCCGTCTGCTTTC-3 ') 和 CAN1-R (5 '-CAGAGTTCTTCAGACTTC-3 ') 为放大和 CAN1-AR (5 '-TGAAATGTGAAGGCAGCGTT-3 '), CAN1-9R (5 '-CCTGCAACACCAGTGATA-3 '), CAN1-10R (5 '-GAGGATGTAACAGGGATGAAT-3 ') 和 CAN1-BR (5 '-CGGTGTATGACTTATGAGGG-3 ') 用于测序的 1800 bp CAN1报告基因。</li>
		<li>为了进行菌落 PCR, 并用重悬在 5 ul2O 的一个单一的殖民地, 并使用 1 ul 作为模板。添加 1 ul 每一个底漆 (5 微米股票), 2 ul 5x KAPA2G 缓冲器 (Mg2 +加), 0.5 ul dNTP (2.5 毫米), 0.5 ul KAPA2G 快速 DNA 聚合酶, 12 ul 的 H2O。</li>
		<li>对于URA3基因的扩增, 运行以下 PCR 程序:95 °c 5 分钟;5周期95°c 为十年代, 61 °c 十五年代, 72 °c 为三十年代;35周期95°c 为十年代, 61 °c 十五年代, 72 °c 为三十年代;72°c 为2分钟并且保持在4°c。对于CAN1基因的扩增, 运行以下 PCR 程序:95 °c 5 分钟;5周期95°c 为十年代, 61 °c 十五年代, 72 °c 为四十五年代;35周期95°c 为十年代, 61 °c 十五年代, 72 °c 为四十五年代;72°c 为2分钟并且保持在4°c。</li>
		<li>使用程序 (如 DNASTAR Seqman Pro 或 Sequencher) 将排序结果与参考序列对齐以识别突变。</li>
		<li>按突变类型 (图 4) 或突变位置 (图 5) 编译突变数据。根据突变的频率计算特定的突变率 (观察到的事件数除以突变序列的数量) 乘以总的突变率。</li>
	</ol>
</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+roles+of+DNA+polymerases+epsilon+and+delta+at+the+eukaryotic+replication+fork+are+evolutionarily+conserved.">The major roles of DNA polymerases epsilon and delta at the eukaryotic replication fork are evolutionarily conserved.</a> PLoS Genetics. 7 (12), e1002407 (2011).</li><li>Lujan, S. A., Williams, J. S., Clausen, A. R., Clark, A. B., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonucleotides+are+signals+for+mismatch+repair+of+leading-strand+replication+errors.">Ribonucleotides are signals for mismatch repair of leading-strand replication errors.</a> Molecular Cell. 50 (3), 437-443 (2013).</li><li>Williams, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topoisomerase+1-mediated+removal+of+ribonucleotides+from+nascent+leading-strand+DNA.">Topoisomerase 1-mediated removal of ribonucleotides from nascent leading-strand DNA.</a> Molecular Cell. 49 (5), 1010-1015 (2013).</li><li>Clausen, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+replication+enzymology+in+vivo+by+genome-wide+mapping+of+ribonucleotide+incorporation.">Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation.</a> Nature Structural &amp; Molecular Biology. 22 (3), 185-191 (2015).</li><li>Foster, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+determining+spontaneous+mutation+rates.">Methods for determining spontaneous mutation rates.</a> Methods Enzymology. 409, 195-213 (2006).</li><li>Williams, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+processing+of+ribonucleotides+in+DNA+by+topoisomerase+1+is+leading-strand+specific.">Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific.</a> Nature Structural &amp; Molecular Biology. 22 (4), 291-297 (2015).</li><li>Pavlov, Y. I., Shcherbakova, P. V., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+consequences+of+putative+active+site+mutations+in+yeast+DNA+polymerases+alpha,+epsilon,+delta,+and+zeta.">In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta.</a> Genetics. 159 (1), 47-64 (2001).</li><li>Nick McElhinny, S. A., Kissling, G. E., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+correction+of+lagging-strand+replication+errors+made+by+DNA+polymerases+{alpha}+and+{delta}.">Differential correction of lagging-strand replication errors made by DNA polymerases {alpha} and {delta}.</a> Proceedings of the National Acaddemy of Sciences U S A. 107 (49), 21070-21075 (2010).</li><li>Clark, A. B., Lujan, S. A., Kissling, G. E., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mismatch+repair-independent+tandem+repeat+sequence+instability+resulting+from+ribonucleotide+incorporation+by+DNA+polymerase+epsilon.">Mismatch repair-independent tandem repeat sequence instability resulting from ribonucleotide incorporation by DNA polymerase epsilon.</a> DNA Repair (Amst). 10 (5), 476-482 (2011).</li><li>Lujan, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mismatch+repair+balances+leading+and+lagging+strand+DNA+replication+fidelity.">Mismatch repair balances leading and lagging strand DNA replication fidelity.</a> PLoS Genetics. 8 (10), e1003016 (2012).</li><li>Reijns, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+removal+of+ribonucleotides+from+DNA+is+essential+for+mammalian+genome+integrity+and+development.">Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development.</a> Cell. 149 (5), 1008-1022 (2012).</li><li>Lujan, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+polymerase+fidelity+and+mismatch+repair+bias+genome+variation+and+composition.">Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition.</a> Genome Research. 24 (11), 1751-1764 (2014).</li></ol>

作者没有什么可透露的。

在这里, 我们描述了两组试验的协议, 这些实验经常用于对 DNA 复制过程中 ribonucleotides 的半定量分析, 以及失修 ribonucleotides 的诱变效应。虽然这些方法涉及模型真核细胞的酵母, 这些技术可以很容易地适应其他微生物, 甚至更高的真核生物。
碱性琼脂糖电泳失修 ribonucleotides 在 DNA 中的应用探讨与南方印迹相结合, 提供了关于 ribonucleotides 的数量和它们被纳入其中的链的?...

在真核 dna 复制过程中, ribonucleotides 由所有三主要 dna replicases、dna 聚合酶 (年 floss-pols) α、ε和δ1、2组成基因组。RNase H2-dependent 核苷酸切除修复 (3) 去除大多数这些嵌入式 ribonucleotides。
DNA 中的核苷酸易水解, 因为 2 ' 羟基的糖基团可以攻击相邻的磷酸二酯键, 释放一个末端与 2 '-3 ' 循环磷酸盐和另一个与 5 '-OH4。碱性条件可以大大加快这种反应。因此, 在碱性溶液的孵化过程中, 嵌入 ribonucleotides 的水解会导致基因组 DNA 的碎片化, 这可以通过碱-琼脂糖电泳5进行可视化。这种 DNA 可以转移到一个膜, 并通过南方印迹分析, 利用链特定的探针, 允许识别碱敏感的网站所造成的 ribonucleotides 纳入新生的领先或滞后链 DNA,分别。
在缺乏 RNase H2 活性的酵母细胞中, 当拓扑异构酶 I (Top1) 在嵌入的核苷酸6、7上刻痕 DNA 时, 可以启动 ribonucleotides 的去除。然而, 当 Top1 在 3 ' 侧核苷酸, 这产生 5 '-OH 和 2 '-3 ' 循环磷酸盐 DNA 末端是顽固的对 religation。不修复或异常处理这些 ' 肮脏的末端 ' 可能导致基因组不稳定。此外, 如果切口发生在重复 DNA 序列中, 修复过程可能导致删除突变。这对于串联重复是特别成问题的, 在 RNase H2-deficient 细胞中通常观察到短的删除 (介于两和五基对之间)。在 Top1-dependent 酵母 RNase H2 活性缺乏的情况下, DNA 聚合酶ε突变体 (pol2-M644G) 混杂在新生的主导链合成过程中会加剧核苷酸的有害影响。
DNA 中 ribonucleotides 的处理导致自发的突变, 这种突变可以通过适当的报告基因和选择相应的表型变化来检测。波动测试或 Luria 和德尔布吕克实验是最常用的方法之一, 以测量自发突变率使用可选择的报告基因8,9。在酵母中, URA3和CAN1基因可以作为一个正向突变检测的记者, 它允许检测所有导致基因功能丧失的突变类型。自发突变率被估计为多平行培养所观察到的中位数, 从单个菌落开始, 而目标报告基因中没有突变。酵母 RNase H2-deficient 菌株如rnh201Δ具有适度升高的整体自发突变率, 这主要是由于串联重复序列的 2-5 bp 删除率升高引起的。因此, 为了充分描述 ribonucleotides 在基因组中的诱变效应, 需要确定特定的突变率。在这种情况下, URA3或CAN1的报告基因可以被放大和排序, 以确定的类型和地点的突变, 和特定的突变率可以计算。在多个独立URA3或CAN1突变体中发现的突变可以用来产生突变谱。

<table>
	<tbody>
		<tr>
			<td>YPD media</td>
			<td></td>
			<td></td>
			<td>20 g dextrose, 20 g peptone, 10g yeast extract, in deionized H2O up to 1 L, add 20 g Bacto agar for solid media, autoclave.</td>
		</tr>
		<tr>
			<td>COM plates</td>
			<td></td>
			<td></td>
			<td>1.3 g SC dropout mix, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar deionized H2O up to 1 L. Adjust pH to 5.8. Autoclave and 30 &#8211; 35 ml per plate.</td>
		</tr>
		<tr>
			<td>CAN plates</td>
			<td></td>
			<td></td>
			<td>1.3 g SC-Ura dropout powder, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar, 25 mg uracil and H2O up to 1 L. Autoclave for 15 mins at 121 &#176;C and cool down to 56 &#176;C. Add 6 mL of filter-sterilized 1% canavanine sulfate solution.</td>
		</tr>
		<tr>
			<td>5-FOA plates</td>
			<td></td>
			<td></td>
			<td>1.3 g SC-Ura dropout powder, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar, 25 mg uracil and H2O up to 800 mL. Autoclave for 15 mins at 121 &#176;C and cool down to 60 &#176;C. Add 200 mL of filter-sterilized 0.5% 5-FOA solution.</td>
		</tr>
		<tr>
			<td>L-Canavanine sulfate</td>
			<td>US Biological</td>
			<td>C1050</td>
			<td></td>
		</tr>
		<tr>
			<td>5-FOA</td>
			<td>US Biological</td>
			<td>F5050</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass culture tube</td>
			<td></td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr>
			<td>Culture rotator in 30 &#176;C incubator</td>
			<td></td>
			<td></td>
			<td>Shaker incubator can be used instead</td>
		</tr>
		<tr>
			<td>96 well round bottom plates</td>
			<td></td>
			<td></td>
			<td>Sterile, any brand</td>
		</tr>
		<tr>
			<td>3 mm glass beads</td>
			<td>Fisher Scientific</td>
			<td>11-312A</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>12-channel pipettes</td>
			<td></td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr>
			<td>Ex Taq DNA Polymerase</td>
			<td>TaKaRa</td>
			<td>RR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Epicentre MasterPure Yeast DNA Purification Kit</td>
			<td>Epicenter</td>
			<td>MPY80200</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S7899</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 Fluorometer</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td>Newer model available</td>
		</tr>
		<tr>
			<td>dsDNA BR Assay kit</td>
			<td>Invitrogen</td>
			<td>Q32850</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma-Aldrich</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Dot Scientific Inc.</td>
			<td>DSF10400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromocresol green</td>
			<td>Eastman</td>
			<td>6356</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene cyanol FF</td>
			<td>International Technologies Inc.</td>
			<td>72120</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl (pH 8.0)</td>
			<td>Teknova</td>
			<td>T5080</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Gold Nucleic Acid Gel Stain</td>
			<td>Invitrogen</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>UV transilluminator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Nylon membrane Hybond-N+</td>
			<td>GE Healthcare</td>
			<td>RPN303B</td>
			<td></td>
		</tr>
		<tr>
			<td>3 MM CHR Chromotography paper</td>
			<td>Whatman</td>
			<td>3030-392</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Caledon</td>
			<td>7560-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stratalinker 1800</td>
			<td>Stratagene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>G-25 spin column</td>
			<td>GE Healthcare</td>
			<td>27-5325-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Sodium phosphate buffer (pH 7.2)</td>
			<td>Sigma-Aldrich</td>
			<td>NaH2PO4 (S9638); 
			Na2HPO4 (S9390)</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L4522</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma-Aldrich</td>
			<td>47671</td>
			<td></td>
		</tr>
		<tr>
			<td>Geiger counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

studying ribonucleotide incorporation: strand-specific detection of ribonucleotides in the yeast genome and measuring ribonucleotide-induced mutagenesis

ribonucleotides 在核 DNA 中的存在被证明是基因组不稳定的来源。核苷酸的范围可以用碱性水解和凝胶电泳来评定, 因为 RNA 在碱性条件下很容易水解。这与南方污点分析结合在一起可以用来确定 ribonucleotides 的位置和链。然而, 这一过程只是半定量的, 可能不够敏感, 以检测核苷酸内容的小变化, 虽然链特定的南方印迹探测提高灵敏度。作为衡量 ribonucleotides 在 DNA 中最显著的生物学后果之一, 自发诱变可以用正向突变法进行分析。使用适当的报告基因, 可以选择导致功能丧失的罕见突变, 并通过将波动实验数据与报告基因的 DNA 测序相结合来测量整体和特异的突变率。波动分析法适用于在特定的遗传背景或生长条件下检测各种诱变过程。

碱凝胶电泳法处理基因组 dna, 允许对 dna 碎片进行半定量检测, 因为 ribonucleotides 的丰度稳定。图 2显示了用或不带5KOH 治疗酵母基因组 DNA 的凝胶图像。Polε的催化亚单位 Pol2 的 M644L 变异降低了吸收 ribonucleotides 的能力, M644G 突变体的 ribonucleotides 比聚酶多。如《议定书》所述, 碱性凝胶可以通过链特定的南方印迹分析进一步探讨。随?...

Watch this Scientific Journal Video about Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis at Jo

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

Ribonucleotides 是在核 DNA 复制过程中纳入基因组的最丰富的非规范核苷酸之一。如果没有正确去除, ribonucleotides 会导致 DNA 损伤和突变。在这里, 我们提出了两个实验方法, 用于评估核苷酸的丰度纳入 DNA 及其诱变效应。

研究核苷酸合并: 酵母基因组 Ribonucleotides 的链特异检测及核苷酸诱变的检测

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

studying-ribonucleotide-incorporation-strand-specific-detection

Research

JoVE Journal

Biology

7.7K 次观看.  National Institute of Environmental Health Sciences, NIH, DHHS. Ribonucleotides 是在核 DNA 复制过程中纳入基因组的最丰富的非规范核苷酸之一。如果没有正确去除, ribonucleotides 会导致 DNA 损伤和突变。在这里, 我们提出了两个实验方法, 用于评估核苷酸的丰度纳入 DNA 及其诱变效应。

视频: Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

科研

生物学

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

格罗斯和精细解剖内耳感觉上皮细胞在成年斑马鱼（斑马鱼）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

在甲醇酵母表达的重组蛋白毕赤酵母</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

单卵母细胞的亚硫酸氢钠诱变

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞:隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

药物与DNA相互作用的细胞全基因组映射与COSMIC（小分子交联，染色质隔离）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...