该项目的资金支助由科学和信息和通信技术部 (2016R1A2B2006354) 资助的韩国国家研究基金会 (NRF) 提供。

1. 媒体的准备工作
<ol><li>用补充剂 (是的), 没有腺嘌呤 (是低艾德), 粟谷氨酸培养基 (普纳12) 和普纳, 没有腺嘌呤 (普纳), 通过混合组件, 如表 1中所述, 准备酵母提取物。是-艾德 (低艾德) 和普纳板有所有相同的成分, 但腺嘌呤是从后者省略。<ol>
<li>使用以下盐 (50x) 库存: 52.5 克/升氯化镁2· 6H2O, 2 克/升 CaCl2· 2H2O, 50 克/升氯化钾, 2 克/升 Na2所以4在蒸馏水。过滤消毒使用0.22 µm 孔径过滤器和存储在4摄氏度。</li>
		<li>使用以下维生素 (1,000x) 股票: 1 克/升泛酸, 10 克/升烟酸, 10 克/升肌醇, 10 毫克/升水生物素在蒸馏水。过滤消毒使用0.22 µm 孔径过滤器和存储在4摄氏度。</li>
		<li>使用以下矿物 (10,000x) 库存: 5 克/升硼酸, 4 克/升 MnSO4, 4 克/升 ZnSO4· 7H2O, 2 克/升 FeCl2· 4H2O, 0.4 克/升 MoO3, 1 克/升, 0.4 克/升丘索4· 5H2o, 10 克/升柠檬酸在蒸馏水中。过滤消毒使用0.22 µm 孔径过滤器和存储在4摄氏度。 注: 是液体培养基可通过省略琼脂制成。</li>
	</ol>
</li>
	<li>蒸压介质, 冷却到60摄氏度以下, 搅拌时用搅拌棒, 速度为 200 rpm。</li>
	<li>对于 YES+G418 (geneticin) 板, 请将1毫升/升 G418 库存解决方案添加到 "是" 介质中。<ol>
<li>使用以下 G418 (1,000x) 库存: 100 毫克/毫升 G418 蒸馏水。过滤消毒使用0.22 µm 孔径过滤器, 整除1.5 毫升离心管, 并存储在-20 摄氏度。</li>
	</ol>
</li>
	<li>再搅拌 5-10 分钟, 将介质整除成90毫米培养皿 (中等整除数的1升至大约40培养皿。把盘子存放在4摄氏度。</li>
</ol>2. 克隆rpt4+及其 5 '/3 ' UTRs
<ol><li>用引物 p1 和 p2 (图 1A) 进行 PCR, 以裂变酵母基因组 DNA (gDNA) 作为模板, 总体积为50µL (~ 1 µg DNA, 20 U 型酶, 1x 反应缓冲器), 并应用表 2中描述的反应周期放大3315基对 (bp) 片段, 包括rpt4+及其 5 '/3 ' UTRs。使用制造商 (图 1A) 指示的纯化套件13提纯 PCR 产品。</li>
	<li>摘要 pBlueScript KS (-) 向量14和包含rpt4+的放大 DNA 片段, 其 5 '/3 ' UTRs 与BamH1和Xho1在总容积20µL (~ 1 µg DNA, 20 U 酵素, 1x 消化缓冲) 在37°c 在水浴为 16-18h (图 1B)。</li>
	<li>凝胶纯化消化载体 (1.2% 琼脂糖凝胶) 和 DNA 插入通过执行泰基琼脂糖凝胶电泳 (100 V, 1 h), 切除所需尺寸的 dna 带, 并将 dna 与凝胶萃取套件15隔离。</li>
	<li>结扎使用 T4 dna 连接酶的载体和 dna 插入, 执行 20-µL 结扎反应, 含有 50-100 ng 的载体 dna, 3 倍摩尔超过插入 dna, 400 U T4 DNA 连接酶, 1x 结扎缓冲。孵化这种混合物在18°c 16-18 小时。</li>
	<li>应用热休克七十年代42摄氏度, 将结扎后的 DNA 转化为100µL 的大肠杆菌DH5α。添加1毫升磅和孵育1小时在37摄氏度, 以恢复。执行离心 (13800 x g), 丢弃上清, 在 LB 氨苄西林 (LA) 板上全部转化的大肠杆菌细胞, 并在37摄氏度孵育16小时。</li>
	<li>选择 4-8 殖民地与牙签和增长隔夜在37°c 在3毫升 LA 媒介。</li>
	<li>根据制造商的协议, 用质粒纯化套件16隔离粒度, 并执行分析限制酶消解与5µL 的分离质粒, 5 U 的每种限制酶 (BamH1和Xho1) 和1x 限制缓冲区。在37摄氏度的水浴中孵育反应混合物3小时. 通过序列候选质粒对克隆进行确认。</li>
</ol>3. 引入无声变异 (Xho1限制网站)
<ol><li>设计一对 33-bp 引物 (p3 和 p4), 其中包含所需的突变在其他补充序列。</li>
	<li>使用表 10中列出的循环参数, 用高保真聚合酶17 (图 1C) 的总体积25µL (10 ng 克隆质粒, 2 µL 2 pmol 底漆混合, 3 U 酶, 1x 反应缓冲器) 进行 PCR。</li>
	<li>将模板质粒与DpnI 一起消化, 总容积为20µL (20 U 酵素, 1x 消化缓冲) 在37°c 在水浴中1小时。</li>
	<li>如步骤 2.5-2.7 所述, 对含有无声突变的质粒进行变换、传播、分离和序列化。 注意:这种无声突变也可以使用一种克隆方法来引入, 它允许在单个反应18中加入多个 DNA 片段。</li>
</ol>4. 随机突变的rpt4+由易出错 PCR
<ol><li>执行易出错的 PCR, 使用质粒与无声突变 (Xho1站点) 作为模板, 引物 p5 和 p6, 总容量50µL (在步骤4.1.1 定义的模板量, 2 µL 10 pmol 底漆混合, 2.5 U 酵素, 1x 反应缓冲)以及用于生成高错误率19 (图 1D) 的特殊聚合酶。按照表 2中的说明执行 PCR。<ol>
<li>使用 4-5 µg 模板质粒控制突变频率为 1 bp/kb (kilobase)。 注意:一个高浓度 (> 500 ng/µL) 的质粒, 可以获得使用迷你准备套件20, 是必需的。</li>
	</ol>
</li>
	<li>用凝胶电泳确认 2670 bp (1.2% 琼脂糖凝胶) 的 PCR 产物。凝胶纯化此 PCR 产品, 如步骤2.5 所述。</li>
</ol>5. 合成 PCR 片段 (菅直人, 3 ' UTR)
<ol><li>使用 pFA6a-KANMX621作为模板、引物 p7 和 p8, 以及表4中列出的条件来获取标记 (菅直人) 片段, 执行 PCR。凝胶纯化产生的 1480 bp 片段, 如步骤 2.5 (图 1E) 所述。<ol>
<li>检查以突变为目标的基因的停止密码子和相邻基因的编码区域是否由超过500的 bp 分离。当该区域小于 500 bp 时, 可能不使用内生终结器;相反, 应该使用抗利尿终止符而不是内生终止符。表 5中显示了使用抗利尿终结器所需的协议更改。 注意:仅当使用 pFA6a-3HA-KANMX6 质粒中可用的抗利尿终结器而不是内生终止符时, 这些更改才适用。</li>
	</ol>
</li>
	<li>用克隆的rpt4+包含的向量作为模板和引物 p9 和 p10, 在总容积50µL (20 ng 克隆质粒中) 进行 PCR, 2 µL 10 pmol 底漆混合, 2 U 型酶, 1x 反应缓冲器), 使用表 6中提供的条件。凝胶纯化获得的 506-bp 3 ' UTR 片段 (图 1E)。</li>
</ol>6. 融合 PCR 法生成转换构造 (图 1E)
<ol><li>在 50 ng/µL 上准备3个片段 (mutagenized rpt4+、菅直人和 3 ' UTR)。</li>
	<li>使用底漆 p11 和 p12 进行三片段的融合 PCR, 如表7所述。(融合 PCR 的协议是对原始协议22的修改。净化主要的 4398-bp PCR 产品。<ol>
<li>使用rpt4+: KAN:3'UTR 碎片的比率为 1:3: 1, 以获得最佳结果。 注意:插入 DNA 的总量不应超过 500 ng。建议的rpt4+数量: KAN:3'UTR 是 50 ng:150 ng:50 ng。mutagenized 区域约为 1.6 kb, 因此, 将每个基地的所有可能组合的最低数量的酵母菌落, 将被变异到其他三是 1600 x 3 = 4800 殖民地。由于一个转化反应通常产生 400-500 菌落, 至少有10反应价值的融合 PCR 结构是需要的。这种需要可以通过简单地增加反应量 (即, PCR 管的数量) 来满足, 系数为十。</li>
	</ol>
</li>
</ol>7. 用电穿孔法转化裂变酵母 (图 1F)
<ol><li>接种酵母到10毫升的是培养基饱和通过孵化超过16小时。</li>
	<li>稀释细胞到光学密度在600毫微米 (OD600) 0.2 在200毫升是中等和孵化在30°c 与震动为 5-6 h, 到 OD600 = 0.6-0.8。</li>
	<li>将单元格集中到 OD600 = 30, 免除四50毫升锥形管, 并在冰上冷却10分钟。</li>
	<li>以 1050 x g 为3分钟, 在4摄氏度进行离心, 收获细胞。把管子和10电小试管放在冰上。在冰上执行步骤 7.3-7.8。</li>
	<li>丢弃上清, 加入15毫升1.2 米山梨醇。轻轻摇动管子, 并用重悬细胞。</li>
	<li>离心细胞在 1050 x g 3 分钟在4摄氏度。</li>
	<li>重复步骤7.4 和7.5。放弃上清。添加500µL 1.2 米山梨醇的每管, 并用重悬细胞, 并收集所有的细胞在一个15毫升锥管。</li>
	<li>添加1.2 米山梨醇高达2.4 毫升 (OD600 = 10 每0.2 毫升)。把管子放在冰上。</li>
	<li>添加200µL 山梨醇悬浮细胞 (确保它们完全悬浮) 到含有融合 PCR 结构的 EP 管, 混合好, 并将样品转移到电试管。</li>
	<li>Electroporate 细胞具有以下选择: 真菌, ShS (2.00kV, 1 脉冲)。</li>
	<li>添加600µL 1.2 米山梨醇对每个电试管的总容量800µL, 然后传播细胞在四是板材 (200 µL/板材)。十电小试管将免除40是板材。</li>
	<li>在30摄氏度孵化出24小时的板材。</li>
	<li>对 YES+G418 进行复制, 并在30摄氏度的3天内孵化板材。</li>
</ol>8. 染色质不稳定突变体的选择和检查误报
<ol><li>对于每个 YES+G418 板, 执行副本电镀, 以是艾德 (低艾德) 和普纳 (无艾德) 板。将复制板孵化 1-2 天, 在30摄氏度, 直到一些在 YES 板上的殖民地显示红色着色 (图 1G)。</li>
	<li>比较是艾德和普纳板, 并选择显示粉红色或白色的在 "是" 板上, 也生长在普纳板上的细胞。不要在普纳上选择没有生长的菌落, 因为它们是假阳性 (例如,反映了菅直人在基因组其他地方的非目标站点被集成)。</li>
	<li>从每个菌落中选取大约 1 x 105单元格, 并在10µL 的 SPZ 溶液中孵化, 其中包含2.5 毫克/毫升 zymolyase 100 T, 37 摄氏度, 至少30分钟. 使用此解决方案的1µL 作为菌落 PCR (图 1H) 的起始模板执行。<ol>
<li>使 SPZ (50 毫升) 混合30毫升2米山梨醇, 4.05 毫升1米 Na2HPO4, 0.95 毫升 NaH2PO4和15毫升蒸馏水 (最终 pH, 7.5)。样品 (5 毫升) 可以补充与 zymolyase 和存储在 500-µL 整除数在-20 °c, 直到使用。</li>
	</ol>
</li>
	<li>进行凝胶电泳 (1.2% 琼脂糖凝胶, 180V, 20 分钟), 以可视化的结果 + 菌落 PCR。选择与XhoI (4 U 酵素, 1x 消化缓冲, 37 °c 水浴, 1 h) 一起展示适当的大小的 PCR 带和消化5µL 的反应产品, 以筛出误报 (图 1H)。</li>
	<li>进行凝胶电泳以可视化XhoI文摘 (1.2% 琼脂糖凝胶, 180 V, 20 分钟)。标记其 PCR 产品由XhoI剪切的菌落, 并将它们贴到 YES+G418 板上 (图 1I)。</li>
	<li>将 gDNA 与裂变酵母细胞分离, 并执行 PCR 产品的序列化23。将获得的序列与野类型序列进行比较, 以识别突变 (图 1I)。</li>
	<li>通过将突变细胞的 PCR 扩增的融合结构转化为野生型细胞, 直接将突变引入23。使用斑点来确认新制作的突变细胞中的表型 (图 1J)。<ol>
<li>利用突变体的基因组 DNA 作为模板, 在50µL (~ 1 µg DNA、20 p1 酶、1x 反应缓冲器) 中, 通过 PCR 与引物和 p10, 可以很容易地扩增融合转化结构, 并应用表2中描述的反应周期. .构造的转换可以通过以下步骤 7.1-7.13 完成。</li>
	</ol>
</li>
</ol>

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D., 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=The+19S+proteasome+is+directly+involved+in+the+regulation+of+heterochromatin+spreading+in+fission+yeast.">The 19S proteasome is directly involved in the regulation of heterochromatin spreading in fission yeast.</a> J Biol Chem. , (2017).</li><li>Tang, N. H., 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=Generation+and+characterisation+of+temperature+sensitive+mutants+of+genes+encoding+the+fission+yeast+spindle+pole+body.">Generation and characterisation of temperature sensitive mutants of genes encoding the fission yeast spindle pole body.</a> PeerJ Preprints. 5, e3377v3371 (2017).</li><li>Rasooly, A., Weisz, 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+vitro+antibacterial+activities+of+phloxine+B+and+other+halogenated+fluoresceins+against+methicillin-resistant+Staphylococcus+aureus.">In vitro antibacterial activities of phloxine B and other halogenated fluoresceins against methicillin-resistant Staphylococcus aureus.</a> Antimicrob Agents Chemother. 46 (11), 3650-3653 (2002).</li><li>Wilson, D. S., Keefe, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+mutagenesis+by+PCR.">Random mutagenesis by PCR.</a> Curr Protoc Mol Biol. , (2001).</li></ol>

作者没有什么可透露的。

随机突变通过容易出错的 PCR 是一个强大的工具, 以产生一个不同的突变池在某一地区。这种技术对于那些试图在特定情况下评估蛋白质功能的研究尤其有用。例如, 我们在这里使用了易出错的 PCR 评估19S 蛋白酶体亚基, Rpt4, 在染色质维护的功能。通过改变容易出错的 PCR 和调整筛选条件的区域, 我们能够在感兴趣的基因组区域和屏幕上变异细胞, 以期望的表型。最近, 一种类似的方法被用来隔离在主...

前遗传学是一种经典的方法, 研究人员寻找自然发生的突变体, 显示特定的表型, 并进行遗传分析。在反向遗传学中, 突变被引入到一个感兴趣的基因中, 并检查表型。在后一种情况下, 目标基因的随机突变经常被用来产生一个突变体, 随后被选择为期望的表型, 如温度敏感性或改变的酶活性。各种方法可用于实现随机突变, 包括易出错的 PCR1;紫外线照射2;化学诱变, 如甲基磺酸乙酯 (EMS) 或一氧化二氮3;使用临时突变菌株, 如过度表达mutD5 4;和 DNA 洗牌5。
在这里, 我们描述了一个反向遗传学策略, 我们利用易出错的 PCR 产生突变池的目标基因的裂变酵母。从它的名字可以推测, 这种方法通过在 PCR 过程中故意引入错误来产生突变。与其他突变方法不同, 容易出错的 PCR 允许用户定义要 mutagenized 的区域。这使得它特别有用的努力研究的功能蛋白质/领域的兴趣。
为了演示这个随机的突变过程, 我们在这里使用rpt4+, 它编码19S 蛋白酶体亚基, 作为一个例子。Rpt4 在裂变酵母6、7、8、9之外的有机体中已被证明具有与蛋白质分解无关的功能, 而蛋白质水解的缺陷可能通过改变蛋白而导致间接影响。水平。因此, 我们对突变体进行了筛选, 从而引发了与蛋白水解无关的变化, 目的是研究蛋白酶体对染色质的作用。
通过调整引物的位置, 可将易出错的 PCR 应用于任何基因区域。表现出期望表型变化的突变体可以与适当的记者鉴别。在这里, 我们使用了插入到着丝粒1外部重复 (工程机械)区域10的ade6+ 记者。本构染色质是在该区域11上形成的, 因此在野外类型条件下, ade6+报告器处于静音状态;红色殖民地10表示这一点。在着丝粒上破坏本构染色质的突变将导致ade6+ 的表达式, 该报告被可视为白色的菌落。

<table><tbody><tr><td>&lt;strong&gt;1. Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270-10KG</td><td></td></tr><tr><td>Yeast extract</td><td>BD Biosciences</td><td>212720</td><td></td></tr><tr><td>L-Leucine</td><td>JUNSEI</td><td>87070-0310</td><td></td></tr><tr><td>Adenine sulfate</td><td>ACR</td><td>16363-0250</td><td></td></tr><tr><td>Uracil&amp;nbsp;</td><td>Sigma-Aldrich</td><td>U0750</td><td></td></tr><tr><td>L-Histidine</td><td>Sigma-Aldrich</td><td>H8125</td><td></td></tr><tr><td>KH2PO4</td><td>Sigma-Aldrich</td><td>1.05108.0050</td><td></td></tr><tr><td>NaCl</td><td>JUNSEI</td><td>19015-0350</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>63140</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>12095</td><td></td></tr><tr><td>Potassium phthalate</td><td>Sigma-Aldrich</td><td>P6758-500g</td><td></td></tr><tr><td>Inositol</td><td>Sigma-Aldrich</td><td>I7508-50G</td><td></td></tr><tr><td>Biotin</td><td>Sigma-Aldrich</td><td>B4501-100MG</td><td></td></tr><tr><td>Boric Acid</td><td>Sigma-Aldrich</td><td>B6768-1KG</td><td></td></tr><tr><td>MnSO4</td><td>Sigma-Aldrich</td><td>M7634-500G</td><td></td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>JUNSEI</td><td>83060-031</td><td></td></tr><tr><td>FeCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>KANTO</td><td>CB8943686</td><td></td></tr><tr><td>Sodium molybdate dihydrate</td><td>YAKURI</td><td>31621</td><td></td></tr><tr><td>KI</td><td>JUNSEI</td><td>80090-0301</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>YAKURI</td><td>09605</td><td></td></tr><tr><td>D-myo-inositol</td><td>MP biomedicals</td><td>102052</td><td></td></tr><tr><td>Pantothenate</td><td>YAKURI</td><td>26003</td><td></td></tr><tr><td>Nicotinic Acid</td><td>Sigma-Aldrich</td><td>N4126-500G</td><td></td></tr><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A4418-100G</td><td></td></tr><tr><td>Agarose</td><td>Biobasic</td><td>D0012</td><td></td></tr><tr><td>G418, geneticin</td><td>LPS</td><td>G41805</td><td></td></tr><tr><td>&lt;strong&gt;2. Enzyme reactions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PfuUltra II Fusion HS DNA Polymerase</td><td>Aglient</td><td>600380</td><td>For site-directed mutagenesis</td></tr><tr><td>GeneMorphII Random mutagenesis Kit</td><td>Aglient</td><td>200500</td><td>For error-prone PCR</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>Thermo Fisher</td><td>F-530L</td><td>For fusion PCR</td></tr><tr><td>Ex Taq&amp;nbsp;DNA Polymerase</td><td>Takara</td><td>RR001b</td><td>For general PCR</td></tr><tr><td>BamH1</td><td>New England BioLabs</td><td>R0136S</td><td></td></tr><tr><td>Xho1</td><td>New England BioLabs</td><td>R0146S</td><td></td></tr><tr><td>Dpn1</td><td>New England BioLabs</td><td>R0176S</td><td></td></tr><tr><td>&lt;strong&gt;3. Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Velveteen square, black</td><td>VWR</td><td>89033-116</td><td>For replica</td></tr><tr><td>Replica-plating tool</td><td>VWR</td><td>25395-380</td><td>For replica</td></tr><tr><td>MicroPulser Electroporator</td><td>Biorad</td><td>1652100&amp;nbsp;</td><td>For fission yeast transformation</td></tr><tr><td>Electroporation Cuvettes, 0.2 cm gap</td><td>Biorad</td><td>1652086&amp;nbsp;</td><td>For fission yeast transformation</td></tr><tr><td>Thermal Cycler</td><td>Bioer</td><td>BYQ6078</td><td>For fusion PCR ramp reaction&amp;nbsp;</td></tr></tbody></table>

gene-targeted random mutagenesis to select heterochromatin-destabilizing proteasome mutants in fission yeast

随机突变的目标基因通常是用来确定突变, 产生预期的表型。在可用于实现随机诱变的方法中, 容易出错的 PCR 技术是一种方便而有效的策略, 用于生成多样性的突变体 (i. e, 一个变种人库)。容易出错的 PCR 是一种选择的方法, 当研究人员试图变异一个预先定义的区域, 如编码区域的基因, 而离开其他基因组区域不受影响。突变体库通过易出错的 PCR 扩增后, 必须克隆成合适的质粒。由于克隆步骤的效率的限制, 容易出错的 PCR 所产生的库的大小受到制约。然而, 在裂变酵母中,粟粟中, 克隆步骤可以用高效的一步融合 PCR 来代替, 从而产生转化的构造。然后, 可以使用适当的记者选择所需表型的突变体。在这里, 我们详细描述了这个策略, 以一个例子, 一个记者插入到 centromeric 染色质。

通过遵循图 1中描述的过程, 可以通过评估菌落的颜色来分析获得的 Rpt4 突变体。在减少图 2中的单元格数时, 会在相关的板块上发现菌落的颜色。插入到染色质区域的ade6+报告器在野生类型中被静音, 并显示在 "YES" 板块中的红色菌落。一旦染色质被破坏, 并且ade6+报告器被表达, 可以在 "YES" 板块中观察到白色的菌落, ...

Watch this Scientific Journal Video about Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast at JoVE.com

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

本文介绍了一种用于裂变酵母靶基因随机诱变的详细方法。例如, 我们针对的是rpt4+, 它对19S 蛋白酶体的亚基进行编码, 并筛选出破坏染色质的突变。

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

gene-targeted-random-mutagenesis-to-select-heterochromatin

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

10.6K 次观看.  Korea Advanced Institute of Science and Technology. 本文介绍了一种用于裂变酵母靶基因随机诱变的详细方法。例如, 我们针对的是rpt4+, 它对19S 蛋白酶体的亚基进行编码, 并筛选出破坏染色质的突变。

视频: Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

科研

遗传学

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

目标<em&gt;原位</em&gt;组蛋白突变的基因在芽殖酵母

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

基于交配的酵母超表达文库筛选

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

一种在裂变酵母中的深度固序辅助自发抑制屏

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

生物学

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

绿色怪物承载多基因缺失的酵母菌株的产生过程

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

隔离增效变种的Hsp104利用酵母Proteinopathy模型

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

化学遗传相互作用的快速鉴定<em&gt;酿酒酵母</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

摘要

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光遗传学随机突变用组蛋白miniSOG在

目标

基于交配的酵母超表达文库筛选

一种在裂变酵母中的深度固序辅助自发抑制屏

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌

绿色怪物承载多基因缺失的酵母菌株的产生过程

隔离增效变种的Hsp104利用酵母Proteinopathy模型

化学遗传相互作用的快速鉴定

成长型测定和遗传需求生化确认的蛋白质降解