本出版物得到了 Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica （PAPIIT）、UNAM [IN223623 to XP-M] 的支持。UPD 是 CONAHCYT 研究员 （CVU：883299）。我们要感谢 Ariann Mendoza-Martínez 博士在光学显微镜图像方面提供的技术帮助。生物渲染许可证：DU26OMVLUU（图 2）;BK26TH9GXH（图 3）;GD26TH80R5（图 4）;PU26THARYD（图 7）;ML26THAIFG（图 9）。

注意：我们建议对每个构建体进行六次转化，因为线粒体转化效率通常较低。不同生长培养基的组成如 表 2 所示。
1. 钨颗粒制备
<ol><li>在微管中称取 30 mg 0.7 μm 钨颗粒（WPs、微载体）。加入 1.5 mL 70% 乙醇 （EtOH） 进行灭菌。涡旋 WP 并让它们在室温下静置 10 分钟。</li>
	<li>在室温下以 13,200 x g 离心 15 分钟。向颗粒中加入 1.5 mL 无菌水，涡旋，并在室温下以 13,200 × g 离心 15 分钟来洗涤 WP。丢弃上清液。</li>
	<li>加入 500 μL 无菌 50% 甘油（终浓度为 60 μg WPs/μL）。 注意：WPs 可以在 -20 °C 下储存至少 6 个月。从该原液中，取所需量进行 6 次转化 （100 μL）。</li>
</ol>2. WPs DNA 包被
<ol><li>将 5 μg 2 μ质粒和 15 μg 含有线粒体序列的细菌质粒加入到冰上的微管中。 注：最终体积不得超过 50 μL，以避免进一步稀释 DNA 包被所需的试剂。</li>
	<li>加入 100 μL WP 悬浮液并涡旋 5 秒（6 mg WPs）。</li>
	<li>加入 4 μL 1 M 亚精胺。短暂涡旋。</li>
	<li>加入 100 μL 冰冷的 2.5 M CaCl2，短暂涡旋。在冰上孵育 10 分钟;每 3 分钟短暂涡旋混合物。 注意：CaCl2 溶液经过过滤灭菌，不得高压灭菌。</li>
	<li>在 4 °C 下以 13,200 × g 离心 WP 30 秒，并弃去上清液。通过在 -20 °C 下轻轻重悬于 200 μL 100% EtOH 中来洗涤颗粒。</li>
	<li>在 4 °C 下以 13,200 × g 离心 WP 30 秒，弃去上清液。重复洗涤。</li>
	<li>使用移液器吸头将 WP 重悬于 60-70 μL 室温 100% 乙醇中;不要涡旋。 注：为确保质粒的质量，DNA 包被应在生物转化的同一天进行。</li>
</ol>3. 生物材料制备
<ol><li>轰击前，在高压灭菌器中对六个宏载体支架（图 5）进行消毒并彻底干燥。或者，将它们浸泡在 EtOH 中，并在镊子的帮助下用火焰对它们进行消毒。将它们放在无菌玻璃板上。</li>
	<li>在 DNA 沉淀过程中（在步骤 2.4 中在冰上孵育 10 分钟），在无菌镊子的帮助下，将六个大载体（或飞盘）插入六个大载体支架中的每一个中。 注意： 不要对宏载体进行消毒。</li>
	<li>将 DNA 包被的 WP 与移液管混合。向每个大载体表面添加 10 μL WP，并用移液器吸头分布在整个表面上。如有必要，在大载流子表面额外添加几微升 100% 乙醇，以使 WP 均匀铺展。让悬浮液在室温下干燥 ~20 分钟。</li>
</ol>4. 酵母细胞和轰击板的制备
<ol><li>将 rho0 受体菌株接种在 2 mL YPR 培养基上（表 2）。在 30 °C 下在旋转的轮中生长一晚。</li>
	<li>在 30 mL 新鲜 YPR 培养基中稀释 300 μL 培养物。在旋转振荡器上以 30 °C 和 200 rpm 生长两晚，直到培养物饱和。</li>
	<li>在室温下以 600 × g 离心酵母细胞 5 分钟。弃去上清液，将酵母细胞重悬于 600 μL YPD 培养基中（表 2）。</li>
	<li>使用无菌玻璃手柄，将 100 μL 酵母细胞悬液涂在固体轰击培养基板上。 注：轰击培养基必须缺少将用于转化的 2 μ质粒的选择标记。</li>
	<li>对其他五个板重复步骤 4.4。</li>
	<li>轰击前在室温下孵育 1-3 小时，以确保液体介质在板表面完全吸收。</li>
</ol>5. 通过生物转化生成合成 rho 菌株
注意： 在开始之前，请务必阅读生物保护设备的用户手册。我们将描述参考设备的协议（材料表 和 图 5）。
<ol><li>用 70% 乙醇清洁生物颗粒输送系统的腔室以防止污染，并静置至少 10 分钟。在轰炸之前，用干净的纸巾擦拭表面。</li>
	<li>打开氦气罐阀门，将压力设置为 2,000 psi 左右。</li>
	<li>打开设备电源并打开真空源。</li>
	<li>使用镊子将宏载体正面朝下放在宏载体支架上。 注意： 请勿使用设备随附的停止屏幕。</li>
	<li>将 macrocarrier 盖拧到 macrocarrier 的顶部。所有部件共同构成了微载流子发射系统（图 5）。</li>
	<li>将微载流发射系统放在真空室内从底部开始的第五层。</li>
	<li>将含有 rho0 菌株草坪的板之一放在腔室底部的第二层。确保板朝上且没有盖子。</li>
	<li>使用无菌镊子，将一个爆破片放在固定帽上。拧上加速管上的固定帽;确保固定帽紧贴。 注意：不要按照手册中的建议用异丙醇处理破裂的椎间盘，因为我们观察到转化效率降低。</li>
	<li>关闭真空室的门。</li>
	<li>将 VAC/VENT/HOLD 开关置于 VAC 位置。当达到 25-28 inHg 真空 度时，将 VAC/VENT/HOLD 开关更改为 HOLD 位置。 注意： 请注意，可实现的真空可能会因海平面而异。我们发现成功达到 25 inHg。</li>
	<li>按下 FIRE 开关射击 WP。等待加速管中的压力增加，直到达到 1,300 psi，此时爆破片会破裂，发出爆裂声。</li>
	<li>爆破片破裂后，通过将 VAC/VENT/HOLD 开关更改为 VENT 位置，立即释放 FIRE 开关和真空。</li>
	<li>小心地，用无菌镊子清除板表面存在的任何爆破片碎屑，并用盖子盖住板。</li>
	<li>对其余五个板重复该过程，在每一轮中放置一个新的爆破片和宏载体。</li>
	<li>最后，关闭氦气罐的主阀。</li>
	<li>关闭腔室并通过将 VAC/VENT/HOLD 开关置于 VAC 位置来建立真空。按下并松开 FIRE 开关几次，将系统从任何氦气压力中释放出来。在门打开的情况下重复上述步骤。</li>
	<li>关闭真空泵和轰炸设备。</li>
	<li>用 70% EtOH 清洁输送系统。用 0.1% SDS 溶液清洗 macrocarrier 支架;用无菌蒸馏水和 100% 乙醇冲洗。</li>
</ol>6. 合成 rho- 菌落选择
<ol><li>将轰击板在 30 °C 下孵育 5-7 晚。 注意：每天检查板是否受到污染非常重要。如有必要，可以用无菌刮刀切掉污染区域，以避免进一步污染。</li>
	<li>将相反交配类型的测试菌株接种在 2 mL YPD 培养基中过夜（参见 图 2D、E 中的具体示例）。</li>
	<li>在 YPD 板上接种 150 μL 的测试仪菌株培养物。使用无菌玻璃手柄，将测试仪均匀地铺在板表面。让板干燥至少 5 分钟。</li>
	<li>使用天鹅绒复制板和复制器印章制作轰炸板的复制品。<ol><li>在对应于转化的 2 μ质粒营养缺陷型标记物的缺失培养基板上复制。生长需要在 30 °C 下孵育一夜。 之后，在 4 °C 下储存直至进一步使用。此板是 主板。</li>
		<li>在包含步骤 6.3 中测试人员草坪的 YPD 板上复制。在 30 °C 下孵育两晚。之后，在选择性培养基上复制该板以检测感兴趣的线粒体基因的存在，例如，呼吸培养基或缺乏精氨酸的培养基，并在 30 °C 下孵育过夜。 只有线粒体中含有合成细菌质粒的细胞在与测试菌株交配后才会在选择性培养基上生长。</li>
	</ol></li>
	<li>在主板上（步骤 6.4.1）鉴定在步骤 6.4.2 中生长的相应菌落。 注意：由于轰击板（以及主板）挤满了转化菌落，因此很难识别在测试仪的交配板上观察到的阳性菌落。因此，强烈建议用永久性记号笔在所有印版的每一侧画几条小线。在复制图章的环上绘制类似的线条。这些线将有助于在主板上定位和定位阳性菌落（参见 图 2D 中的示例）。</li>
	<li>如步骤 6.4.1 所示，通过将阳性菌落从主板划线到第二个选择性缺失板来纯化阳性菌落。在 30 °C 下孵育两晚。 注：条纹必须允许单个菌落的生长，以确保菌株的纯度。</li>
	<li>重复选择过程（步骤 6.2-6.5）以确认线粒体中存在细菌质粒。 注意：通常，此过程可以再重复两次以选择稳定的合成 rho- 菌株。</li>
	<li>一旦合成的 rho- 菌株在线粒体中稳定地维持质粒 DNA，选择 3-5 个不同的阳性菌落并将它们接种在 2 mL 的 YPD 中。使用这种培养物制备冷冻管原液（通过将 600 μL 培养物和 600 μL 50% 甘油混合以在 -70 °C 下保存），并通过细胞诱导将质粒序列插入目标 mtDNA 中（步骤 6.7）。 注意：由于没有选择压力来维持来自合成 rho 菌株的线粒中的转化质粒，因此质粒很容易丢失。因此，我们建议尽快进行细胞诱导。</li>
</ol>7. 通过细胞诱导将合成 rho- 菌株中存在的构建整合到线粒体基因组中的靶位点
<ol><li>用 3 mL YPD 培养基接种两管，一根用供体细胞（合成 rho 细胞），另一支用受体细胞（预期最终构建的酵母细胞）。在 30 °C 下孵育过夜。</li>
	<li>在无菌微管中混合 750 μL 供体菌株和 250 μL 受体菌株（比例为 3：1）。</li>
	<li>将混合物以 13,200 × g 离心 1 分钟;弃去上清液。使用末端切掉的 1 mL 移液器吸头，取一滴细胞沉淀物并将其放在 YPD 板上。在 30 °C 下孵育 2-4 小时。 注：为避免交叉污染，不要在单个板中放置两个以上的细胞悬液（参见 图 4A 中的示例）。</li>
	<li>取少量细胞混合物，将其重悬于 15 μL 无菌水中，放在载玻片上，并放置盖玻片。在光学显微镜下用 40 倍或 100 倍物镜检查是否存在 shmoos;当存在两种交配类型时，这种特征形状就会出现（图 3B）。</li>
	<li>如果存在 shmoos，则在 2 mL YPD 培养基中重悬少量 （~0.5 mm3） 细胞团。在 30 °C 下在旋转轮中孵育 2 小时，以使双核受精卵恢复。</li>
	<li>涡旋混合物，并在 990 μL 无菌水（1：100 稀释）中稀释 10 μL 细胞悬液。使用玻璃珠或无菌玻璃手柄将 100 μL 细胞稀释液涂布在只有受体菌株（和散发的二倍体）生长的缺失培养基上。在 30 °C 下孵育 2-3 晚。</li>
	<li>如介绍中所述，在必要的选择性培养基上复制所得菌落（阶段 4;具体示例见 图 4C ）。</li>
	<li>鉴定出阳性菌落后，将它们重新放在相同的选择性培养基上，以纯化携带目标 mtDNA 的单倍体菌株。</li>
	<li>选择至少两个阳性菌落，并在 30 °C 下在 2 mL YPD 培养基上培养过夜。通过将 600 μL 的 50% 甘油与 600 μL 的饱和 YPD 培养物混合，进行低温备份。储存在 -70 °C。</li>
	<li>分离所得菌株的 DNA31,32。通过 PCR 扩增所需区域并测序以确认线粒体突变的存在。 注意：每个阳性菌落都是一个技术复制，必须独立确认以确保线粒体基因组中的正确序列。</li>
</ol>

酵母线粒体的生物转化用于诱变分析

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作者没有任何需要披露的利益冲突。

目前的工作描述了 如何成功地转化 酵母中的线粒体。该过程从高速微弹轰击到纯化预期的酵母菌株，需要 ~8-12 周，具体取决于合成 rho 菌株 需要多少轮纯化。该方法的一些关键步骤如下。首先，线粒体基因构建体中突变位点周围添加的侧翼区域越大，靶突变成功整合的可能性就越高。侧翼区域的最小尺寸为 ~100 nt;但是，强烈建议在突变的每一侧使用超过 500 nt。其次，在选择转化?...

酵母酿酒酵母是一种被广泛认可的用于研究线粒体生物发生的模型。由于酵母是一种厌氧的兼性生物，因此可以广泛研究引入损害呼吸的突变的原因和后果。此外，这种生物体拥有友好的遗传和生化工具来研究线粒体途径。然而，探索呼吸复合物组装和线粒体蛋白质合成机制的最强大资源之一是转化线粒体和修饰细胞器基因组的能力。以前，在线粒体 DNA （mtDNA） 中引入点突变或小缺失/插入 1,2,3,4,5，删除基因 6,7，进行基因重排 7,8，为线粒体蛋白添加表位 9,10，将基因从细胞核重新定位到线粒体11，12，并引入 BarStar13、GFP14,15、荧光素酶16 和最广泛使用的 ARG8m17,18 等报告基因。线粒体基因组修饰使我们能够解剖和识别原本难以理解的机制。例如，插入线粒体 DNA 中 COX1 基因座的 ARG8m 报告基因对于理解 Mss51 在 Cox1 生物发生中具有双重作用至关重要。首先，它是 COX1 mRNA 的翻译激活剂，其次，它是新制备的 Cox1 蛋白的组装伴侣 7,19。这项工作提出了一种转化酿酒酵母线粒体的详细方法。尽管线粒体转化方案较早发布 16,20,21,22,23，但通过视频的视觉方法对于彻底了解该方法的不同阶段和细节至关重要。该方法由各个步骤组成，分为四个一般阶段：
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66856/66856fig01v2.jpg"> 图 1：通过高速微弹轰击进行线粒体转化过程的概述：1） 在 DNA 包被之前对钨颗粒进行消毒和制备。2） 两种不同的质粒沉淀在 WP 表面。一种是含有将被定向到线粒体基质的构建体的细菌质粒。另一种是携带营养缺陷型标记物的核酵母表达载体。3） 缺乏线粒体 DNA （rho0） 的受体酵母菌株生长在半乳糖或棉子糖等发酵碳源上，它不会对线粒体基因表达产生任何葡萄糖抑制作用 34,35。培养物铺布在含有轰击培养基的培养皿上。4） 用质粒包被的 WP 通过高速微弹轰击射向受体菌株。5） 通过与测试菌株交配来选择含有线粒体质粒的阳性合成 rho- 菌落。6） 线粒体构建体通过称为细胞诱导的过程将合成的 rho- 菌株（供体）与受体菌株交配，从而在所需的基因座处整合到线粒体基因组中。7） 选择携带线粒体构建体的阳性受体单倍体菌株并在不同的培养基中纯化。缩写： WPs = 钨颗粒;mtDNA = 线粒体 DNA。<a href="https://www.jove.com/files/ftp_upload/66856/66856fig01largev2.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
用旨在整合到线粒体基因组中的 DNA 构建体转化细胞 （图 1，步骤 1-4） 虽然可以直接转化含有完整线粒体基因组 （rho+） 的酵母，但如果细胞缺乏线粒体 DNA （rho0）24，转化效率会提高 10-20 倍。钨颗粒 （WP） 包被有两种不同的质粒，它们将被共转化。第一个是携带营养缺陷型标志物的酵母 2 μ表达载体，例如 LEU2 或 URA3 （分别为 YEp351 或 YEp352）。如果 DNA 成功生物引入酵母细胞，转化体将在营养缺陷型培养基（即缺乏亮氨酸或尿嘧啶的培养基）上生长。该质粒有助于对获得核质粒的细胞进行首次选择;否则，所得菌落的数量将使板饱和。第二质粒是细菌质粒（如 pBluescript 或类似物），包含旨在整合到线粒体基因组中的线粒体结构。该构建体必须包含至少 100 nt 的 5' 和 3' 侧翼线粒体序列，以便与感兴趣的线粒体区域重组。根据我们的经验，较大的侧翼序列更有可能成功地与线粒体基因组中的靶基因座重组。
一个具体的例子如图 225 所示。在这个例子中，目的是删除编码相应蛋白质 （Cox1ΔC15） 最后 15 个氨基酸的线粒体 COX1 基因区域（图 2A）。携带 COX1ΔC15 突变的细菌质粒分别含有 395 nt 和 990 nt 的基因 5' 和 3' 非翻译区。质粒来源于 pBluescript （pXPM61），并与 2 μ质粒 YEp352 一起在 WP 表面共沉淀（图 2B）。然后通过微弹轰击将 WP 引入选定的受体菌株（图 2C）。该菌株名为 NAB6926，是一种带有 kar1-1 和 ade2 等位基因的 MATa、rho0 菌株（这些特性的重要性将在下面讨论）。将细胞接种在缺乏尿嘧啶的轰炸培养基上，以选择获得 2 μ质粒的细胞（图 2C）。细胞在 30 °C 下生长 5-7 晚。
选择获得含有线粒体构建体的细菌质粒和携带营养缺陷型标记物的核 2 μ质粒的细胞 （图 1， 步骤 5） 阳性菌落将在其线粒体22 中保持细菌质粒的许多拷贝。由于转化的细胞最初是 rho0，因此没有线粒体序列支持质粒中存在的线粒体构建体的翻译;因此，转化的线粒体基因不会表达。有必要将转化体与测试菌株交配，以检测获得线粒体质粒的酵母菌落。测试菌株的线粒体基因组包含目标基因的非功能性突变版本。交配后，线粒体会融合，转化质粒中包含的线粒体序列将与来自测试菌株的突变线粒体基因重组;因此，WT 基因的恢复将重建功能。得到的二倍体将具有可检测的阳性表型（通常在呼吸介质或缺乏精氨酸的介质中生长的能力）。线粒体中携带细菌质粒的阳性细胞称为“合成红细胞”。在图 2D 的具体示例中，携带 COX1ΔC15 构建体（名为 XPM199）的合成 rho 细胞被复制接种在缺乏尿嘧啶的培养基上（这是从中纯化阳性菌落的母板）。他们还在测试菌株 L4527 草坪上进行了复制接种，该菌株包含无功能突变 cox1D369N。交配两晚后，L45 和 XPM199 的线粒体基因组重组，产生功能性 COX1 基因;因此，二倍体恢复了在呼吸介质上生长的能力。从主板中，我们挑选了阳性菌落。它们在缺乏尿嘧啶的平板上划线，并重复选择性测试以获得纯合成的 rho 细胞（图 2E）。需要注意的是，测试板仅用于鉴定合成 rho- 菌落，不能从这些板中回收突变体。
将构建体整合到预期菌株的线粒体基因组中 此步骤是通过称为细胞诱导28,29 的过程实现的（图 1，步骤 6）。在这种方法中，合成的 rho- 菌株（供体菌株）与相反交配类型的预期受体菌株交配。一个基本要求是两种交配菌株中至少有一个携带 kar1-1 突变以阻止核聚变30。因此，两个细胞的交配会产生双核受精卵（图 3）。来自亲本细胞（供体和受体）的线粒体网络融合，线粒体 DNA 分子重组。双核受精卵被孵育/回收以允许出芽，这将产生单倍体细胞。这些单倍体携带其中一个亲本细胞的核背景。同样，单倍体可以携带来自亲本细胞或目标重组线粒体 DNA 的线粒体 DNA。然而，kar1-1 突变并非 100% 有效，在交配过程中会形成一些真正的二倍体28,29。合成 rho- 菌株 （供体，XPM199） 携带示例中的 kar1-1 等位基因。作为选择性标记物，它具有 ade2 等位基因，使其成为腺嘌呤的营养缺陷型细胞（图 4）。受体菌株是 XPM10，一种带有 cox1Δ：：ARG8m 构建体的 rho + 菌株，其中报告基因 ARG8m 取代了线粒体基因组7 中的 COX1 密码子。将两种细胞培养物的混合物作为液滴添加到 YPD 板中，以便进行交配。3-5 小时后，在光学显微镜下观察细胞以检测 shmoos 的形成（图 3B），这是一种与交配相关的细胞形状。孵育/恢复时间后，将双核受精卵接种在缺乏腺嘌呤的培养基上，以防止供体细胞的生长。
选择携带目标线粒体基因组的单倍体菌株 （图 1，步骤 7） 细胞诱导后存在供体、受体和二倍体细胞的混合物。因此，在孵育/回收双核受精卵后，细胞必须在不同的选择性培养基中生长，以鉴定和纯化感兴趣的单倍体。选择性培养基取决于供体的基因型、受体菌株和预期的线粒体结构。然而，一般来说，选择选择性培养基的原因是：i） 孵育/恢复后，细胞诱导混合物在供体亲本菌株无法生长的选择性培养基上生长。在图 4A、B 的具体示例中，供体（合成 rho-菌株）携带无功能的 ade2 等位基因。因此，细胞诱导混合物必须在缺乏腺嘌呤的培养基板上孵育。这是主板。ii） 一旦菌落生长，在缺乏腺嘌呤的培养基上再次复制母板（以生成新的母板，从中纯化感兴趣的阳性菌落），然后，在只有二倍体可以生长的培养基上复制。这是避免进一步无意中纯化二倍体集落所必需的。在图 4C 中的示例中，只有二倍体可以在缺乏亮氨酸的培养基上生长。iii） 母板也在培养基上复制，其中只有那些包含目标线粒体 DNA 的受体单倍体才会生长。在图 4C 的示例中，单倍体在含有乙醇/甘油作为碳源的呼吸培养基上生长，因为 Cox1ΔC15 蛋白具有功能。所得的带有目标 mtDNA 的 rho + 菌株被命名为 XPM20925。 表 2 和图 4 中使用的菌株列在表 1 中。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66856/66856fig02.jpg"> 图 2：描述线粒体转化和阳性 rho 细胞选择的具体示例的图表。 （A） 线粒体基因组的预期修饰是编码 Cox1 亚基最后 15 个氨基酸的区域 （Cox1ΔC15） 的缺失。（B） WPs 包被两个质粒以转化酵母细胞。一个是在细胞核中定向和表达的 2 μ质粒 Yep352。另一个是质粒 pXPM61，它包含 COX1ΔC15 等位基因以及 COX1 5' 和 3'-UTR 的 395 nt 和 990 nt。（C） WPs 从缺乏线粒体 DNA 的菌株 NAB69 （rho0 菌株） 引入细胞中。从轰击板获得的转化菌落在缺乏尿嘧啶的培养基上复制，尿嘧啶是核质粒 YEp352 的营养缺陷型标志物。（D） 为了选择阳性 rho- 菌落，在缺乏尿嘧啶的培养基上复制 -URA 板。这是从中挑选和分离阳性菌落的母板。它还在具有富介质 （YPD） 的板和测试菌株的草坪上进行了复制。在交配过程中，合成的线粒体 DNA 与来自测试菌株 L4527 的线粒体 DNA 重组，该菌株携带 COX1 基因 （D369N） 突变。在呼吸培养基上生长的二倍体包含转化的 DNA。从主板中挑选相应的菌落进行复位和纯化。为了帮助识别所有复制铺板中母板中的阳性菌落，在板的边缘（绿线）上用永久性标记进行标记。（E） 将选定的阳性菌落重新放置在缺乏尿嘧啶的培养基上。这是新的主板。与 D 中一样，又进行了两轮测试以纯化合成的红菌落。缩写： WPs = 钨颗粒;mtDNA = 线粒体 DNA;-URA/Sorb = 缺乏尿嘧啶/含有山梨醇;WT = 野生型。<a href="https://www.jove.com/files/ftp_upload/66856/66856fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66856/66856fig03v2.jpg"> 图 3：描述细胞诱导程序的图表。 （A） 细胞诱导过程中细胞如何交配的一般概述。在细胞诱导过程中，来自供体菌株和受体菌株的线粒体融合，因此来自两种菌株的线粒体 DNA 重新组合。由于 kar1-1 突变30 导致核融合减少，因此形成了双核受精卵。经过一段时间的孵育/恢复时间后，选择包含预期线粒体突变的双核受精卵芽和单倍体受体。（B） 合成的 rho- 菌株（供体）和受体菌株通过细胞诱导交配，可以形成 shmoos（红色箭头），这是交配酵母的一种特征形状。图像是在光学显微镜下用 100 倍物镜拍摄的。比例尺 = 10 μm。 <a href="https://www.jove.com/files/ftp_upload/66856/66856fig03largev2.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66856/66856fig04.jpg"> 图 4：描述细胞诱导以将突变 COX1ΔC15 整合到线粒体基因组中的具体示例的图表。 （A） 将合成 rho- 菌株（供体，XPM199）和目标菌株（受体，XPM10）的液体培养物混合进行交配。shmoo 形成后，将混合物在液体培养物中回收 2-4 小时。接下来，将细胞诱导混合物铺在装有缺乏腺嘌呤的培养基的平板上，以防止供体细胞的生长。（B） 细胞诱导过程中来自供体 （XPM199） 和受体 （XPM10） 的线粒体 DNA 重组事件的示意图。（C） 将母板复制到不同的选择性培养基上，以识别和纯化那些将预期线粒体基因整合到细胞器 DNA 中的单倍体 （XPM209）25。缩写： -ADE = 缺乏腺嘌呤;-LEU = 缺乏亮氨酸。 <a href="https://www.jove.com/files/ftp_upload/66856/66856fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL pipette tips</td>
			<td>Axygen</td>
			<td>T-1000-B</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microtube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L pipette tips</td>
			<td>Axygen</td>
			<td>T-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical bottom tube&#160;</td>
			<td>Axygen</td>
			<td>SCT-25ML-25-S</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L pipette tips</td>
			<td>Axygen</td>
			<td>T-200-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical bottom&#160; tube</td>
			<td>Axygen</td>
			<td>SCT-50ML-25-S</td>
			<td></td>
		</tr>
		<tr>
			<td>AfiII</td>
			<td>New England BioLabs</td>
			<td>R0520S</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>SeaKem</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytic balance</td>
			<td>OHAUS</td>
			<td>ARA520</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>TOMY</td>
			<td>ES-315</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolisitic Macrocarrier holder&#160;</td>
			<td>BIO-RAD</td>
			<td>1652322</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>VWR</td>
			<td>89038-528</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -ADE</td>
			<td>Formedium</td>
			<td>DCS0049</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -ARG</td>
			<td>Formedium</td>
			<td>DCS0059</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -LEU</td>
			<td>Formedium</td>
			<td>DCS0099</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -URA</td>
			<td>Formedium</td>
			<td>DCS0169</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture glass flask</td>
			<td>KIMAX KIMBLE</td>
			<td>25615</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture glass tube</td>
			<td>Pyrex</td>
			<td>9820</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>BD</td>
			<td>215520</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>JT Baker&#160;</td>
			<td>9000</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Millipore</td>
			<td>620006</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>Z265926</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass handle</td>
			<td>Sigma</td>
			<td>S4647</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>JT BAKER</td>
			<td>2136-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Helium tank grade 5 (99.99 %)</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>HSTaq&#160; Kit</td>
			<td>PCR BIO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifugue</td>
			<td>Eppendorf</td>
			<td>022620100</td>
			<td></td>
		</tr>
		<tr>
			<td>NdeI</td>
			<td>New England BioLabs</td>
			<td>R0111L</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>New Brunswick scientific</td>
			<td>NB-G25</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Axygen</td>
			<td>PCR-02-C</td>
			<td></td>
		</tr>
		<tr>
			<td>PDS-1000/He TM Biolistic Particle Delivery System</td>
			<td>BIO-RAD</td>
			<td>165-2257</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (100X10)</td>
			<td>BD</td>
			<td>252777</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Raffinose</td>
			<td>Formedium</td>
			<td>RAF03</td>
			<td></td>
		</tr>
		<tr>
			<td>Replica plater</td>
			<td>Scienceware</td>
			<td>Z363391</td>
			<td></td>
		</tr>
		<tr>
			<td>Rupture discs 1350 Psi</td>
			<td>BIO-RAD</td>
			<td>1652330</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbitol</td>
			<td>Sigma</td>
			<td>S7547</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma</td>
			<td>S0266</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Thermo Scientific</td>
			<td>EL0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Rotator</td>
			<td>Thermo Scientific</td>
			<td>88882015</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten microcarriers M10</td>
			<td>BIO-RAD</td>
			<td>1652266</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccum pump of 100L/min capacity</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Velvet pads</td>
			<td>Bel-Art</td>
			<td>H37848-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex&#160;</td>
			<td>Scientifc Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Wood aplicator stick</td>
			<td>PROMA</td>
			<td>1820060</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen base without aminoacids</td>
			<td>BD</td>
			<td>291920</td>
			<td></td>
		</tr>
	</tbody>
</table>

mitochondrial transformation in baker&#39;s yeast to study translation and respiratory complex assembly

几十年来，面包酵母酿酒 酵母已被广泛 用于了解线粒体生物学。该模型提供了有关真核生物中基本、保守的线粒体途径以及真菌或酵母特异性途径的知识。 酿酒酵母 的众多能力之一是操纵线粒体基因组的能力，到目前为止，这仅在 酿酒酵母 和单细胞藻类 莱茵衣藻中是可能的。酵母线粒体的生物转化使我们能够引入定点突变、进行基因重排并引入报告基因。这些方法主要用于了解线粒体中两个高度协调的过程的机制：线粒体核糖体的翻译以及呼吸复合物和 ATP 合酶的组装。然而，线粒体转化可能用于研究其他途径。在本工作中，我们展示了如何通过高速微弹轰击转化酵母线粒体，选择和纯化预期的转化体，并在线粒体基因组中引入所需的突变。

The yeast Saccharomyces cerevisiae is a widely recognized model used to study mitochondrial biogenesis. Since yeast is an anaerobic, facultative organism, it is possible to extensively study the causes and consequences of introducing mutations that impair respiration. In addition, this organism possesses friendly genetic and biochemical tools to study mitochondrial pathways. However, one of the most powerful resources to explore the mechanisms of respiratory complex assembly and mitochondrial protein synthesis is the ability to transform mitochondria and modify the organelle&#39;s genome. Previously, it has been helpful to introduce in the mitochondrial DNA (mtDNA) point mutations or small deletions/insertions1,2,3,4,5, delete genes6,7, make gene rearrangements7,8, add epitopes to mitochondrial proteins9,10, relocate genes from the nucleus to the mitochondria11,12, and introduce reporter genes like BarStar13, GFP14,15, luciferase16, and the most widely used ARG8m17,18. Mitochondrial genome modifications have allowed us to dissect and identify mechanisms that otherwise would have been difficult to comprehend. For example, the ARG8m reporter gene inserted at the COX1 locus&#160;in the mitochondrial DNA was crucial to understanding that Mss51 has a dual role in Cox1 biogenesis. First, it is a translational activator of the COX1 mRNA, and second, it is an assembly chaperone for the newly made Cox1 protein7,19. This work presents a detailed method to transform S. cerevisiae mitochondria. Although the mitochondrial transformation protocol was published earlier16,20,21,22,23, a visual approach through a video is essential to thoroughly understand the different stages and details of the method. The method consists of various steps and is divided into four general stages:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66856/66856fig01v2.jpg" /> 
Figure 1: Overview of the mitochondrial transformation procedure by high-velocity microprojectile bombardment: 1) The tungsten particles are sterilized and prepared before DNA coating. 2) Two different plasmids are precipitated on the surface of the WPs. One is a bacterial plasmid containing the construct that will be directed to the mitochondrial matrix. The other is a nuclear yeast expression vector carrying an auxotrophy marker. 3) A receptor yeast strain lacking mitochondrial DNA (rho0) is grown on a fermentative carbon source like galactose or raffinose, which does not exert any glucose repression of mitochondrial gene expression34,35. The culture is spread on petri dishes containing the bombardment medium. 4) WPs coated with plasmids are shot to the receptor strain by high-velocity microprojectile bombardment. 5) Positive synthetic rho- colonies containing the mitochondrial plasmid are selected by mating to a tester strain. 6) The mitochondrial construct is integrated into the mitochondrial genome at the desired locus by mating the synthetic rho- strain (donor) with the acceptor strain by a process known as cytoduction. 7) The positive receptor, haploid strain carrying the mitochondrial construct is selected and purified in different media. Abbreviations: WPs = tungsten particles; mtDNA = mitochondrial DNA. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Transformation of cells with the DNA construct intended to integrate into the mitochondrial genome (Figure 1, steps 1-4) 
Although it is possible to directly transform a yeast containing a complete mitochondrial genome (rho+), the transformation is 10-20x more efficient if the cells lack mitochondrial DNA (rho0)24. Tungsten particles (WPs) are coated with two different plasmids that will be co-transformed. The first one is a yeast 2 &#181; expression vector carrying an auxotrophy marker, such as LEU2 or URA3 (e.g., YEp351 or YEp352, respectively). If the biolistic introduction of DNA into the yeast cells is successful, the transformants will grow on the auxotrophy medium (i.e., a medium lacking leucine or uracil). This plasmid is helpful in making the first selection of the cells that acquired the nuclear plasmid; otherwise, the number of resulting colonies would saturate the plate. The second plasmid is a bacterial plasmid (such as pBluescript or similar) containing the mitochondrial construction intended to be integrated into the mitochondrial genome. The construct must contain at least 100 nt of 5&#39; and 3&#39; flanking mitochondrial sequences for recombination with the mitochondrial region of interest. In our experience, larger flanking sequences are more likely to successfully recombine with the target locus in the mitochondrial genome.
A specific example is shown in Figure 225. In this example, the aim was to delete the region of the mitochondrial COX1 gene coding for the last 15 amino acids of the corresponding protein (Cox1&#916;C15) (Figure 2A). The bacterial plasmid carrying the COX1&#916;C15 mutation contained 395 nt and 990 nt of the gene&#39;s 5&#39; and 3&#39; untranslated regions, respectively. The plasmid was derived from pBluescript (pXPM61) and, together with the 2 &#181; plasmid YEp352, they were coprecipitated on the surface of WPs (Figure 2B). The WPs were then introduced by microprojectile bombardment into the selected recipient strain (Figure 2C). This strain, named NAB6926, is a MATa, rho0 strain bearing the kar1-1 and ade2 alleles&#160;(the importance of these characteristics is discussed below). The cells were plated on bombardment media lacking uracil to select those that acquired the 2 &#181; plasmid (Figure 2C). Cells were grown for 5-7 nights at 30 &#176;C.
Selection of the cells that acquired the bacterial plasmid containing the mitochondrial construct and the nuclear 2 &#181; plasmid carrying the auxotrophy marker (Figure 1, step 5) 
The positive colonies will maintain many copies of the bacterial plasmid in their mitochondria22. Since the transformed cells are originally rho0, no mitochondrial sequences support the translation of the mitochondrial construct present in the plasmid; hence, the transformed mitochondrial gene will not be expressed. It is necessary to mate the transformants with a tester strain to detect the yeast colonies that acquired the mitochondrial plasmid. The mitochondrial genome of the tester strain contains a nonfunctional mutated version of the gene of interest. After mating, the mitochondria will fuse, and the mitochondrial sequence included in the transformed plasmid will recombine with the mutated mitochondrial gene from the tester strain; consequently, the recovery of the WT gene will reconstitute function. The resulting diploid will have a detectable positive phenotype (usually the capacity to grow in respiratory medium or a medium lacking arginine). The positive cells carrying the bacterial plasmid in mitochondria are named &#34;synthetic rho- cells&#34;. In the specific example of Figure 2D,&#160;synthetic rho- cells carrying the COX1&#916;C15 construct (named XPM199) were replica-plated on a medium lacking uracil (this is the master plate from which positive colonies were purified). They were also replica-plated on a tester strain L4527 lawn, which contains the nonfunctional mutation cox1D369N. After mating for two nights, the mitochondrial genome of L45 and XPM199 recombined, resulting in a functional COX1 gene; therefore, the diploids recovered the ability to grow on respiratory media. From the master plate, we picked the positive colonies. They were streaked on plates lacking uracil, and the selective tests were repeated to obtain pure synthetic rho- cells (Figure 2E). It is important to note that the testing plate is only for the identification of synthetic rho- colonies, and mutants cannot be recovered from these plates.
Integration of the construct into the mitochondrial genome of the intended strain 
This step is achieved by a process called cytoduction28,29 (Figure 1, step 6). In this approach, the synthetic rho- strain (donor strain) is mated to the intended acceptor strain of the opposite mating type. An essential requirement is that at least one of the two mating strains carries the kar1-1 mutation to impede nuclear fusion30. Hence, mating of the two cells produces a binucleated zygote (Figure 3). The mitochondrial network from the parental cells (donor and acceptor) fuse and the mitochondrial DNA molecules recombine. The binucleated zygotes are incubated/recovered to allow budding, which will produce haploid cells. These haploids carry the nuclear background of one of the parental cells. Similarly, haploids can carry the mitochondrial DNA from either the parental cell or the recombined mitochondrial DNA of interest. However, the kar1-1 mutation is not 100% effective, and some true diploids will be formed during the mating28,29. The synthetic rho- strain (donor, XPM199) carried the kar1-1 allele in the example. As a selective marker, it had the ade2 allele, making it an auxotroph for adenine (Figure 4). The acceptor strain was XPM10, a rho+ strain bearing the cox1&#916;::ARG8m&#160;construct, where the reporter ARG8m&#160;replaced the COX1 codons in the mitochondrial genome7. The mixture of both cell cultures was added to a YPD plate as a drop to allow mating. After 3-5 h, the cells were observed under a light microscope to detect the formation of shmoos (Figure 3B), a cell shape associated with mating. After the incubation/recovery time, the binuclear zygotes were plated on a medium lacking adenine to prevent the growth of the donor cells.
Selection of the haploid strain carrying the mitochondrial genome of interest (Figure 1, step 7) 
A mixture of donor, acceptor, and diploid cells is present after cytoduction. Therefore, after the incubation/recovery of the binucleated zygotes, cells must be grown in different selective media to identify and purify the haploids of interest. The selective media depends on the genotypes of the donor, acceptor strains, and the intended mitochondrial construction. However, in general, the reasoning for choosing the selective media is: i) after incubation/recovery, the cytoduction mix is grown on selective medium where the donor parental strain cannot grow. In the specific example in Figure 4A,B, the donor (synthetic rho- strain) carries the nonfunctional ade2 allele. Thus, the cytoduction mix must be incubated on a medium plate lacking adenine. This is the master plate. ii) Once the colonies grow, the master plate is replicated again on a medium lacking adenine (to generate a new master plate from where the positive colonies of interest will be purified), and next, on a medium where only diploids can grow. This is necessary to avoid further inadvertent purification of diploid colonies. In the case of the example from Figure 4C, only diploids can grow on media lacking leucine. iii) The master plate is also replicated on media where only those acceptor haploids that incorporated the mitochondrial DNA of interest will grow. In the example of Figure 4C,&#160;haploids grow on respiratory media containing ethanol/glycerol as a carbon source since the Cox1&#916;C15 protein is functional. The resulting rho+ strain bearing the mtDNA of interest was named XPM20925. The strains used in Figure&#160;2 and Figure 4 are listed in Table 1.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66856/66856fig02.jpg" /> 
Figure 2: Diagram describing a specific example of mitochondrial transformation and selection of positive rho- cells. (A) The intended modification of the mitochondrial genome was a deletion of the region coding for the last 15 amino acids of the Cox1 subunit (Cox1&#916;C15). (B) WPs were coated with two plasmids to transform yeast cells. One was the 2 &#181; plasmid Yep352 that was directed and expressed in the nucleus. The other was plasmid pXPM61, which contains the COX1&#916;C15 allele plus 395 nt and 990 nt of the COX1 5&#39; and 3&#39;-UTRs, respectively. (C) The WPs were introduced into cells from the strain NAB69, which lacks mitochondrial DNA (rho0 strain). Transformant colonies obtained from the bombardment plates were replicated on a medium lacking uracil, the auxotrophic marker of the nuclear plasmid YEp352. (D) To select the positive rho- colonies, the -URA plate was replicated on a media lacking uracil. This was the master plate from which the positive colonies were picked and isolated. It was also replicated on plates with rich media (YPD) and a lawn of a tester strain. During mating, the synthetic rho- mitochondrial DNA was recombined with the mitochondrial DNA from the tester strain, L4527, which carries a mutation in the COX1 gene (D369N). The diploids that grew on respiratory media contained the transformed DNA. The corresponding colonies were picked from the master plate to restreak and purify. To help identify the positive colonies in the master plate throughout all replica plating, marks were made with a permanent marker on the edges of the plates (green lines). (E) The selected positive colonies were restreaked on a medium lacking uracil. This was the new master plate. As in D, two more rounds of testing were carried out to purify the synthetic rho- colonies. Abbreviations: WPs = tungsten particles; mtDNA = mitochondrial DNA; -URA/Sorb = lacking uracil/ containing Sorbitol;&#160;WT = wild type. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66856/66856fig03v2.jpg" /> 
Figure 3: Diagram describing the cytoduction procedure. (A) General overview of how cells mate during cytoduction.&#160;During cytoduction, mitochondria from the donor and acceptor strains fuse so mitochondrial DNA from both strains recombine. Since nuclear fusion is reduced due to the kar1-1 mutation30,&#160;binuclear zygotes are formed. After some incubation/recovery time, the binuclear zygotes bud and haploid acceptors containing the intended mitochondrial mutation are selected. (B) Mating of the synthetic rho- strain (donor) and the acceptor strain through cytoduction allows the formation of shmoos (red arrows), a characteristic shape of mating yeast. The image was taken under a light microscope with a 100x objective. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66856/66856fig04.jpg" /> 
Figure 4: Diagram describing a specific example of cytoduction to integrate the mutation COX1&#916;C15 in the mitochondrial genome. (A) Liquid cultures of the synthetic rho- strain (donor, XPM199) and the strain of interest (acceptor, XPM10) were mixed to mate. After shmoo formation, the mix was recovered in a liquid culture for 2-4 h. Next, the cytoduction mix was spread on a plate with a medium lacking adenine to prevent the growth of the donor cells. (B) Schematic representation of the recombination events of the mitochondrial DNA from the donor (XPM199) and the acceptor (XPM10) during cytoduction. (C) The master plate was replicated on different selective media to identify and purify those haploids that integrated the intended mitochondrial gene in the organelle&#39;s DNA (XPM209)25. Abbreviations: -ADE = lacking adenine; -LEU = lacking leucine. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

作者聚焦：通过创新的线粒体研究推进蛋白质合成和组装的技术和发现

Baker 中的线粒体转化

Our research explores the regulation of mitochondrial protein synthesis and its connection to inner membrane assembly, crucial for oxidative phosphorylation. Using the model, we study the molecular mechanism of mitochondrial function, leveraging its suitability for detailed molecular and genetic investigations. Many technologies are used for research in our field, like RNA deep sequencing, multiomic technologies, cryo-electron microscopy, high-velocity bombardment to transform gene mitochondria, among others.We have made important contributions to understand the role and mechanisms of action of many proteins involved in mitochondrial translation regulation and on respiratory complexes III and IV assembly. This is the case for proteins like Mss51, Pet309, Pet54, mS38, CPP3, CPP6, among others. This protocol is an indispensable tool for understanding how mitoribosomes find the STARD-CHOL mitochondrial mRNAs.It has been the key to understanding the role of specific nucleotides of mitochondrial mRNAs in translation regulation. These are some examples of what this technique can address. The only available resource to mitochondrial sequences and to use translation of the in the mitochondrial DNA is our protocol, high-velocity bombardment transformation of gene mitochondria.This method has provided invaluable information regarding translation and respiratory complexes assembly that otherwise we could not obtain.

本节介绍了线粒体转化不同阶段的一些代表性结果。图 6 显示了轰炸程序。合成的 rho-cell 携带带有报告基因 ARG8m 的细菌质粒，它将取代线粒体基因的编码序列（图 6A）。轰击后，在缺乏尿嘧啶 （-URA） 的培养基上复制板;这是主板（图 6B）。生长的菌落具有带有 URA3 营养缺陷型标记物的?...

这项工作解释了如何使用生物方法转化酵母线粒体。我们还展示了如何选择和纯化转化体，以及如何在线粒体基因组中的靶位置引入所需的突变。

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about ...

mitochondrial-transformation-baker-s-yeast-to-study-translation

Research

JoVE Journal

Genetics

Mitochondrial Transformation in Baker&#39;s Yeast to Study Translation and Respiratory Complex Assembly

465 次观看.  Universidad Nacional Autónoma de México. 我们的研究探讨了线粒体蛋白合成的调节及其与内膜组装的联系，这对氧化磷酸化至关重要。使用该模型，我们研究了线粒体功能的分子机制，利用其对详细分子和遗传学研究的适用性。我们领域的许多技术被用于研究，如 RNA 深度测序、多组学技术、冷冻电子显微镜、高速轰炸以转化基因线粒体等。我们为了解许多参与线粒体翻译调节以及呼?...