Expression of Barstar as a selectable marker in yeast mitochondria
Expression of Barstar as a selectable marker in yeast mitochondria
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DOI：
10.1007/s00438-003-0879-2
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发表时间：
2003-09-01
影响因子：
3.1
通讯作者：
Fox, TD
中科院分区：
文献类型：
--
作者：
Mireau, H;Arnal, N;Fox, TD
我们描述了一种基于细菌基因的线粒体转化的新的和潜在的通用选择系统，并证明了其在酿酒酵母中的可行性。我们首先发现细胞质合成的Barnase（一种RNase）在靶向细胞器时干扰线粒体基因的表达，当表达在适当水平时不会造成致命。接下来，我们合成了一个基因，该基因利用酵母线粒体遗传密码来指导特异性巴纳酶抑制剂Barstar的合成，并证明了该基因BARSTM的表达整合在mtDNA中，保护呼吸功能免受进口巴纳酶的侵害。最后，我们发现对线粒体靶向藤蔓酶的抗性筛选可用于鉴定线粒体DNA中含有BARSTM的罕见线粒体转化子。讨论了在其他生物中采用这种策略的可能性。
We describe a new and potentially universal selection system for mitochondrial transformation based on bacterial genes, and demonstrate its feasibility in Saccharomyces cerevisiae. We first found that cytoplasmically synthesized Barnase, an RNase, interferes with mitochondrial gene expression when targeted to the organelle, without causing lethality when expressed at appropriate levels. Next, we synthesized a gene that uses the yeast mitochondrial genetic code to direct the synthesis of the specific Barnase inhibitor Barstar, and demonstrated that expression of this gene, BARSTM, integrated in mtDNA protects respiratory function from imported barnase. Finally, we showed that screening for resistance to mitochondrially targeted barnase can be used to identify rare mitochondrial transformants that had incorporated BARSTM in their mitochondrial DNA. The possibility of employing this strategy in other organisms is discussed.