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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes
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High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes

机译：衣原体线粒体的高效生物弹转化可用于在复杂的I基因中插入突变

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Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100-250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This Δnd4 mutation causes a misassembly of complex I, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex I. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex I, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex I.
机译：莱茵衣藻的线粒体转化已通过使用粒子枪装置和克隆的线粒体DNA或PCR片段进行了优化。一个呼吸不足的菌株缺乏一个1.2kb的线粒体DNA区域，包括左端粒和部分cob基因，可以挽救，也可以挽救线粒体cox1和nd1基因的双移码突变体。已实现高转化效率（每微克DNA 100-250个转化子），使用线性化质粒DNA可获得最佳结果。转化子的分子分析表明，可以复制右端粒序列以通过重组来重建左端粒。另外，可以引入无害和有害突变。通过将核苷酸取代引入cob基因中，已经产生了抗噻噻唑抗性的转化体。同样，在缺失和移码受体菌株的nd4线粒体基因中均已在框架内缺失了23个密码子。据信这23个密码子编码ND4蛋白的第一个跨膜片段。这种Δnd4突变会导致复合物I的错配，其亚复合物的积累比野生型复合物I小250 kDa。衣藻中有效线粒体转化的可用性为研究线粒体的生物发生提供了宝贵的工具，并且更具体地说，对于线粒体编码的复合体I的亚基的定点诱变，由于酵母Saccharomyces cerevisiae的线粒体基因组实际上可以随意操作，因此缺乏复合体I，这引起了特别的兴趣。

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