BIOLISTIC TRANSFORMATION OF YEAST MITOCHONDRIA

我们最近成功地在生命中改变了线粒体 带有外源DNA的酵母细胞使用“biolistic”(生物 弹道)技术，即用弹道轰击细胞 包裹着DNA的微弹。拟议的研究是 意在提炼技术，开发一套便捷的 酵母转化载体和受体菌株的研究 线粒体，并利用这些发展来研究选定的 线粒体分子生物学和生物化学中的问题。 载体和受体菌株将被构建成允许 转化子的选择、线粒体内的维持 自主表达载体，并引入修饰后的 线粒体DNA序列与同源位点的关系 线粒体基因组。的功能和顺序要求 保守的线粒体转录启动子和推定 我们将研究复制的起源。这些数据不会 只对设计更好的线粒体有贡献 转型系统，但将促进我们对 线粒体基因组的表达和传递 提供重要的体内相关体外数据，这已经 到目前为止都是不可能的。一种系统的分析方法 线粒体编码的细胞色素b蛋白将被携带到 进一步了解配体结合和其他功能域 通过定点突变获得蛋白质。最后，我们建议 转化编码复合体III的两个核编码基因 多肽进入线粒体并测定其能力 他们的蛋白质产品将在功能上并入 复合体III。

We have recently succeeded in transforming mitochondria in living yeast cells with exogenous DNA using the "biolistic" (biological ballistic) technique, which is the bombardment of cells with microprojectiles coated with DNA. The proposed research is intended to refine the technique, to develop a set of convenient vectors and recipient strains for transformation of yeast mitochondria, and to exploit these developments to study selected problems in mitochondrial molecular biology and biochemistry. Vectors and recipient strains will be constructed to allow selection of transformants, the maintenance of intramitochondrial autonomous expression vectors, and to introduce modified mitochondrial DNA sequences into homologous sites in the mitochondrial genome. The function and sequence requirements of conserved mitochondrial transcriptional promotors and putative origins of replication will be examined. These data will not only contribute to the design of better mitochondrial transformation systems, but will advance our understanding of the expression and transmission of the mitochondrial genome and provide important in vivo correlates to in vitro data, which has been impossible up to now. A systematic analysis of the mitochondrial-encoded cytochrome b protein will be carried out to understand further, ligand binding and other functional domains of the protein by site-directed mutagenesis. Finally, we propose to transform two nuclear-encoded genes encoding complex III polypeptides into mitochondria and to determine the ability of their protein products to be functionally incorporated into complex III.