BIOLISTIC TRANSFORMATION OF YEAST MITOCHONDRIA

我们最近成功地将线粒体 酵母细胞与外源DNA使用“生物射弹”（生物 弹道）技术，这是轰击细胞与 包裹着DNA的微粒 拟议的研究是 打算细化技术，开发一套方便的 用于转化酵母的载体和受体菌株 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的问题。 将构建载体和受体菌株以允许 选择转化体，维持线粒体内的 自主表达载体，并引入修改后的 线粒体DNA序列中的同源位点， 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假定的 将检查复制起点。 这些数据将不会 只会有助于设计更好的线粒体 转换系统，但将促进我们对 线粒体基因组的表达和传递， 提供了重要的体内相关体外数据， 到目前为止是不可能的。 的系统分析 将进行细胞色素编码的细胞色素B蛋白， 进一步了解配体结合和其他功能域 蛋白质的定点突变。 最后提出 将两个核编码基因 多肽进入线粒体，并确定的能力， 将它们的蛋白质产物功能性地并入 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.