YEAST MITOCHONDRIAL DNA RECOMBINATION

拟议研究的长期目标是在分子和 细胞术语重组和遗传的基础过程 酵母线粒体基因组的行为。特别强调的是 着重分析了小说中的重组事件及其认定 以及参与这些进程的关键组成部分的特征。 本提案侧重于三个主要调查领域：1)a) 参与I组内含子迁移性和剪接的蛋白质，2)移动性 富含GC的回文(GC簇)和3)线粒体的细胞生物学 遗传学。移动内含子的研究集中在详细的生化和 开放基因编码的双功能蛋白的反向遗传学研究 细胞色素氧化酶亚基I内含子4α阅读框(ORF) 吉恩。AI4α是已知的酵母两个可移动内含子之一 线粒体基因组。其ORF产物既是一种核酸内切酶，也是 内含子移动性和RNA剪接所需的潜在成熟酶。我们会 纯化的AI4α编码蛋白的相互作用特征 核酸内切酶I-SceII与DNA结合，进一步确定其识别位点。 我们将使用新开发的线粒体转化方法来 确定核酸内切酶所需蛋白质的重要结构域 成熟酶活性。我们将检验核酸内切酶和 成熟酶的活性共享蛋白质的结构域。我们将测试一个模型 激活潜在的成熟酶活性需要相互作用 在AI4α编码的蛋白质和核的突变形式之间- 编码线粒体亮氨酰tRNA合成酶。线粒体转化 传递遗传学将被用来评估捐赠者和接受者 GC簇移动性的序列要求及其在体内的作用 发生在这些分子边界的双链断裂。突变者 将寻求影响GC集群迁移率的因素。最后，我们会 使用我们研发的荧光显微镜和试剂 受精卵和受精卵中线粒体和线粒体的原位混合 异质单倍体细胞及其向萌发芽的分离。那些 实验旨在建立管理这些过程的规则， 它必须最终确定观察到的重组模式 线粒体DNA。将进行突变筛选，以确定核基因 参与线粒体分离的基因。

The long term goal of the proposed research is to define in molecular and cellular terms the processes underlying the recombination and genetic behavior of the yeast mitochondrial genome. Particular emphasis will be placed on the analysis of novel recombination events and the identification and characterization of key components that participate in those processes. The present proposal focuses in three major areas of investigation: 1) a protein involved in group I intron intron mobility and splicing, 2) mobile GC-rich palindromes (GC clusters) and 3) the cell biology of mitochondrial genetics. Studies of mobile introns focus on detailed biochemical and reverse genetics studies of a bifunctional protein encoded by the open reading frame (ORF) of intron 4alpha of the cytochrome oxidase subunit I gene. aI4 alpha is one of two known mobile introns of the yeast mitochondrial genome. Its ORF product is both an endonuclease required for intron mobility and a latent maturase required for RNA splicing. We will characterize the interaction of the purified aI4 alpha encoded endonuclease, I-SceII, with DNA and define further its recognition site. We will use newly developed methods of mitochondrial transformation to define important domains of that protein required for endonuclease and maturase activities. We will test the hypothesis that endonuclease and maturase activities share domains of the protein. We will test a model that activation of the latent maturase activity requires an interaction between the aI4 alpha-encoded protein and a mutant form of a nuclear- encoded mitochondrial leucyl tRNA synthetase. Mitochondrial transformation and transmission genetics will be used to evaluate the donor and recipient sequence requirements for GC cluster mobility and the role of in vivo double strand breaks that occur at the borders of those elements. Mutants affecting the mobility of GC clusters will be sought. And finally, we will use fluorescence microscopy and reagents that we have developed to follow in situ the mixing of mitochondria and mtDNAs in zygotes and in heteroplasmic haploid cells and their segregation to emerging buds. Those experiments are designed to establish the rules governing those processes, which must ultimately determine the observed recombination patterns of mtDNA. Mutant screens will be undertaken aimed at defining nuclear genes that participate in mitochondrial segregation.