YEAST MITOCHONDRIAL DNA RECOMBINATION

酵母线粒体 DNA 重组

• 批准号: 2177934
• 负责人: RONALD A BUTOW
• 金额: $ 37.35万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-05 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177934
• 关键词: DNA damage; RNA splicing; Saccharomyces cerevisiae; antibody; cell transformation; cytochrome oxidase; endonuclease; enzyme mechanism; fluorescence microscopy; fluorescent dye /probe; fungal genetics; genetic recombination; laboratory mouse; laboratory rabbit; ligase; microorganism genetics; mitochondrial DNA; molecular genetics; mutant; nucleic acid hybridization; nucleic acid probes; open reading frames; protein reconstitution; purine nucleotides; site directed mutagenesis; western blottings

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.

拟议研究的长期目标是在分子和 细胞术语是重组和遗传的基础过程 酵母线粒体基因组的行为。 将特别强调 放在分析新的重组事件和鉴定 以及参与这些过程的关键组件的特性。 目前的建议集中在三个主要的调查领域： 参与I组内含子内含子移动和剪接的蛋白，2）移动的 富含GC的回文酶（GC簇）和3）线粒体的细胞生物学 遗传学 移动的内含子的研究集中在详细的生物化学和 一种由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4 α的阅读框 基因 aI 4 α是酵母中两个已知的移动的内含子之一 线粒体基因组 它的ORF产物既是一种核酸内切酶， 内含子迁移率和RNA剪接所需的潜伏成熟酶。 我们将 表征纯化的aI 4 α编码的 核酸内切酶I-SceII与DNA结合，并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， 确定核酸内切酶所需蛋白质的重要结构域， 成熟的活动。 我们将检验核酸内切酶和 成熟酶活性共享蛋白质的结构域。 我们将测试一个模型 潜在成熟酶活性的激活需要一种相互作用， aI 4 α编码的蛋白质和突变形式的核- 编码线粒体亮氨酰tRNA合成酶。 线粒体转化 和传递遗传学将用于评估供体和受体 GC簇迁移率的序列要求和体内 发生在这些元件边界的双链断裂。 突变体 将寻求影响GC簇的流动性的方法。 最后，我们将 使用荧光显微镜和我们开发的试剂， 线粒体和mtDNA在受精卵和胚胎中的原位混合 异质单倍体细胞和它们分离成出芽。 那些 设计实验以建立管理这些过程的规则， 这最终必须决定观察到的重组模式， 线粒体DNA 将进行突变筛选，目的是确定核基因 参与线粒体分离。