ORGANIZATION, EXPRESSION, AND INHERITANCE OF YEAST MTDNA

拟议研究的长期目标是：1)了解 基因内含子4编码的新蛋白(P31)的双功能特性 酵母线粒体COXr基因(AI4pha)，既有DNA内切酶又有DNA内切酶 内含子定位和潜在成熟酶(剪接)所需的活性 2)了解酵母细胞是如何组织和调节 线粒体DNA(MtDNA)的拷贝数，从而将mtDNA传递给 子代细胞，并确保呼吸正常状态。在上一次 在授权期内，我们确定了p3l的不同临界域 其核酸内切酶活性和成熟酶功能所必需的。一个 当前提案目标一的关键要素是检验假设 激活p3l的潜在成熟Se活性需要一个 与另一种双功能蛋白Nam2p的高效相互作用 通过核NAM2基因，它既是线粒体的亮氨酸tRNA 合成酶和一个剪接因子。这些对p3l和nam2p的研究旨在 在蛋白质依赖的AI4pha剪接的体外重建中。 第二个目标将利用这一发现--发现类HMO线粒体 蛋白质Abf2p是维持野生型(p+)mtDNA所必需的 并且似乎参与了线粒体DNA的复制控制。我们将描述 Abf2零突变体和意外多拷贝的自发抑制子 抑制基因ILV5，我们发现它编码线粒体 催化Ile、Leu和Ile生物合成的一步的蛋白质(AHAR) 瓦尔。我们将检验AHAR是一种双功能蛋白质的假设 合成支链氨基酸和线粒体DNA所必需的 稳定性。最后，我们将进行系统的生化和遗传学研究 分析与线粒体DNA类核相关的蛋白质以关联 线粒体DNA-蛋白质复合体的组织特性与线粒体DNA的维持。 这些研究的最终目的是了解 线粒体DNA分离和拷贝数控制，并提供对 可能是线粒体DNA分离装置。细胞如何传输的问题和 控制线粒体DNA的数量是基本的，几乎适用于所有 真核细胞，但对此知之甚少。现在很明显，有一个数字 遗传性人类疾病涉及分离和复制的各个方面 突变型和野生型线粒体DNA的数量。我们已经开发出一种易驾驭的 用来研究这些重要问题的实验系统。

The long term goals of the proposed research are 1) to understand the bifunctional nature of a novel protein (p3l) encoded by intron 4 of the yeast mitochondrial COXr gene (aI4alpha) that has both a DNA endonuclease activity required for intron homing and a latent maturase (splicing) activity; and 2) to understand how yeast cells organize and regulate the copy number of their mitochondrial DNA (mtDNA) so as to transmit mtDNA to progeny cells and insure a respiratory competent state. In the previous grant period we identified separate critical domains of p3l that are required for its endonuclease activity and for its maturase function. A key element of goal I of the current proposal is to test the hypothesis that activation of the latent maturaSe activity of p3l requires a productive interaction with another bifunctional protein, Nam2p, encoded by the nuclear nam2 gene that is both a mitochondrial leucyl tRNA synthetase and a splicing factor. These studies of p3l and Nam2p are aimed at an in vitro reconstitution of protein-dependent splicing of aI4alpha. The second goal will exploit the -finding that the HMO-like mitochondrial protein, Abf2p, is required for the maintenance of wild-type (p+) mtDNA and appears to be involved in mtDNA copy control. We will characterize spontaneous suppressors of an abf2 null mutant and an unexpected multicopy suppressor gene, ILV5, which we identified that encodes a mitochondrial protein (AHAR) that catalyzes a step in the biosynthesis of Ile, Leu and Val. We will test the hypothesis that AHAR is a bifunctional protein required for the synthesis of branched chain amino acids and for mtDNA stability. Finally, we will carry out a systematic biochemical and genetic analysis of the proteins associated with mtDNA nucleoids to correlate the organizational properties mtDNA-protein complexes with mtDNA maintenance. The ultimate objective of these studies is to understand the mechanisms of mtDNA segregation and copy number control and to provide insight into the putative mtDNA segregation apparatus. The issue of how cells transmit and control the amount of their mtDNA is fundamental and applies to almost all eukaryotic cells but is poorly understood. It is now clear that a number of inherited human disorders involve aspects of the segregation and copy number of mutant and wild-type mtDNAs. We have developed a tractable experimental system in which to investigate these important issues.