Organization, Expression, and Inheritance of Yeast mtDNA

描述(由申请人提供)： 这项研究的总体目标是以发芽酵母酿酒酵母为模式生物，了解遗传油线粒体DNA(MtDNA)的机制。我们的研究将结合遗传、生化、分子和细胞生物学方法来表征在营养生长过程中有助于线粒体DNA忠实传递的各种成分。为此，我们提出了以下具体目标：1)探索线粒体DNA类核的结构和组织。目的是了解类核蛋白如何与线粒体DNA相关，以及它们如何对线粒体DNA的组织和遗传做出贡献；2)阐明线粒体DNA分离装置的组成部分。我们将使用各种生化和细胞生物学方法来检测mtDNA类核蛋白和某些突变形式的类核蛋白Ilv5p是如何与线粒体内膜以及可能成为mtDNA分离装置组成部分的候选蛋白相互作用的；3)阐明RTG逆行反应途径在mtDNA稳定性和遗传中的作用。我们发现，这一途径通过控制代谢类核蛋白与线粒体DNA遗传密切相关。我们将评估逆行途径如何控制mtDNA的稳定性和遗传，并绕过主要mtDNA包装蛋白Abf2p的要求；4)建立mtDNA类核分裂的规则，以及Hsp60、活性ORI序列和mtDNA转录在类核分裂中的作用。这些目标源于我们的发现，即类核分裂是一个受调控的过程。我们建议同时开发异步和同步类核分裂系统。这些研究将与区分活性和非活性ORI元件的顺式作用元件的分析相结合。总而言之，这些研究对了解人类线粒体疾病具有直接意义。

DESCRIPTION (provided by applicant): The overall objective of the proposed research is to understand the mechanism of inheritance oil mitochondrial DMA (mtDNA) using the budding yeast, Saccharomyces cerevisiae, as a model organism. Our studies will combine genetic, biochemical, molecular and cell biological approaches to characterize the various components that contribute to the faithful transmission of mtDNA during vegetative growth. To these ends, we propose the following specific aims: 1) to probe the structure and organization of mtDNA nucleoids. The goal is to understand how the nucleoid proteins are associated with mtDNA, and how they contribute to mtDNA organization and inheritance; 2) to elucidate components of the mtDNA segregation apparatus. We will use a variety of biochemical and cell biological methods to examine how mtDNA nucleoids and certain mutant forms of the nucleoid protein, Ilv5p, which behaves according to all criteria tested like mtDNA nucleoids, interact with the inner mitochondrial .membrane and with candidate proteins that are likely to be components of the mtDNA segregation apparatus; 3) to elucidate the role of the RTG-retrograde response pathway in mtDNA stability and inheritance. We have found that this pathway is intimately involved in mtDNA inheritance through its control of 'metabolic' nucleoid proteins. We will evaluate how the retrograde pathway controls the stability and inheritance of mtDNA, and circumvents the requirement for the major mtDNA packaging protein, Abf2p; 4) to establish the rules of mtDNA nucleoid division and the roles of Hsp60, active ori sequences and transcription of mtDNA in nucleoid division. These objectives follow from our discovery that nucleoid division is a regulated process. We propose to develop both asynchronous and synchronous nucleoid division systems. These studies will be coupled with the analysis of cis-acting elements that distinguish active from inactive ori elements. Altogether, these studies have direct implications for understanding mitochondrial diseases in humans.