POSITIONAL CLONING OF THE FR LOCUS

描述：本提案涉及核线粒体的主题 交互. 核基因组如何维持正常的 线粒体功能在高等真核生物的解决。 特别是， 该提案侧重于可能的机制， 线粒体的分离和传递 人口，以及那些排除线粒体积累的人， 突变。 正在研究的系统是一个有趣的核- 菜豆线粒体的相互作用 线粒体突变 是一种细胞质雄性不育（CMS）突变， 植物不能产生有活力的花粉。 菜豆CMS性状 与线粒体基因组中的插入有关， 产生新的开放阅读帧，指定为PVS-ORF 239。 的 线粒体重排与新的 蛋白质是植物CMS系统中的常见主题。 但是，Bean CMS 系统具有一些独特的功能。 pvs蛋白的表达 在花粉母细胞的发育过程中， 这种异常的蛋白质并不像预期的那样存在于线粒体中，而是存在于 在发育中的胼胝质层中的细胞外围。 这难道 干扰胞质分裂事件，导致生产 正常的花粉粒 在含有该酶的菌株中， 由几种核恢复因子之一的作用引起的线粒体突变 基因. 其中一个基因，Fr基因座， 提议 Fr恢复不育系的育性， 从而实现线粒体突变的定向消除。 到 阐明Fr作用的分子基础，该提案概述了 一系列旨在定位克隆Fr. Physical 定位基因座，构建大插入文库， 从紧密连锁的标记中进行染色体步移， 概述了来自推定的Fr基因的cDNA克隆。

DESCRIPTION: This proposal deals with the topic of nuclear-mitochondrial interactions. The question of how the nuclear genome maintains normal mitochondrial function in higher eukaryotes is addressed. In particular, the proposal focuses on the possible mechanisms that regulate segregation and transmission of mitochondria in a heteroplasmic population, and those that preclude the accumulation of mitochondrial mutations. The system under study is an interesting nuclear- mitochondrial interaction in common bean. The mitochondrial mutation is a cytoplasmic male sterility (CMS) mutation that results in the inability of the plant to produce viable pollen. The CMS trait in bean is associated with an insertion in the mitochondrial genome which generates a novel open reading frame designated pvs-orf239. The association of a mitochondrial rearrangement and production of novel proteins are a common theme in plant CMS systems. However, the bean CMS system has some unique features. The expression of the pvs protein during development of the pollen meiocytes leads to the accumulation of this aberrant protein not in mitochondria as expected but at the periphery of the cell in the developing callose layer. This presumably interferes with the cytokinesis events that lead to the production of normal pollen grains. Fertility is restored in strains containing the mitochondrial mutation by the action of one of several nuclear restorer genes. One of these genes, the Fr locus, is the focus of this proposal. Fr restores fertility to the CMS strain apparently by effecting the directed elimination of the mitochondrial mutation. To elucidate the molecular basis of Fr action, the proposal outlines a series of experiments aimed at the positional cloning of Fr. Physical mapping of the locus, the construction of a large-insert library for use in chromosome walking from tightly linked markers, and the isolation of cDNA clones derived from the putative Fr gene are outlined.