POSITIONAL CLONING OF THE FR LOCUS

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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.