The yeast spindle plaque and associated microtubules mediate the fusion of nuclei during mating. By identifying the genes and gene products responsible for nuclear fusion we aim to identify the components of the spindle plaque and the mechanism of its function. The spindle plaque is of wide interest as it is the sole organizer of microtubules in the yeast cell. Little is known about the mechanism of function of any microtubule organizer. Therefore understanding of the mechanism of nuclear fusion is likely to be important to the understanding of a broad set of phenomena common to all eukaryotic cells such as organelle movement, membrane and organelle fusion, and the regulation and function of microtubules in mitosis and meiosis. Yeast is an organism which is easily manipulated genetically, which should allow rapid analysis of these very complex processes. We will address the following specific experimental aims: 1) The full spectrum of functions on one nuclear fusion gene, KARI, in mitosis, nuclear fusion and sporulation, will be determined by a combination of the genetic analysis of mutations in the gene and the subcellular localization of the gene product. Genes that interact with the KARI gene will be identified by mutations that suppress different KARI mutations. 2) The mechanism of nuclear fusion will be examined by the development of an in vitro system for nuclear fusion. The proteins that are required for nuclear fusion will be characterized in the in vitro nuclear fusion assay. 3) The pathway of nuclear fusion will be dissected by the isolation of mutations in the other genes that are required for nuclear fusion. The functions of the different genes will be ordered by reciprocal shift experiments and cytology of the different mutants. Nuclear fusion genes will be isolated and analyzed by in vitro mutagenesis and gene replacement to determine their role in the mitotic functions of the spindle plaque.
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