The proposed project's goals are to understand the role of the MPS1 protein kinase in S. cerevisiae spindle pole body (SPB) duplication and in the spindle assembly checkpoint, and to determine if this kinase is widely conserved in the control of centrosome duplication. The formation of mitotic and meiotic spindles responsible for chromosome transmission is dependent upon proper centrosome duplication and function. Understanding centrosome duplication will be pertinent to dissecting the mechanisms of nondisjunction involved in chromosome imbalance and in the expression of recessive mutations in tumor- suppressing genes. Mutations in the MPS1 gene cause defects in SPB duplication and the spindle assembly checkpoint pathway. The investigator's are now able to monitor Mps1p (epitope-tagged) protein kinase activity isolated from yeast at endogenous levels. He will use this reagent to study how the kinase activity of Mps1p is regulated during the cell cycle. Mutagenesis to separate the two functions of Mps1p and the analysis of Mps1p phosphorylation will be performed with the goal of understanding how Mps1p executes its two roles during the cell cycle. To identify genes whose products interact with Mps1p, they plan to identify suppressors of the distinct dosage phenotypes of overexpressing either active or catalytically inactive MPS1. These will be analyzed to determine how they interact with MPS1 and to determine their role in SPB duplication or the spindle assembly checkpoint. The investigator's have identified mutations that are inviable in combination with mutations in the MPS1 gene. One he will continue to study is the novel, essential MOE1 gene that appears to be required for normal spindle function. They also will isolate the genes corresponding to several other synthetic lethal mutations they have identified. Molecular analysis of these newly identified genes should indicate how the encoded gene product interacts with Mps1p, and demonstrate if the new genes is involved in SPB duplication or the spindle checkpoint. Sequence homologues of MPS1 have been identified in various organisms. In mice a potential homologue is encoded by the esk gene. They propose to study the expression and activity of esk in mouse embryonic fibroblasts. He also will overexpress both active and inactive esk in mouse cells and determine if the cells exhibit phenotypes indicative of defects in centrosome duplication or the spindle assembly checkpoint. The results should indicate if the MPS1 kinase encodes a conserved function in either of its known roles.
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