The proposed project's goals are to understand the role of the MPS1- encoded protein kinase in S. cerevisiae spindle pole body (SPB) duplication and the coordination of this event with the cell cycle, 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 mechanism of nondisjunction involved in chromosome imbalances and in the expression of recessive mutations in tumor-suppressing genes. Mutations in the MPS1 gene cause defects in SPB duplication and cell cycle control. MPS1 encodes a protein kinase homolog, and there is preliminary evidence suggesting that the MPS1 gene product has protein kinase activity. The in vivo regulation of the MPS1-encoded kinase will be studied to determine if its activity is regulated in a cell cycle dependent fashion. Mechanisms of regulation will be identified and their importance verified by mutagenesis of the MPS1 gene to observe the consequences of defects in a given regulatory event. Two different genes that suppress mutations in MPS1 have been identified. These genes will be analyzed in the hopes of understanding how they interact with MPS1 and to determine their role in SPB duplication. Other genes that interact with MPS1 will be sought by using the MPS1 gene in a """"""""two-hybrid"""""""" screen to identify yeast genes whose products bind the MPS1 protein, and will also be sought by a synthetic lethal analysis to identify mutations that are inviable in combination with mutations in the MPS1 gene. The """"""""two-hybrid"""""""" screen may identify genes encoding substrates for the kinase, or genes encoding proteins that bind the kinase and control its activity; the synthetic lethal screen may identify genes that interact with MPS1 less directly but act in the same pathway. Molecular analysis of these newly identified genes should indicate how these genes interact with MPS1 and demonstrate if the new genes are involved in SPB duplication. Sequence homologs of MPS1 gene have been identified in mammals. These genes are being tested to determine if they are functional homologs as well. To determine if MPS1 kinase activity is a conserved feature of SPB duplication, a S.pombe homolog is being sought. Once in hand, the role of the S. pombe MPS1 gene in SPB duplication will be determined. A separate tactic is to determine if the MPS1 kinase can function in clam oocyte extracts that assemble centrosomes de novo. The results of these disparate approaches should indicate if MPS1 kinase is widely conserved in the duplication of centrosomes and, with the above analysis in S. cerevisiae, suggest how the kinase activity controls this process.
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