Errors in chromosome segregation are a major cause of the genetic instability of tumor cells and birth defects. These errors are normally prevented by a checkpoint that keeps cells from initiating chromosome segregation until the mitotic or meiotic spindle has been correctly assembled. The ultimate goal of the proposed work is a molecular explanation of the spindle assembly checkpoint. This system must contain sensing mechanisms that detect defects in the structure of the spindle and a signal transduction pathway that relays information from these sensors to prevent the activation of the biochemical machinery that normally initiates chromosome segregation and cell division. This application proposes genetic and biochemical studies of the spindle assembly checkpoint. The genetic approach uses budding yeast mad (mitotic arrest deficient) mutations that can no longer arrest in mitosis when their spindle is disrupted. The biochemical approach uses frog egg extracts that recapitulate the cell cycle in vitro, and which can be arrested in mitosis by depolymerizing the spindle. Experiments are proposed to: 1) Continue the characterization of the MAD genes by determining the function of the MAD1-MAD3 genes, and isolating and characterizing the MAD4-MAD7 genes. 2) Investigate the MAD-dependent signal transduction pathway by isolating and characterizing dominant gain of function alleles of the MAD genes and suppressors of the mad mutants, by using biochemical and genetic methods to detect interactions between the Mad proteins, and by determining the interactions between the Mad proteins and the machinery that destroys cyclin at the end of mitosis. 3) Determine how the MAD-dependent checkpoint monitors the structure of the spindle by using a potent genetic selection to isolate recessive temperature sensitive mutations that arrest cells in mitosis, and analyzing the interactions of these and existing mitotic mutants with the mad mutations. 4) Use frog egg extracts to determine the components of the spindle that are needed to activate the spindle assembly checkpoint and determine how these lead to the activation of MAP kinase, and how active MAP kinase produces a mitotic arrest. 5) Clone vertebrate homologs of the budding yeast MAD genes and use these and biochemical activities characteristic of mitotically arrested cells to analyze human tumor cell lines for defects in the spindle assembly checkpoint.
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