The goal of this project is to study the composition and assembly of the vertebrate kinetochore, and to determine the role of individual kinetochore proteins in chromosome segregation. Many of the studies will be performed with Xenopus egg extracts in conjunction with chromatin beads. The beads, magnetic spheres coupled to plasmid DNA, serve as """"""""artificial chromosomes"""""""" in extracts to mediate formation of a bipolar spindle. First, several known kinetochore proteins will be coupled to chromatin beads and incubated in extracts, in order to nucleate assembly of kinetochore complexes. Proteins which bind selectively to beads will be isolated and any novel proteins cloned. The beads will be assayed, in vitro, for the formation of functional kinetochores. Additionally, centromeric DNA will be coupled to beads to determine the precise DNA sequences that are required for recruitment of kinetochore proteins. The second objective is to study the coordination of kinetochore assembly relative to DNA replication and spindle formation. Kinetochore proteins will be fused to green fluorescent protein and their localization monitored in egg extracts and Xenopus tissue culture cells. The final objective is to immunodeplete individual kinetochore proteins from egg extracts containing sperm nuclei in order to determine the role of the proteins in chromosome segregation and spindle formation. Disruption of spindle microtubules preferentially kills many abnormally dividing cells, and therefore anti- mitotic drugs are widely used in the treatment of cancer. Thus, a molecular understanding of spindle assembly and function should lead to a better understanding of cancer and the development of new anti-cancer therapies.
