This proposal aims at further understanding the molecular composition, the higher-order structure and the biological function of the budding yeast kinetochore have been cloned and several protein components have been identified. However, much less is known about its higher-order structure and the mechanisms of its functions. In this proposal, two in vitro assays have been designed: 1. By a cross-linking/PCR, specific protein/DNA interaction can be detected. This assay will be applied to the study of the kinetochore and the cell cycle to study the role of the DNA and protein elements in the assembly of the kinetochore and the cell cycle regulation of the kinetochore assembly, to define its higher-order structure and to confirm the localization of putative kinetochore proteins. 2. By the combination of the yeast chromosome tagging, minichromosome isolation and an in vitro chromosome/microtubule interaction assay techniques, the biochemical activities of the yeast kinetochore on a minichromosome can me measured in vitro. This assay can be applied to the study of the kinetochore functions, specifically, the kinetics of the kinetochore functions, specifically, the kinetics of the kinetochore/microtubule interaction, the cell cycle regulation of its activities and the contribution of specific DNA and protein elements to the kinetochore activities.
