STRUCTURAL ANALYSIS OF AN EUKARYOTIC CENTROMERE

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Centromeres of eukaryotic chromosomes are specific regions along the chromatin fiber that play a fundamental role in chromosome movement during cell division. Centromere DNA sequences isolated from the yeast, Saccharomyces cerevisiae, enable foreign DNA introduced into yeast to function as ordinary yeast chromosomes during cell division. We will examine how the centromere DNA sequence interacts with chromatin components in the cell nucleus, including nuclear proteins and microtubules, to give rise to a functional kinetochore. We will determine when the kinetochore is assembled during the cell cycle, and how this structure is maintained during cell division. We will examine the biochemical and molecular constitution of the kinetochore by using molecular techniques to detach this complex from the chromatin fiber. The proteins that are associated with this structure in vivo will then be identified. We will raise antibodies against the proteins to determine their intracellular localization, as well as to isolate the protein coding DNA sequence. We will isolate mutants in centromere function as an alternative method to identify gene products required for proper chromosome segregation. The identification of trans-acting factors required for chromosome movement will enable us to determine how these factors interact with the centromere DNA to give rise to a functional unit involved in chromosome locomotion. Knowledge of the factors that control the organization of the centromere may provide fundamental principles governing the molecular mechanism of cell division. Cell division is a central process of living organisms. Changes in the control of cell division may be an important mechanism that results in the production of aberrant cell populations, including cancer cells. Centromeric abnormalities, premature centromere separation and chromosome instability have been found in cases of Roberts'- SC phocomelia, ataxia telangiectasia, and a number of other syndromes. An understanding of the factors controlling cell division may be fundamental for the further understanding of these dysfunctions.