Accurate chromosome segregation is essential for the faithful transmission of genetic information. The linkage between sister chromatids directs them to attach to opposite poles of the mitotic spindle, and the prompt dissolution of this linkage allows chromosomes to segregate to the poles at anaphase. Mitotic chromosome movements depend on kinetochores, which also act as signaling centers to delay anaphase until all the chromosomes have been correctly aligned on the spindle. This proposal describes experiments to investigate kinetochore assembly and function, the nature of the linkage between sister chromatids, and the mechanisms that regulate this linkage during the eukaryotic cell cycle. Frog egg extracts will be used to investigate how the Xenopus homolog of the conserved CENP-A, B, and C proteins contribute to the assembly and function of the kinetochore. Antibodies to CENP-A will be used to enrich kinetochore-containing chromosome fragments, making it possible to screen large pools ol expressed cDNAs for novel kinetochore-binding proteins. Sister chromatid separation will be studied in budding yeast using green fluorescent protein (GFP) marked chromosomes. This technique will be used to, 1) investigate the length, DNA sequence, and topological features of chromosomes that are needed to establish and maintain sister chromatid linkage, 2) test the role of proximity in establishing the linkage between sisters, and 3) investigate the relationship between topological and cohesin-dependent mechanisms of sister linkage. DNA microarrays will be used to determine why topoisomerase II activity is required during anaphase to resolve sister chromatid linkage. These experiments will combine the genetic tractability of yeast and the complex in vitro reactions that are possible in frog extracts in an integrated approach to understanding mitotic chromosome behavior. This information will be directly relevant to understanding how aneuploidy is generated in Down syndrome and tumor progression.