In higher eukaryotic cells, DNA presents as a chromatin fiber, a complex with various kinds of proteins, and chromatin fibers are highly condensed to form metaphase chromosomes during mitosis. Formation of metaphase chromosome structure is essential for equal segregation of replicated chromosomal DNA into the daughter cells. So far, information on the higher order structure of metaphase chromosome remains to be limited. Thus, we have carried out proteome analysis of isolated human metaphase chromosomes, provided proteinous view of the chromosomes (Uchiyama et al., J. Biol. Chem., 2005). In this study, in order to identify proteins involved in formation and/or maintenance of metaphase chromosome, the critical points of NaCl concentration of the solution at which the morphology of the isolated metaphase chromosomes dramatically changes has been determined and the proteins dissociated from the chromosomes at that points were identified. Under 0.4 M NaCl condition, the size of the chromosomes became significantly larger than those in the lower concentrations, with DNA haloes appearing around the chromosomes, and the separation of each sister chromatid at the arm region. The dissociated proteins were identified using ESI-LC MS/MS and MALDI-TOF MS, followed by classification into four groups based on their functional information and localization. Some of the identified proteins whose functions on mitotic chromosomes have not reported are now being analyzed for characterization.
