Molecular mecbanism for determination ofdynamics and positioning in nucleus of vertebrates

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The single-strand formation and transmolecular triplex formation are thought to enable sequences spaced distantly along the genome to associate with each other and organize nuclear DNA into ordered configurations. Triplex-forming DNAs in the human interphase nucleus were analyzed by combining immunodetection by antitriplex antibodies and fluorescence in situ "nondenaturing " hybridization employing polypurine/polypyrimidine (PuPy) -tract probes. The "nondenaturing" hybridization technique, which has been used to detect RNA, could detect single-stranded DNAs in nondenatured nuclei. Triplexes visualized differentially with distinct PuPy-tract probes were associated spatially with centromere sequences in the interphase nucleus in a sequence-specific manner. Therefore, we were interested in centromore formation and function. To determine the precise functions of centromere proteins, we examined their roles by generating a conditional loss-of-function mutant in the chicken DT40 cell line. We concluded that both CENP-I and CENP-H are necessary for localization of CENP-C but not CENP-A to the centromere. We also performed living cell analysis of cells expressing GFP-or YFP- tagged centromere proteins to observe the dynamics of chromosomes.