Molecular mecbanism for determination ofdynamics and positioning in nucleus of vertebrates

单链形成和跨分子三链体形成被认为使基因组中沿着分布的序列能够相互关联并将核DNA组织成有序的构型。三链体形成的DNA在人类间期核进行了分析相结合的免疫检测抗三链体抗体和荧光原位“非变性“杂交采用聚嘌呤/聚嘧啶（PuPy）道探针。用于检测RNA的“非变性”杂交技术可以检测非变性核中的单链DNA。三链体可视化差异与不同的蛹道探针在空间上与着丝粒序列在间期核中的序列特异性的方式。因此，我们对中心点的形成和功能感兴趣。为了确定着丝粒蛋白的精确功能，我们通过在鸡DT 40细胞系中产生条件性功能丧失突变体来研究它们的作用。我们的结论是CENP-I和CENP-H都是CENP-C定位所必需的，而CENP-A则不是。我们还对表达GFP或YFP标记的着丝粒蛋白的细胞进行了活细胞分析，以观察染色体的动态。

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.