Molecular mecbanism for determination ofdynamics and positioning in nucleus of vertebrates

单链形成和跨分子三链形成被认为能够使沿基因组相距较远的序列相互关联，并将核DNA组织成有序的构型。用抗三链抗体的免疫检测和多嘌呤/多嘧啶(PuPy)探针的荧光原位“非变性”杂交相结合的方法分析了人间期核中形成三链的DNA。已被用于检测RNA的“非变性”杂交技术，可以在未变性的细胞核中检测到单链DNA。用不同的PuPy-Track探针差异显示的三联体与间期核中的着丝粒序列以序列特异性的方式在空间上相关。因此，我们对中心区的形成和功能很感兴趣。为了确定着丝粒蛋白的精确功能，我们通过在鸡DT40细胞系中产生一个条件功能丧失突变体来检查它们的作用。我们的结论是CENP-I和CENP-H都是CENP-C定位所必需的，但CENP-A不是定位于着丝粒的必要条件。我们还对表达GFP或YFP标记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.