Methods to analyze nuclear organizition of mammalian chromosomes in interphase nuclei

染色体DNA有序排列在细胞核的特定结构域中，在基因表达和DNA复制以及重组中起着重要作用，特别是在减数分裂细胞核中。非b DNA结构（如三联体）的形成被认为是决定细胞核空间组织的重要分子机制。三联体的形成很容易发生在聚苯胺/聚嘧啶序列中，留下单链未配对的dna，可以与其他具有显著互补性的单链dna杂交。这将使基因组序列中间隔较远的dna在间期细胞核中相互结合。间隔较远的dna也可以形成跨分子三联体，将自己组织成有序的阵列。为了研究控制人类和小鼠细胞核中染色体DNA空间组织的机制，我们开发了具有特定特征的DNA探针的多色原位结合测定方法，例如在人类有丝分裂和小鼠减数分裂细胞核中形成三联体。该方法类似于用于检测单链rna的荧光原位杂交，因此能够检测未变性细胞核中的单链dna。Polyporine/ polypy嘧啶序列，如（GA/TC）n, (GAA/TTC)n，以及人类和小鼠基因组中存在的Polyporine/ polypy嘧啶序列，在不同的探针序列下给出明确的结合信号，导致不同的信号模式。利用微机控制的5对优化的激发和发射光滤光片，在不与冷却CCD相机混合的情况下，拍摄了5种不同颜色的荧光。

Ordered arrangement of chromosomal DNAs into specific domains in nuclei has been proposed to fulfilll important roles in proper gene expression and DNA replication, as well as recombination especially in meiotic nuclei. Formation of non-B DNA structures such as triplex is thought important as a molecular mechanism determining spatial organization in nuclei. Triplex formation occurs readily in polypouine/polypyrimidine sequences leaving single-strand unpaired DNAs that can hybridize with other single-strand DNAs with significant complementarity. This will enable distantly spaced DNAs in genome sequences to associate with each other in interphase nuclei. Distantly spaced DNAs may also form transmolecular triplexes organizing themselves into an ordered array. To investigate mechanisms governing spatial organization of chromosomal DNAs in human and mouse nuclei, we develop multi-colors in situ binding assay methods for DNA probes with specific characteristics such as triplex formation, both in human mitotic and mouse meiotic nuclei. The method resembled fluorescence in situ hybridization used for detecting single-strand RNAs, thus was able to detect single-strand DNAs in nondenatured nuclei. Polyporine/polypyrimidine sequences, such as (GA/TC)n, (GAA/TTC)n and those present in human and mouse genomes, gave clear binding signals with different probe sequences resulting in different signal patterns. Using five pairs of filters optimized for excitation and emission lights that were controlled by a microcomputer, 5 colors of fluorescences were taken without mixing the colors with a cooled CCD camera.