权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Methods to analyze nuclear organizition of mammalian chromosomes in interphase nuclei

分析间期细胞核中哺乳动物染色体核组织的方法

基本信息

批准号：
08554033
负责人：
IKEMURA Toshimichi
金额：
$ 5.5万
依托单位：
NATIONAL INSTITUTE OF GENETICS
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

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.

染色体DNA在细胞核中的有序排列在基因的正常表达、DNA的复制和重组中起着重要的作用，尤其是在减数分裂的细胞核中。非B DNA结构如三链体的形成被认为是决定核中空间组织的分子机制。三链体形成容易发生在聚嘌呤/聚嘧啶序列中，留下单链未配对DNA，其可以与具有显著互补性的其他单链DNA杂交。这将使基因组序列中间隔较远的DNA在间期核中相互结合。间隔较远的DNA也可以形成跨分子三链体，将它们自己组织成有序阵列。为了研究人类和小鼠细胞核中染色体DNA空间组织的机制，我们开发了多色原位结合分析方法，用于具有特定特征的DNA探针，例如在人类有丝分裂和小鼠减数分裂细胞核中形成三链体。该方法类似于用于检测单链RNA的荧光原位杂交，因此能够检测非变性细胞核中的单链DNA。多孔蛋白/多嘧啶序列，如（GA/TC）n，（GAA/TTC）n和那些存在于人类和小鼠基因组中，与不同的探针序列产生不同的信号模式的明确的结合信号。使用由微计算机控制的针对激发光和发射光优化的五对滤光器，用冷却的CCD照相机拍摄5种颜色的荧光而不混合颜色。