NUCLEAR MATRIX STRUCTURES AND GENOMIC FUNCTION

细胞核是包括人类在内的所有真核生物细胞遗传信息的储存库。尽管在定义DNA复制、转录、RNA剪接和加工等主要基因组功能的基本分子特性方面取得了相当大的进展，但直到最近，我们对这些过程如何在功能性细胞核范围内组织和协调的了解仍然非常有限。分子探针的应用以及三维显微镜和计算机图像分析的发展正在迅速纠正这一缺陷，并基于核结构的功能动力学导致了细胞核的新观点。原位标记与三维显微镜和计算机成像技术相结合，揭示了DNA复制和转录位点在细胞核中被组织成更高阶的单位或“区域”。在细胞周期中，复制和转录区域的动态相互作用和“重新分区”可能构成哺乳动物细胞中复制和转录程序复杂的全球协调的结构基础。作为这种功能组织的一个基础，基因组在间期细胞核中排列在离散的染色体区域中。在保持DNA完整的条件下，提取细胞核基质细胞后，这种组织得到了显著的维持。核基质内部成分的适当提取导致染色体区域的相应破坏，同时释放一个离散的蛋白质子集，这些蛋白质是染色体区域锚定蛋白的候选者。在此基础上，我们提出以下具体目标：(1)进一步发展三维显微镜的多维计算机成像方法；(2)阐明s期早期基因复制的DNA复制和转录位点的三维时空特性；(3)研究染色体区域与DNA复制和转录位点的高阶结构域的关系，并表征候选染色体区域锚定蛋白；(4)研究活细胞中DNA复制和转录位点的三维动力学。

The cell nucleus is the repository for the genetic information of all eucaryotic cells including those of man. Despite considerable progress in defining basic molecular properties of the primary genomic functions of DNA replication, transcription, and RNA splicing and processing, our knowledge of how these processes are organized and coordinated within the confines of the functional cell nucleus has been until recently very limited. Impressive progress in applying molecular probes along with developments in 3-D microscopy and computer image analysis is rapidly correcting this deficiently and is leading to a new view of the cell nucleus based on the functional dynamics of nuclear architecture. In situ labeling coupled with three dimensional microscopy and computer imaging techniques reveal that DNA replication and transcription sites are organized into higher order units or "zones" in the cell nucleus. The dynamic interplay and "re-zoning" of replication and transcription regions during the cell cycle may form the structural basis for the elaborate global coordination of replicational and transcriptional programs in the mammalian cell. As one basis for this functional organization, the genome is arranged in discrete chromosome territories in the interphase nucleus. This organization is strikingly maintained following extraction of cells for nuclear matrix under conditions which maintained the DNA intact. Appropriate extraction of the nuclear matrix internal components leads to a corresponding disruption of chromosome territories along with the release of a discrete subset of proteins that are candidates for being chromosome territory anchoring proteins. With these previous studies as a basis we propose the following specific aims: (1) Further develop multi-dimensional computer imaging approaches to 3-D microscopy; (2) Elucidate the 3-D spatio-temporal properties of DNA replication and transcription sites for genes replicating in early S-phase; (3) Study chromosome territories in relations to higher order domains of DNA replication and transcription sites and characterize candidate chromosome territory anchor proteins; (4) Investigate the 3-D dynamics of DNA replication and transcription sites in living cells.