LARGE SCALE CHROMATIN STRUCTURE AND FUNCTION

在高等真核染色体中，DNA折叠成多个 组织层次。最高水平的染色质折叠， 由30 nm染色质纤维的折叠组成，占高达 250：1的包装比例，并涉及整个转录的组织 和复制功能结构域及其在有丝分裂和 间期染色单体正是这种大规模的染色质结构 我们研究的重点。我们的长期目标是确定 大规模染色质结构的基本折叠基序， 染色体结构，和染色质结构的关系， 这一层次的组织，以调节DNA功能，包括 转录和复制。 我们的具体目标是回答以下问题：l）早做 有丝分裂染色体浓缩（解浓缩）事件对应于 a染色丝纤维的折叠（展开）; 2）什么是压实 染色丝纤维的比率;染色质折叠基序是什么 染色丝纤维的结构3)什么是大规模的可重复性- 规模染色质折叠？4)大规模染色质的变化 组织伴随DNA复制和转录？5)是 大规模染色质限制了反式因子的可及性 组织？ 最近发展的一种原位定位的方法， 使用lac操纵子-阻遏物结合的特定染色体区域 提供了极好的超微结构保存，并将使我们能够 通过光学显微镜观察活细胞中的特定染色体区域 并且使用电子显微镜以高分辨率进行观察。 褶皱分析 大规模染色质组织的基础基序将利用三个- 间期染色体结构的三维重建. 利用光学切片光产生的重叠分辨率 显微镜，电子显微镜断层摄影术，连续切片电子 显微镜，以及连续半厚连续切片和 电子显微断层摄影术 这项拟议的研究将提供一个基本的描述折叠 大规模染色质和染色体组织的基础图案。 我们今后的工作方向将集中在结构和机制 转录和大规模染色质组织之间的联系， 特别是在染色质结构域的水平上， 长度

Within the higher eukaryotic chromosome, DNA is folded into multiple levels of organization. The highest levels of chromatin folding, consisting of the folding of 30 nm chromatin fibers, account for up to a 250:1 packing ratio and involve the organization of entire transcription and replication functional domains and their compaction within mitotic and interphase chromatids. It is this large-scale chromatin structure which is the focus of our research. Our long term objectives are to determine the basic folding motifs underlying large-scale chromatin structure and chromosome architecture, and the relationship of chromatin structure at this level of organization to regulation of DNA function, including transcription and replication. Our specific aims are to answer the following questions: l) Do early events in mitotic chromosome condensation (decondensation ) correspond to a folding (unfolding) of chromonema fibers; 2) What is the compaction ratio for chromonema fibers; what chromatin folding motifs underlie the structure of chromonema fibers? 3) What is the reproducibility of large- scale chromatin folding? 4) What changes in large-scale chromatin organization accompany DNA replication and transcription? 5) Is accessibility of trans factors limited by large-scale chromatin organization? A recently developed method for in situ localization of specific chromosome regions using lac operator - repressor binding provides excellent ultrastructural preservation and will allow us to visualize specific chromosome regions in living cells by light microscopy and at high resolution using electron microscopy. Analysis of folding motifs underlying large-scale chromatin organization will exploit three- dimensional reconstructions of interphase chromosome structure at overlapping resolution generated using optical sectioning light microscopy, electron microscopy tomography, serial sectioning electron microscopy, and a combination of serial semi-thick serial sections and electron microscopy tomography. This proposed research will provide a basic description of the folding motifs underlying large-scale chromatin and chromosome organization. Future directions of our work will focus on the structural and mechanistic links between transcription and large-scale chromatin organization, particularly at the level of chromatin domains, tens to hundreds of kb in length.