LARGE SCALE CHROMATIN STRUCTURE AND FUNCTION

在高等真核生物的染色体中，dna折叠成多个 组织层级。染色质折叠的最高水平， 由30纳米染色质纤维的折叠组成，占最大 250：1包装比例，涉及整个转录的组织 和复制功能域及其在有丝分裂和有丝分裂中的紧凑 间期染色单体。正是这种大规模的染色质结构 这是我们研究的重点。我们的长期目标是确定 大范围染色质结构和基本折叠基序 染色体构型及其与染色质结构的关系 这一级别的组织对DNA功能的调节，包括 转录和复制。 我们的具体目的是回答以下问题：L)早做 有丝分裂染色体凝聚(解凝集)中的事件对应于 染色质纤维的折叠(展开)；2)紧密程度是什么 染色质纤维的比率；什么染色质折叠基序隐藏在 染色单体纤维的结构？3)大纤维的重现性是什么？ 大规模染色质折叠？4)大规模染色质有什么变化 伴随DNA复制和转录的组织是什么？ 受大规模染色质限制的反式因子的可及性 组织？一种新开发的原位定位方法 利用Lac操纵子-抑制子结合的特定染色体区域 提供极佳的超微结构保存，并将使我们能够 用光学显微镜观察活细胞中的特定染色体区域 并在高分辨率下使用电子显微镜。关于折叠的分析 大规模染色质组织的潜在主题将利用三个- 间期染色体结构的空间重构 利用光学分割光产生的重叠分辨率 显微镜、电子显微镜层析成像、连续切片电子 显微镜，以及连续的半厚连续切片和 电子显微镜断层摄影术。 这项拟议的研究将提供折叠的基本描述 大范围染色质和染色体组织的潜在主题。 我们今后的工作方向将集中在结构和机械方面 转录和大规模染色质组织之间的联系， 尤其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.