Chromatin Structure

这个提议解决了染色质如何 组织在真核细胞核中，通过检查一种新的 假说. 这一假说是，与蛋白质相关的蛋白质， 端粒，或染色体末端（端粒蛋白，或TP） 是核纤层，它们又是一大群 中间丝蛋白（IF）。 的能力 组装和分解成细丝， 维持核组织。 使用的微生物 在这些研究中的是下毛纤毛虫广口游仆虫， 这是特别有利的，因为 大核中单个染色体元件的长度， 导致大约1亿个携带端粒的DNA 片段，其中端粒DNA序列都是相同的。 此外，大核包含明确定义的区域， 浓缩、分散和复制的染色质。 越高 秩序的组织，决定了每个人的结构， 这些功能区是未知的。 这些特性使 游仆虫是研究端粒的极好系统， 真核生物核结构和功能。 实验 本研究的建议涉及TP的表征， 克隆该基因并测定其序列。 述序列将 与已知的IF序列进行比较。 免疫荧光 定位和STEM质量测定将用于 确定TP核位点和与DNA的化学计量。 原位 将使用电子显微镜分析和免疫定位 以表征10 nm的细丝。 复溶实验 以确定影响染色质的因素 organization. 预计这项研究将阐明其中一项职能， 端粒和核纤层蛋白在组织染色质中的作用 真核细胞核中的结构。 因此，该项目解决了 细胞生物学的核心问题之一，即， 细胞核内基因组的结构组织。

This proposal addresses the question of how chromatin is organized in the eukaryotic nucleus, by examining a novel hypothesis. The hypothesis is that proteins associated with the telomeres, or ends, of chromosomes (telomeric proteins, or TP) are nuclear lamins, which in turn are members of a large group of proteins called intermediate filaments (IF). Their ability to assemble and disassemble into filaments would then be responsible for maintenance of nuclear organization. The organism to be used in these studies is the hypotrichous ciliate Euplotes eurystomus, which is particularly advantageous because of the unusually short lengths of individual chromosomal elements in the macronuclei, resulting in approximately 100,000,000 telomere-bearing DNA pieces, in which the telomeric DNA sequences are all the same. Furthermore, the macronucleus contains clearly defined regions of condensed, dispersed, and replicating chromatin. The higher order organization which determines the structure of each of these functional regions is not known. These properties make Euplotes an excellent system for the study of telomeres and eukaryotic nuclear structure and function. The experiments proposed for this study involve characterization of TPs by cloning the gene and determining its sequence. The sequence will be compared with sequences of known IFs. Immunofluorescent localization and STEM mass determination will be used to determine TP nuclear sites and stoichiometry with DNA. In situ electron microscopic analysis and immunolocalization will be used to characterize the 10 nm filaments. Reconstitution experiments will be performed to determine factors which affect chromatin organization. It is expected that this study will clarify one of the functions of telomeres and the role of lamins in organizing chromatin structure in a eukaryotic nucleus. Thus, the project addresses one of the central issues of cell biology, namely, the architectural organization of the genome within the nucleus.