Chromatin Structure

这一建议解决了染色质是如何在真核生物细胞核组织的问题，通过检查一个新的假设。假设是与染色体端粒或末端相关的蛋白质（端粒蛋白，或TP）是核层蛋白，而核层蛋白又是称为中间细丝（IF）的一大组蛋白质的成员。它们组装和拆卸成细丝的能力将负责维持核组织。在这些研究中使用的生物是次毛纤毛Euplotes eurystomus，这是特别有利的，因为宏核中单个染色体元素的长度异常短，导致大约1亿个端粒DNA片段，其中端粒DNA序列都是相同的。此外，宏核包含明确定义的浓缩、分散和复制染色质区域。决定这些功能区结构的高级组织尚不清楚。这些特性使整位体成为研究端粒和真核生物核结构和功能的一个极好的系统。本研究拟进行的实验包括通过克隆基因和确定其序列来表征TPs。该序列将与已知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。原位电镜分析和免疫定位将用于表征10nm细丝。重组实验将进行，以确定影响染色质组织的因素。期望本研究能够阐明真核细胞中端粒的功能之一和层粘连蛋白在染色质结构组织中的作用。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.