CHROMOSOME STRUCTURE AND CONDENSATION MECHANISMS

染色体结构和缩合机制

• 批准号: 3278887
• 负责人: WILLIAM C EARNSHAW
• 金额: $ 18.86万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3278887
• 关键词: DNA binding protein; DNA gyrase; DNA topoisomerases; HeLa cells; cell bank /registry; cell fusion; chickens; chromatin; density gradient ultracentrifugation; electron microscopy; erythropoiesis; fluorescence microscopy; freezing; genetic manipulation; guinea pigs; image processing; immunoelectron microscopy; immunofluorescence technique; laboratory mouse; laboratory rabbit; monoclonal antibody; nonhistone nucleoprotein; nucleosomes; protein structure; radiotracer; surface antigens

The heirarchy of packing interactions in chromatin begins with nucleosomes and culminates in mitotic chromosome bands. Lower levels of packing are apparently determined by DNA-histone and histone-histone interactions, but higher levels (such as organization of chromatin into 100 kb topological domains) are probably regulated by non-histone proteins. Our long term goal is to understand the role of non-histone proteins in determining mitotic chromosome architecture. The best candidates for structural non-histone proteins are components of the poorly characterized chromosome scaffold fraction (proteins which remain associated after extraction of 99% of the chromosomal DNA and 95% of the protein). We have recently shown that DNA topoisomerase II is a component of this fraction. We will continue to characterize this and other scaffold components. 1) Antibodies (polyclonal, monoclonal, autoimmune) recognizing specific scaffold antigens will be obtained. These will be used to determine the efficiency of recovery of specific antigens in the scaffold fraction, to localize these antigens in intact chromosomes and whole cells by immunofluorescence and immunoelectron microscopy, to quantitate the antigens across the cell cycle and in non-dividing cells by photometry and immunoprecipitation, and to map the interactions between scaffold components by gradient sedimentation and immunoprecipitation. 2) The role of scaffold proteins in chromosome condensation will be assayed by studying the effect of the antibodies on premature chromosome condensation (induced when mitotic and interphase cells are fused). 3) Highly extended prematurely condensed chromosomes will also be used for structural studies. The folding of the chromatin fiber will be examined using fluorescence microscopy coupled with computer image processing methods. 4) The activity of topoisomerase II in mitotic cells will be examined. We describe an in situ assay for DNA gyrase activity in deproteinized mitotic chromosomes. 5) Finally, we describe a developmental system (chicken erythropoiesis) where scaffold proteins are progessively lost. We will examine the changes in chromatin domain structure which accompany this loss in order to deduce the function of various scaffold components in establishment of nuclear structure. Techniques used will include immunofluorescence, immunoblotting, scanning calorimetry and quantitative fluorimetry (used to measure the size of chromatin domains).

染色质中包装相互作用的继承结构始于核小体 并在有丝分裂染色体带中达到顶点。 较低的包装是 显然是由DNA-HISTONE和组蛋白 - 酮相互作用确定的，但是 较高的水平（例如，将染色质组织成100 kb拓扑 域）可能受非启用蛋白的调节。 我们的长期 目的是了解非历史蛋白在确定中的作用 有丝分裂染色体建筑。 结构性的最佳候选人 非历史蛋白是染色体表征不佳的成分 脚手架分数（提取99％后保持相关的蛋白质 染色体DNA和95％的蛋白质）。 我们最近显示了 DNA拓扑异构酶II是该部分的组成部分。 我们将 继续表征该和其他脚手架成分。 1）抗体 （多克隆，单克隆，自身免疫）识别特定的脚手架抗原 将获得。 这些将用于确定 恢复脚手架分数中的特定抗原，以本地化这些 通过免疫荧光和完整染色体中的抗原和全细胞 免疫电子显微镜，以量化整个细胞周期的抗原 并通过光度法和免疫沉淀在非分裂细胞中，并绘制 通过梯度沉积和脚手架成分之间的相互作用和 免疫沉淀。 2）支架蛋白在染色体中的作用 通过研究抗体对 过早的染色体凝结（诱导有丝分裂和相间时诱导 细胞被融合）。 3）高度扩展过早凝结的染色体 也将用于结构研究。 染色质的折叠 将使用荧光显微镜与计算机相结合检查纤维 图像处理方法。 4）拓扑异构酶II在有丝分裂中的活性 将检查细胞。 我们描述了DNA回旋酶的原位测定 脱蛋白有丝分裂染色体的活性。 5）最后，我们描述了一个 脚手架蛋白的发育系统（鸡肉红细胞生成） 逐渐失去。 我们将检查染色质结构域的变化 伴随这种损失的结构，以推断出的功能 建立核结构的各种脚手架成分。 所使用的技术将包括免疫荧光，免疫印迹，扫描 量热法和定量荧光法（用于测量 染色质域）。