Combined Micromechanical-Biochemical Study of Mitotic Chromosome Structure

本项目是对动物细胞有丝分裂染色体结构的研究。对于这项工作，研究者将使用显微操作和生化技术的独特组合。生物物理力测量将用于测定有丝分裂染色体中生物化学引入的结构变化。通过这种方式，可以定量研究特定类型的分子在确定有丝分裂染色体结构中的作用。初步结果表明，DNA本身提供了折叠有丝分裂染色体的结构完整性。这些结果挑战了经典的有丝分裂染色体结构的蛋白质支架模型，并表明该方法在排除染色体结构的特定模型方面的价值。该项目的第一个目标是通过这种方式进一步研究有丝分裂染色体中的DNA连通性，并将这些测量扩展到非洲爪蟾染色体。另外的酶实验将研究RNA和蛋白质在有丝分裂染色体结构中的作用。第二个目的是比较使用爪蟾卵提取物重建的染色单体和爪蟾染色体的物理性质。第三个目标是描述有丝分裂期间染色体从细胞中移除时观察到的染色体间细丝，特别是量化它们的DNA含量。有丝分裂染色体内是否存在连续的蛋白质骨架是细胞生物学中一个基本而开放的话题。考虑到前一系统是染色体的精确模型这一普遍假设，理解细胞中重组染色体与染色体的关系是极其重要的。最后，将有丝分裂染色体间丝的存在问题置于坚实的基础之上，将需要对有丝分裂染色体结构的许多假设进行新的思考。这项研究的更广泛的影响将是开发新的生物物理技术（纳米牛顿尺度的力测量，微流体酶处理）来研究染色体结构。与此同时，计划的活动将集中在物理学和生物学相结合的博士生研究教育上。目前，全国缺乏受过这类训练的年轻科学家。因此，该项目将独特地加强这一增长领域的科学人员和技术储备。

This project is a study of the structure of the mitotic chromosome of animal cells. For this work the investigator will use a unique combination of micromanipulation and biochemical techniques. Biophysical force measurements will be used to assay biochemically-introduced structural changes in mitotic chromosomes. In this way the role of specific types of molecules in defining mitotic chromosome structure can be quantitatively studied. Preliminary results indicate that DNA itself provides the structural integrity of the folded mitotic chromosome. These results challenge the classical protein scaffold model of mitotic chromosome structure, and indicate the value of this approach in ruling out specific models of chromosome structure. The first objective of this project is further study of DNA connectivity in the mitotic chromosome in this way, and to extend these measurements to Xenopus chromosomes. Additional enzyme experiments will study the role of RNA and protein in mitotic chromosome structure. A second objective is to compare the physical properties of Xenopus chromosomes from cells with chromatids reconstituted using Xenopus egg extracts. A third objective is to characterize the interchromosome filaments observed when chromosomes are removed from cells during mitosis, and in particular to quantify their DNA content. The question of whether or not there is a contiguous protein skeleton inside the mitotic chromosome is a basic and open topic in cell biology. Understanding the relation of reconstituted chromosomes to chromosomes in cells is extremely important given the general assumption that the former system is an accurate model of chromosomes. Finally, putting the question of existence of mitotic interchromosome filaments on solid ground will demand new thinking about many assumptions about mitotic chromosome structure. Broader impacts of this research will be the development of new biophysical techniques (nanonewton-scale force measurement, microfluidic enzymatic treatments) for the study of chromosome structure. Along with this, the proposed activities will focus on research education for Ph.D. students, combining physics and biology. There is currently a national shortage of young scientists with this kind of training. This project will therefore uniquely strengthen the scientific personnel and technique pools in this growth area.