CHROMOSOME MOVEMENT IN MEIOSIS AND MITOSIS

减数分裂和有丝分裂中的染色体运动

• 批准号: 6342846
• 负责人: Sharyn A. Endow
• 金额: $ 30.86万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6342846
• 关键词: Drosophilidae; X ray crystallography; adenosinetriphosphatase; cell cycle; chromosome movement; conformation; cryoelectron microscopy; green fluorescent proteins; growth /development; intermolecular interaction; kinesin; laboratory rabbit; meiosis; microtubule associated protein; microtubules; mitotic spindle apparatus; polymerase chain reaction; protein structure function; tubulin; video microscopy; western blottings

The long-term objectives of this project are to identify and characterize the forces that underlie chromosome movement in meiosis and mitosis, including interactions of chromosomes with the spindle and microtubule dynamics involved in spindle assembly and function. The proposed studies focus on Ncd, a kinesin family microtubule motor protein in Drosophila. Ncd is a minus end-directed motor that plays an essential role in spindle assembly and function in meiosis and mitosis. The proposed studies focus on 1) the mechanism by which the 'neck', the region of the protein adjacent to the conserved motor domain, functions to determine motor directionality, 2) conformational changes in the motor that occur during motor function, and 3) the effects of minus-end motility and motor uncoupling on Ncd function in the cell. The specific aims of the proposed studies are to: 1. Determine the mechanism of neck function in motor directionality. Functional tests of the neck in determining motor polarity will be performed by constructing mutant motors and analyzing polarity in vitro. 2. Identify regions of the motor involved in changes of conformation during motor function. Mutants that uncouple ATP hydrolysis from force generation or trap the motor in different states of ATP hydrolysis will be selected or designed, and tested in vitro to identify structural elements that move during motor function. 3. Determine the effect of minus-end polarity and motor uncoupling on Ncd function in vivo. Truncated mutants and motor uncoupling mutants will be introduced into Drosophila as gfp gene fusions to determine the effects of the mutated motors on Ncd function and dynamics in live cells. These studies address the molecular basis of force generation in the spindle and the role of microtubule motors in chromosome movement during cell division. Defective chromosome distribution causes aneuploidy and genetic abnormalities, and is associated with malignancies in humans.

该项目的长期目标是确定和表征减数分裂和有丝分裂中染色体运动的作用力，包括染色体与纺锤体的相互作用以及参与纺锤体组装和功能的微管动力学。拟议的研究重点是 Ncd，果蝇中的一种驱动蛋白家族微管运动蛋白。 Ncd 是负端定向马达，在纺锤体组装以及减数分裂和有丝分裂功能中发挥重要作用。拟议的研究重点关注 1) “颈部”（与保守运动结构域相邻的蛋白质区域）确定运动方向性的机制，2) 运动功能期间发生的运动构象变化，以及 3)负端运动和运动解偶联对细胞 Ncd 功能的影响。拟议研究的具体目标是： 1. 确定颈部功能在运动方向性方面的机制。将通过构建突变电机并在体外分析极性来进行确定电机极性的颈部功能测试。 2. 识别运动功能期间参与构象变化的运动区域。将选择或设计将 ATP 水解与力产生分离或将电机捕获在不同 ATP 水解状态的突变体，并进行体外测试，以识别在运动功能期间移动的结构元件。 3.确定负端极性和运动解偶联对体内Ncd功能的影响。截短的突变体和运动解偶联突变体将作为 gfp 基因融合引入果蝇，以确定突变的运动对活细胞 Ncd 功能和动力学的影响。这些研究探讨了纺锤体中力产生的分子基础以及微管马达在细胞分裂过程中染色体运动中的作用。染色体分布缺陷会导致非整倍体和遗传异常，并与人类恶性肿瘤有关。