THE STRUCTURE AND SEQUENCE OF NON-MUSCLE MYOSIN GENES

非肌肉肌球蛋白基因的结构和序列

• 批准号: 3966530
• 负责人: J A HAMMER
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3966530
• 关键词: Acanthamoeba; cell biology; cytoskeleton; enzyme structure; genes; genetic library; genetic translation; immunoprecipitation; messenger RNA; molecular cloning; nucleic acid sequence

The purpose of this project is to isolate the genes encoding the non-muscle myosins of Acanthamoeba and to use the genes as tools to investigate myosin structure/function relationships and the in vivo functions of these cytoplasmic myosins. This project is part of the general effort in the Laboratory of Cell Biology to understand the organization and function of the cytoskeleton, using as a model system the soil amoeba Acanthamoeba. Acanthamoeba expresses simultaneously at least three distinct myosin enzymes, myosin IA, myosin IB and myosin II. Using molecular cloning techniques, we have isolated and purified a myosin II heavy chain gene and a myosin IB heavy chain gene. This study will provide, for the first time, the complete amino acid sequence of a non-muscle myosin. While non-muscle and muscle myosins share many common features, non-muscle myosins do possess unique structural, enzymatic, and regulatory properties. The amoeba myosin sequence data will be of great value in furthering our understanding of the unique structural and functional aspects of the amoeba myosins, and hopefully provide insight into the properties of cytoplasmic myosins in general. The significance of this work is that by using the tools of molecular biology we can approach the study of these myosins in novel ways which are not possible using the classical techniques of protein chemistry. For example, we can use the genes to (1) make single determinant antibodies to synthetic peptides as probes of myosin function, (2) assign function sites in the 1 degree sequence in combination with the amino acid composition of chemically crosslinked peptides, (3) search for cytoplasmic myosin genes in higher eukaryotes, (4) alter the intracellular levels of myosin in the living cell as a way to study their roles in cell physiology, and (5) study structure/function relationships via site-directed mutagenesis of the gene.

该项目的目的是隔离编码非肌肉的基因 阿甘达莫巴的肌球蛋白，并使用这些基因作为研究肌球蛋白的工具 结构/功能关系和这些的体内功能 细胞质肌球蛋白。 该项目是一般努力的一部分 细胞生物学实验室了解的组织和功能 细胞骨架，用作模型系统，土壤变形虫阿斯塔梅巴。 Acanthamoeba同时表达至少三个不同的肌球蛋白 酶，肌球蛋白IA，肌球蛋白IB和肌球蛋白II。 使用分子克隆 技术，我们已经孤立并纯化了肌球蛋白II重链基因和 肌球蛋白IB重链基因。 这项研究将首次提供 非肌肉肌球蛋白的完整氨基酸序列。 而非肌肉 和肌肉肌球蛋白具有许多常见特征，非肌肉肌球蛋白会做 具有独特的结构，酶和调节性能。 这 Amoeba肌球蛋白序列数据在推动我们的 了解变形虫的独特结构和功能方面 肌球蛋白，并希望能够深入了解细胞质的特性 一般而言。 这项工作的重要性是，使用分子工具 生物学我们可以以新颖的方式对这些肌动物进行研究 不可能使用蛋白质化学的经典技术。 为了 例如，我们可以使用基因来（1）制作单一的决定抗体 合成肽作为肌球蛋白功能的探针，（2）分配功能位点 在1度序列中与氨基酸组成的结合 化学交联的肽，（3）在搜索细胞质肌球蛋白基因 较高的真核生物，（4）改变了细胞内肌球蛋白的肌球蛋白水平 活细胞是研究其在细胞生理学中作用的一种方式，（5）研究 通过基因的位置定向诱变的结构/功能关系。