STRUCTURE AND FUNCTION OF CYTOPLASMIC DYNEIN

由微管组成的亚细胞器负责许多 细胞内和细胞内产生的运动。的功能和调节 这些细胞器对两个个体的生命和成长都至关重要。 细胞和多细胞生物体。这些以微管为基础的故障 细胞器影响细胞行为的全局方面，包括染色体 分离、细胞分裂、细胞分化和组织形态发生； 并反映在许多医学问题上，包括癌症，先天性 染色体综合征和出生缺陷。我们的远景目标 研究是为了阐明控制该功能的分子机制 基于微管的细胞器。 在基础水平上，微管的功能和调节 细胞器依赖于驱动它们的马达。微管马达是 转化来自核苷酸的能量的能量转换酶 微管的滑动和/或运动中的结合和水解 相对于微管的囊泡或细胞器。一个微管马达， 在果蝇胚胎及其运动中已发现胞质动力蛋白 用微管滑动试验表征体外活性。分子 遗传策略将被用来研究细胞和 细胞质动力蛋白的发育功能及鉴定 动力蛋白重链多肽的结构和功能域 是它的功能所必需的。 编码胞质动力蛋白重链多肽的DNA序列 将通过表达克隆技术使用单特异性 识别动力蛋白重链的抗体。重叠克隆 包含整个蛋白质编码序列的基因将被分离和分析 预测多肽的一级序列并进行鉴定 与其他机械力化学酶共有的保守基序。重组 动力蛋白多肽将从分离的编码序列中产生 将在体外和体内进行测试，以确定结构和功能 动力蛋白多肽的结构域。抗重组动力蛋白抗体 片段将被用来将初级氨基酸序列与 用电子显微镜观察到的二维结构，以及 描述果蝇胚胎胞质动力蛋白的分布。 分离的动力蛋白克隆将提供开始突变的手段 细胞质动力蛋白在细胞和发育中的作用分析 活着。

Subcellular organelles composed of microtubules are responsible for many of the motions produced by and within cells. The function and regulation of these organelles is critical to the life and growth of both individual cells and multicellular organisms. Malfunctions in these microtubule-based organelles influence global aspects of cell behavior including chromosome segregation, cell division, cell differentiation, and tissue morphogenesis; and are reflected in numerous medical problems including cancer, congenital chromosomal syndromes, and birth defects. The long range goal of our research is to elucidate the molecular mechanisms that govern the function of microtubule-based organelles. At a basic level, the function and regulation of microtubule-based organelles depends on the motors that drive them. Microtubule motors are energy-transducing enzymes that convert the energy derived from nucleotide binding and hydrolysis into the sliding of microtubules and/or the movement of vesicles or organelles relative to the microtubule. A microtubule motor, cytoplasmic dynein, has been identified in Drosophila embryos and its motor activity characterized in vitro by microtubule gliding assays. Molecular and genetic strategies will be used to investigate the cellular and developmental functions of cytoplasmic dynein and to identify the structural and functional domains of the dynein heavy chain polypeptide that are required for its functions. DNA sequences that encode the heavy chain polypeptide of cytoplasmic dynein will be isolated by expression cloning techniques using a monospecific antibody that recognizes the dynein heavy chain. Overlapping clones containing the entire protein coding sequence will be isolated and analyzed to predict the primary sequence of the polypeptide and to identify conserved motifs shared with other mechano-chemical enzymes. Recombinant dynein polypeptides will be produced from the isolated coding sequences and will be assayed in vitro and in vivo to identify structural and functional domains of the dynein polypeptide. Antibodies against recombinant dynein fragments will be used to relate the primary amino acid sequence to the two-dimensional structure observed by electron microscopy, and to characterize the distribution of cytoplasmic dynein in Drosophila embryos. The isolated dynein clones will provide the means to begin a mutational analysis of the cellular and developmental roles of cytoplasmic dynein in vivo.