STRUCTURE AND FUNCTION OF CYTOPLASMIC DYNEIN

基于微管的运动对两者的生命和生长至关重要 单个细胞和多细胞生物体。的故障。 提供细胞内转运的基于微管的机器 影响包括染色体在内的细胞行为的全局方面 分离、细胞分裂、纤毛和鞭毛运动、内吞 和分泌囊泡的运输，细胞质的组织 隔室和细胞极性的建立。这个 这些细胞行为和机制之间的相互关系 细胞分化和组织形态发生持续不断地 被承认并反映在许多医疗问题上，包括 癌症、先天性染色体综合征和出生缺陷。最基本的 层面，构建了微管机的功能和规范 转化成为它们提供动力的相关马达酶。这项建议旨在 了解无处不在的微管电机的结构和功能， 胞质动力蛋白。 在过去的资助期间，我们进行了一项生化、分子、 果蝇细胞质动力蛋白重链的遗传特征。 在下一批资助期内，我们建议进一步研究 动力蛋白马达固有成分的功能域 复合体，包括重链、中链和轻链多肽。 我们的Dhc64C基因的克隆和鉴定，以及 隐性致死突变的分离，定义了一种功能性动力蛋白 重链(DHC)转录单位。我们现在将对此进行修改 转录单位，在体内表达改变的DHC转基因。这个 将分析改变后的DHs的功能和生化特性 体内和体外提供动力蛋白的分子结构域图 多肽。此外，动力蛋白突变的DNA损伤以前 通过基因筛选在体内分离将揭示新的功能域 在DHC中的意义。 果蝇74kD中间体和50-60kD的拟议分析 发育背景下的轻链基因将阐明其作用 这些亚基在调节细胞的组装和亚细胞靶向中的作用 动力蛋白运动复合体。我们将克隆基因，分析 分发他们的成绩单，并启动对 基因产品。基因的细胞遗传学定位和基因的遗传 将继续研究基因座的特征，作为必要的背景 未来的突变分析。 基因分析将被用来阐明动力蛋白的全部范围 功能，并识别新的基因和基因产品 动力蛋白功能所必需的。我们将分离和分析外源基因 粘性和/或粗眼表型的增强剂和抑制剂 Dhc64C突变。拟议中的屏幕将利用 为眼睛发育中的缺陷评分提供了一个绝佳的机会 探讨动力蛋白运动功能在脑血管疾病中的作用 协调的核运动，细胞分裂的调节，和/或 符合和/或指导适当的信号转导机制 眼的分化。

Microtubule-based motility is critical to the life and growth of both individual cells and multicellular organisms. Malfunctions of the microtubule-based machines that provide for intracellular transport influence global aspects of cell behavior including chromosome segregation, cell division, ciliary and flagellar motility, endocytotic and secretory vesicle traffic, the organization of the cytoplasmic compartment, and the establishment of cell polarity. The interrelationships between these cellular behaviors and mechanisms of cellular differentiation and tissue morphogenesis are continually being recognized and are reflected in numerous medical problems including cancer, congenital chromosomal syndromes and birth defects. At a basic level, the function and regulation of microtubule-based machines is built into the associated motor enzymes that power them. This proposal seeks to understand the structure and function of the ubiquitous microtubule motor, cytoplasmic dynein. During the past grant period, we have conducted a biochemical, molecular, and genetic characterization of Drosophila cytoplasmic dynein heavy chain. In the next grant period, we propose to further study the structural and functional domains of the intrinsic components of the dynein motor complex, including the heavy, intermediate, and light chain polypeptides. Our cloning and characterization of the Dhc64C gene, as well as the isolation of recessive lethal mutations, has defined a functional dynein heavy chain (DHC) transcription unit. We will now modify this transcription unit to express altered DHC transgenes in vivo. The functional and biochemical properties of altered DHCs will be analyzed in vivo and in vitro to provide a molecular domain map of the dynein polypeptide. In addition, the DNA lesions of dynein mutations previously isolated in vivo by genetic screens will reveal new domains of functional significance within the DHC. The proposed analysis of the Drosophila 74 kD intermediate-, and 50-60 kD light chain genes in a developmental context will shed light on the role of these subunits in regulating the assembly and subcellular targeting of the dynein motor complex. We will clone the genes, analyze the distribution of their transcripts, and initiate a domain analysis of the gene products. The cytogenetic mapping of the genes and the genetic characterization of the loci will be pursued as necessary background for the future mutational analyses. Genetic analysis will be used to elucidate the full range of dynein function and to identify to novel genes and gene products that are required for dynein function. We will isolate and analyze extragenic enhancers and suppressors of the rough eye phenotype of the Glued and/or Dhc64C mutations. The proposed screens will capitalize on the ease of scoring defects in eye development and provide an excellent opportunity to investigate the participation of the dynein motor function in the coordinated nuclear movements, the regulation of cell division, and/or the signal transduction mechanisms that coincide with and/or direct the proper differentiation of eye.