STRUCTURE AND FUNCTION OF CYTOPLASMIC DYNEIN

基于微管的能动性对两者的生命和生长都至关重要 单个细胞和多细胞生物。的故障 提供细胞内运输的微管机器 影响细胞行为的全局方面，包括染色体 分离，细胞分裂，纤毛和鞭毛运动，内吞 和分泌囊泡运输，细胞质的组织 隔室，和细胞极性的建立。的 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不断地 认识到并反映在许多医疗问题，包括 癌症、先天性染色体综合征和出生缺陷。在一个基本 水平，建立了基于微管的机器的功能和调节 转化为驱动它们的相关运动酶。这项建议旨在 了解无处不在的微管马达的结构和功能， 细胞质动力蛋白 在过去的资助期间，我们进行了一项生物化学，分子， 和果蝇细胞质动力蛋白重链的遗传特征。 在下一个资助期内，我们建议进一步研究 动力蛋白马达内在成分的功能域 复合物，包括重链、中间链和轻链多肽。 我们的Dhc 64 C基因的克隆和表征，以及 分离隐性致死突变，定义了功能性动力蛋白 重链（DHC）转录单位。我们现在将修改此 转录单位以在体内表达改变的DHC转基因。的 将在中分析改变的DHCs的功能和生化特性 提供动力蛋白的分子结构域图谱 多肽。此外，以前动力蛋白突变的DNA损伤 通过遗传筛选在体内分离将揭示新的功能结构域， 在DHC中的重要性 拟分析果蝇74 kD的中间体，和50-60 kD 轻链基因在发育过程中的作用 这些亚单位在调节组装和亚细胞靶向 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，并启动一个域分析， 基因产物基因的细胞遗传学作图和遗传学 基因座的表征将作为必要的背景， 未来的突变分析 遗传分析将被用来阐明动力蛋白的全部范围 功能，并确定新的基因和基因产物， 动力蛋白功能所必需的。我们会分离并分析基因外的 Glued和/或Glued的粗糙眼表型的增强子和抑制子 Dhc 64 C突变。拟议中的屏幕将利用 对眼睛发育缺陷进行评分， 研究动力蛋白运动功能参与 协调的核运动，细胞分裂的调节，和/或 信号转导机制，符合和/或指导适当的 眼睛的区别

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.