NUCLEOCYTOPLASMIC TRANSPORT--NEW FACTORS AND PATHWAYS

核质转运--新因素和途径

• 批准号: 6386900
• 负责人: AURELIAN RADU
• 金额: $ 19.23万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386900
• 关键词: cell nucleus; gene expression; genetic library; immunoprecipitation; intracellular transport; northern blottings; nuclear membrane; nucleic acid sequence; protein localization; protein structure function; transport proteins; western blottings; yeast two hybrid system

Various classes of proteins and nucleic acids are imported into and exported out of the cell nucleus through separate pathways, defined by independent sets of carrier molecules and docking sites ad the nuclear pore complexes. For some transported species the components of their transport machineries are partially or totally unknown. Our aim is to identify and characterize new nucleocytoplasmic transport factors, based on two complementary strategies. The first approach involves obtaining the full DNA sequences of potential transport factors whose existence is inferred from the presence in the GenBank EST database of shot, partial cDNA sequences, showing significant homology with known transport factors. The second strategy is based on the fact that all known transport factors dock temporarily, during passage across the nuclear pore, to proteins at the nuclear pore complexes. One of these docking site proteins, Nup98, which we isolated and shown to be implicated in multiple transport pathways, will be used for retention and isolation of new transport factors from the nucleoplasm. The proteins isolated by these two approaches will be characterized in terms of full cDNA sequence, subcellular localization, and level of expression in various tissues and developmental stages. Most importantly, the proteins interacting with these transport factors will be identified by co-immunoprecipitation, screening of expression libraries, and two-hybrid assays. The ensemble of these data, and especially the set of interacting molecules, will shed light on the specific functions of these proteins as transport factors. The relevance of these studies for human health is linked to the fact that a vast number of proteins and nucleic acids cannot exert their function unless they are imported in or exported out of the nucleus in a precise manner, and in fact there are known cases when tumors arise due to defects in nucleocytoplasmic transport pathways are used by viruses, knowing in detail these mechanisms could also offer opportunities for new anti-viral therapies and for improved gene therapy vectors.

各种类型的蛋白质和核酸被输入到细胞中， 通过不同的途径从细胞核输出，定义为 独立的载体分子组和与核的对接位点 孔隙复合体对于某些被运输的物种， 运输机械部分或全部未知。我们的目标是 鉴定和表征新的核质转运因子， 两个互补的战略。第一种方法涉及获得 潜在转运因子的完整DNA序列，其存在是 从GenBank EST数据库中存在的镜头，部分 cDNA序列，与已知的转运因子显示出显着的同源性。 第二种策略是基于这样一个事实，即所有已知的运输因素 在穿过核孔的过程中暂时停靠在蛋白质上， 核孔复合体。其中一个对接点蛋白Nup98， 我们分离出了它，并证明它与多种运输有关， 通道，将用于保留和隔离新的运输 来自核质的因子。这两个人分离出的蛋白质 方法的特征在于全cDNA序列， 亚细胞定位和在各种组织中的表达水平， 发育阶段最重要的是，蛋白质与 这些转运因子将通过免疫共沉淀来鉴定， 表达文库的筛选和双杂交测定。述全体 这些数据，尤其是相互作用的分子， 了解这些蛋白质作为转运因子的特殊功能。 这些研究与人类健康的相关性与以下事实有关： 大量的蛋白质和核酸不能发挥它们的功能， 除非它们以精确的方式输入或输出核 方式，事实上，有已知的情况下，当肿瘤出现由于缺陷 在核质转运途径中， 详细说明这些机制也可以为新的抗病毒药物提供机会。 治疗和改进的基因治疗载体。