FUNCTIONAL STUDY OF NATIVE AND SYNTHETIC NUCLEI

我们研究的长期目标是了解 核孔控制所有进出核的交通。 在 近年来，人们对级联反应的认识激增， 在从质膜传递信号时发生的事件 到细胞核。 越来越多的信号转导问题， 发育决定、致癌转化和HIV-1病毒 传染性集中在核进口或出口上。 不幸的是我们 我对孔的分子性质以及它是如何 控制着复杂的交通 在过去的资助期内，我们开发和 使用的系统，通过该系统，核孔和核被膜可以 重组。 经过生化改变的核孔现在可以被创造出来， 在系统的免疫耗竭后，并分析功能。 使用 在这样的系统中，我们已经鉴定了一种关键的核内和孔糖蛋白， nup 97，核生长、DNA复制和RNA 导出. 我们还在划定大会的范围方面取得了良好进展， 核孔 最后，我们实现了长期目标，转录 从克隆的模板组装成合成的细胞核， 体外检测RNA输出。 我们提出的目标集中在三个方面：1）详细分析 新发现的核孔蛋白nup 97 具体来说，我们将克隆nus 97 基因，创建缺失和突变的nup 97蛋白，并使用蛋白质来询问 哪些结构域是恢复细胞核生长和DNA复制所必需的， 97-耗尽核 我们将继续进行体内研究，以确定 nup 97的结构域参与RNA输出，并将识别孔 蛋白质和胞质因子通过强有力的 酵母遗传系统 2)第二个主要目标是从分子水平上 使用两个系统剖析核孔组装的步骤， 环层（AL）组装系统，以及一种新的锚定核 汇编系统 这些研究将有助于阐明 在组装过程中加入孔蛋白，最近的邻居 相互作用，以及必须发生的膜融合的新机制 穿过双层核膜形成核孔 复溶 环形薄板也将作为第一个起点， 脊椎动物核孔的分离。 孔的纯化应 允许产生用于鉴定新孔的单克隆抗体 蛋白质，并提供以前无法实现的洞察孔结构。 3)在补助金的最后一部分，我们描述了实现pol II的计划 和pol III转录，并设计一种测定方法， 这些转录物的体外RNA输出。 一旦实现这一目标， 系统将使我们能够分析RNA输出的机制和 特异性蛋白质，如NUP 97，在免疫耗竭和组装后， 合成核 总之，我们将继续研究孔隙功能， 孔组装和RNA输出，目的是阐明它们在 细胞功能

The long term objective of our research is to understand the way in which the nuclear pore controls all traffic entering the exiting the nucleus. In recent years, there has been an explosion in knowledge of the cascade of events which occur in the transduction of a signal from the plasma membrane to the nucleus. Increasingly, questions of signal transduction, developmental determination, oncogenic transformation, and HIV-1 viral infectivity are focusing on nuclear import or export. Unfortunately, we know relatively little of the molecular nature of the pore and how it controls this complex traffic. In the past grant period, we developed and used systems by which the nuclear pore and nuclear envelope can be reconstituted. Biochemically altered nuclear pores can now be created, following immunodepletion of the systems, and analyzed for function. Using such systems, we have identified a key intranuclear and pore glycoprotein, nup97, that is required for nuclear growth, DNA replication, and RNA export. We have also made good progress on delineating the assembly of the nuclear pore. Lastly, we have achieved as long-term goal, transcription from cloned templates assembled into synthetic nuclei, and ar now ready to examine RNA export in vitro. Our proposed goals focus on three areas: 1) A detailed analysis of the newly discovered nucleoporin, nup97. Specifically, we will clone the nus97 gene, create deleted and mutant nup97 proteins, and use the proteins to ask which domains are required to restore nuclear growth and DNA replication to 97-depleted nuclei. We will pursue in vivo studies to determine which domains of nup97 are involved in RNA export, and will identify the pore proteins and cytosolic factors that interact with nup97 through a powerful yeast genetic system. 2) A second major goal will be to molecularly dissect the steps in assembly of the nuclear pore using two systems, an annulate lamellae (AL) assembly system, and a novel anchored nuclear assembly system. These studies will help to elucidate the order of addition of pore proteins during assembly, the nearest-neighbor interactions, and the novel mechanism of membrane fusion which must occur across the double nuclear membrane to create a nuclear pore. Reconstituted annulate lamellae will also be used as a starting point for the first isolation of the vertebrate nuclear pore. Purification of the pore should allow production of monoclonal antibodies for identification of new pore proteins and provide previously unattainable insight into pore structure. 3) In the last section of the grant, we describe plans to achieve pol II and pol III transcription in synthetic nuclei, and to devise an assay for the in vitro RNA export of these transcripts. Once this is achieved, this system will allow us to analyze the mechanism of RNA export and the role of specific proteins, such as nup97, following immunodepletion and assembly of synthetic nuclei. In summary, we will pursue studies of pore function, pore assembly, and RNA export, with the goal of elucidating their role in cellular function.