FUNCTIONAL STUDY OF NATIVE AND SYNTHETIC NUCLEI

我们研究的长期目标是了解 核孔控制着所有进入和离开细胞核的交通。在……里面 最近几年，人们对一连串的 发生在质膜信号转导过程中的事件 到原子核。越来越多的信号转导问题， 发育决定、致癌转化与HIV-1病毒 传染性集中在核进口或出口上。不幸的是，我们 对毛孔的分子性质以及它是如何形成的知之甚少 控制这一复杂的流量。在过去的授权期内，我们开发了 核孔和核膜可以被使用的系统 重新组合。生物化学改变的核孔现在可以被创造出来， 在免疫系统耗尽后，进行功能分析。vbl.使用 在这样的系统中，我们已经确定了一种关键的核内和孔道糖蛋白， Nup97，这是核生长、DNA复制和RNA所必需的 出口。此外，我们在划定香港特别行政区议会 核孔。最后，我们已经实现了长期目标--转录 从组装成人造核的克隆模板，Ar现在准备好 在体外检测RNA输出。 我们提出的目标集中在三个方面：1)详细分析 新发现的核孔素，nup97。具体地说，我们将克隆nus97 基因，创建缺失和突变的nup97蛋白，并使用这些蛋白来询问 需要哪些结构域来恢复核生长和DNA复制 97耗尽的原子核。我们将继续进行体内研究，以确定 Nup97的结构域参与RNA输出，并将识别孔洞 与nup97相互作用的蛋白质和胞浆因子 酵母遗传系统。2)第二个主要目标将是从分子上 使用两个系统剖析核孔组装的步骤，一个 环状薄层(AL)组装系统和一种新的锚定核 装配系统。这些研究将有助于阐明 在组装过程中添加最近邻的孔蛋白 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。重组 环状薄片也将被用作第一个 脊椎动物核孔的分离。毛孔的净化应该是 允许生产用于识别新毛孔的单抗 蛋白质，并提供了以前无法获得的孔结构洞察力。 3)在赠款的最后一节，我们描述了实现POL II的计划 和PolIII在人造核中的转录，并设计一种分析方法 这些转录本的体外RNA输出。一旦实现了这一点，这 系统将使我们能够分析RNA输出的机制和 免疫耗竭和组装后的特定蛋白质，如nup97 人造原子核。总而言之，我们将继续研究毛孔功能， 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.