PILOT STUDY--NUCLEOLAR CYTOPLASMIC TRANSPORT

核仁和细胞质之间的大分子交换是一种 所有真核细胞的基本过程。我们之前确定了 Nopp140，一种穿梭的核定位信号结合蛋白 核仁和细胞质之间的高度定域化的轨迹。这个 这项赠款建议的长期目标是了解该机制 以及对Nopp140介导的核仁-细胞质转运的调节。 具体地说：1.p57的特性和鉴定 额外的Nopp140相关蛋白。我们发现了一种Nopp140 大鼠肝核抽提物中定位于 NOPP 140类似于核内踪迹，并克隆了其cDNA。我们建议： (A)确定p57是否也穿梭于细胞核和细胞质之间。(B) 内源性和细菌表达的p57与p57相互作用的研究 不是140。(C)通过以下方式确定额外的NOPP 140相互作用蛋白 细胞组分的亲和层析 重组Nopp140和p57。2.NOPP 140的结构和动力学 脚印。开发一种能够更容易地检测到 轨道，这将使我们能够同时操纵细胞 孵化条件，我们将尝试在灯光下将其可视化 激光扫描共聚焦显微镜的显微水平和三个 光学切片的三维重建：(A)免疫定位 Nopp140和P57，以及(B)荧光标记的重组NOPP 140和 P57，在显微注射后或在体外进行核进口检测。3. Nopp140基因的分子分析。Nopp140由两个mRNAs编码 在不同的细胞类型中差异表达但转录的 来自单一基因。要深入了解的表达模式 Nopp140基因将从大鼠基因组Lamba dash中克隆 文库及其启动子和内含子-外显子结构分析。最终， 这些研究将有助于对上调的理解。 核糖体合成和由此导致的核仁过度活动 每一个癌细胞的主要特征。事实上，重要的是 核仁在调节细胞生长中的作用一直受到 核仁癌蛋白LYAR的最新发现。

Exchange of macromolecules between the nucleolus and the cytoplasm is an essential process of all eukaryotic cells. We previously identified Nopp140, a nuclear localization signal binding protein that shuttles between the nucleolus and the cytoplasm on highly localized tracks. The long-term objective of this grant proposal is to understand the mechanism and regulation of Nopp140 mediated nucleolar-cytoplasmic transport. Specifically: 1. Characterization of p57 and identification of additional Nopp140 associated proteins. We have identified a Nopp140 associated protein (p57) in rat liver nuclear extracts that localized to Nopp 140 like intranuclear tracks, and cloned its cDNA. We propose to: (a) Determine if p57 also shuttles between nucleus and cytoplasm. (b) Study the interaction of endogenous and bacterially expressed p57 with Nopp140. (c) Identify additional Nopp 140 interacting proteins by affinity chromatography of cellular fractions using columns of recombinant Nopp140 and p57. 2. Structure and dynamics of Nopp 140 tracks. To develop a system that allows an easier detection of the tracks and that will enable us to simultaneously manipulate cellular incubation conditions, we will try to visualize them on a light microscopical level by laser scanning confocal microscopy and three dimensional reconstruction of optical sections of: (a) immunolocalized Nopp140 and p57, and (b) fluorescently labeled recombinant Nopp 140 and p57, after microinjection or in in vitro nuclear import assays. 3. Molecular Analysis of the Nopp140 gene. Nopp140 is encoded by two mRNAs that are differentially expressed in different cell types but transcribed from a single gene. To gain insight into the expression pattern of the two mRNAs, the Nopp140 gene will be cloned from a rat genomic lamba DASH library and its promoter and intron-exon structure analyzed. Ultimately, these studies will contribute to the understanding of the upregulation of ribosome synthesis and the resulting nucleolar hyperactivity which are major characteristics of every cancer cell. In fact, the importance of the nucleolus in the regulation of cell growth has been emphasized by the recent discovery of the nucleolar oncoprotein LYAR.