NF2 BINDING PROTEINS

描述：NF 2基因的种系突变易导致 多种类型的肿瘤以及眼部特征如白内障， 视网膜错构瘤 虽然NF 2种系突变相对罕见， NF 2基因是散发性良性肿瘤中最常见的单一突变基因， 脑瘤 除了作为隐性肿瘤抑制因子的功能外 基于基因分析和与ERM蛋白的序列同源性， 已知NF 2基因产物的功能，称为merlin或 施旺诺明 他们已经确定了七种神经鞘蛋白/梅林蛋白结合 蛋白质（SBkPs）使用酵母双杂交系统。 除了 BII-血影蛋白或胞衬蛋白，六种新的蛋白质已被鉴定。 提出了三个具体目标来评估这些蛋白质： 1)神经鞘蛋白和SBP的相互作用将在 体外结合测定和体内免疫共沉淀。 交互 结构域将通过测试雪旺诺明的部分融合物和 使用B-半乳糖苷酶过滤试验和体外结合试验的SBP。 2)获得SBkPs的全长cDNA并将其定位到人 染色体区域使用FISH和辐射杂交作图。 组织和 通过北方和Western印迹分析， 以及免疫细胞化学进行分析。 亚细胞共定位 将通过共聚焦显微镜测定。 3)SBPs的功能重要性将通过分析 与含有天然存在的神经鞘素的结合减少 错义突变 将SBP cDNA转染细胞系后， 人神经鞘瘤细胞，细胞形态，增殖及 将观察神经鞘蛋白的细胞内定位。 基于 雪旺蛋白在STS 26 T雪旺细胞中的核仁定位及其作用 RNA聚合酶I转录的影响将在体外使用核分析仪进行测定。 提取物、纯化的转录因子和重组蛋白。

DESCRIPTION: Germline mutations in the NF2 gene predispose to tumors of multiple types as well as ocular features such as cataracts and retinal hamartomas. Although NF2 germline mutations are relatively rare, the NF2 gene is the single most commonly mutated gene in sporadic benign brain tumors. Other than its function as a recessive tumor suppressor based on genetic analyses and sequence homology to ERM proteins, little is known about the function of the NF2 gene product, called merlin or schwannomin. They have identified seven schwannomin/merlin binding proteins (SBkPs) using the yeast two-hybrid system. In addition to BII-spectrin or fodrin, six novel proteins have been identified. Three specific aims are proposed to evaluate these proteins: 1) The interaction of schwannomin and SBPs will be verified using in vitro binding assays and in vivo co-immunoprecipitation. Interacting domains will be determined by testing partial fusions of schwannomin and SBPs using filter assays for B-galactosidase and in vitro binding assays. 2) Full length cDNAs for SBkPs will obtained and mapped to human chromosome regions using FISH and radiation hybrid mapping. tissue and subcellular distribution of SBPs by Northern and Western blot analyses as well as immunocytochemistry will be analyzed. Subcellular co-localization will be determined by confocal microscopy. 3) The functional importance of SBPs will be determined by analyzing decrease of binding to schwannomins containing naturally occurring missense mutations. After transfection of SBP cDNAs into cell lines and human schwannoma cells, changes of cell morphology, proliferation and schwannomin intracellular localization will be observed. Based on nucleolar localization of schwannomin in STS26T Schwann cells, its effect on RNA polymerase I transcription will be assayed in vitro using nuclear extracts, purified transcription factors and recombinant proteins.