NF2 BINDING PROTEINS

描述：NF2基因的胚系突变易患 多种类型的肿瘤以及眼部特征，如白内障和 视网膜错构瘤。尽管NF2胚系突变相对较少， NF2基因是散发性良性疾病中最常见的单一突变基因 脑瘤。除了它作为隐性肿瘤抑制因子的功能之外 根据对ERM蛋白的遗传分析和序列同源性，很少 已知NF2基因产物的功能，称为Merlin或 施瓦诺明。他们已经确定了七种Schwannomin/Merlin结合 利用酵母双杂交系统进行蛋白质(SBkP)的分离。除了……之外 BII-Spectrin或Fodrin，已鉴定出六种新的蛋白质。 我们提出了三个具体目标来评估这些蛋白质： 1)Schwannomin和SBPs的相互作用将使用 体外结合试验和体内免疫共沉淀试验。互动 结构域将通过测试Schwannomin和Schwannomin的部分融合来确定 使用B-半乳糖苷酶滤过试验和体外结合试验。 2)将获得SBkP的全长cDNA，并将其映射到人类 染色体区域的FISH和辐射杂交作图。组织和 Northern和Western印迹分析SBPs的亚细胞分布 以及免疫细胞化学分析。亚细胞共定位 将通过共聚焦显微镜来确定。 3)通过分析确定SBPS的功能重要性 减少与含有天然产物的神经鞘蛋白的结合 错义突变。将SBP cDNAs导入细胞系后， 人神经鞘瘤细胞形态、增殖及细胞周期的变化 将观察神经鞘蛋白在细胞内的定位。基于 神经鞘蛋白在STS26T雪旺细胞中的核仁定位及其作用 关于RNA聚合酶I的转录将在体外用核进行检测 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.