The functional analysis of !6q-linled autosomal dominant cerabeller ataxia associated gene.

本研究的一个目的是分析与第16条染色体长度相关的小脑易性症相关的先孕基因。对于本病，我们进行了致病基因座的鉴定、物理图谱的制作、候选结构域的限定等工作。此外，在一个白藜芦醇素-1基因中，链式反应的不平衡发现了很高的变化。当我们为患者大脑制作了一种抗体，并对其进行了免疫组织化学分析时，该蛋白与患者小脑浦肯野细胞的细胞骨架蛋白结合在一起。它是一种基因变化，或者这是患者特有的唯一变化，是本病发病的直接原因，或者与其关系极为密切。我们利用文库中的双杂交系统方法，克隆了与该蛋白形成相互作用或复合体的蛋白的cDNA，以检测该蛋白产物的特性。将筛选的人胎脑基因文库与酵母转录因子的转录激活域连接后，构建了紫杉酚-1和酵母转录基因诱饵载体，并将其引入宿主酵母中。我们对在营养培养基上生长的阳性菌落进行了筛选，并从酵母细胞中收集了质粒DNA，将阳性克隆保存在其中。碱基序列通过直接测序法对其进行解析，并鉴定出蛋白质，但与细胞骨架蛋白质有关的事情并不清楚。我们现在正在检查包括多克隆抗体的制造，原位杂交。可能在其他地方有这种疾病的致病基因，并详细检查候选区域，并对结果做出贡献。

A purpose of this study is to analyze it about an associated gene of prepotence to chain the 16th chromosome length arm-related cerebellum transsexualism. As for this disease, we performed identification of a cause gene seat, manufacture of physical map, limitation of a candidate domain till now. Furthermore, in a puratrophin-1 gene, chain reaction non-equilibrium found a high change. When we made an antibody for puratrophin-1 and analyzed it in a patient brain immunohistochemically, the protein cohered with cytoskeletal protein with a patient cerebellum Purkinje cell. It is a gene change or this is an only change peculiar to a patient, and it is a direct cause of the onset of this disease or it was related to extremely closely.From a background such as the above, we inspected it whether this change was etiology. We used two-hybrid system method from a library and did cloning of cDNA of the protein which formed interaction or a complex to this protein to examine a characteristic of a product of a puratrophin-1 gene. We built puratrophin-1 and yeast transcription gene bait plasmid to include and introduced it into host yeast together when I tied the Homo sapiens fetus brain cDNA library which became an object of screening to a transcription activation domain of a yeast transcription factor and produced it. We did screening of the positive colony which we grew on a nutrient medium and collected plasmid DNA from a yeast cell to hold a positive clone in. Base sequence parses this by a direct sequence method and identifies protein, but a thing related to cytoskeletal protein does not become clear. We are examining including manufacture of a polyclonal antibody, in situ hybridization now. There may be a cause gene of this disease elsewhere and examines a candidate domain in detail and is contributing it about the result.