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Molecular analysis of Fukuyama muscular dystrophy and functional analysis of the gene product fukutin.

福山性肌营养不良症的分子分析及基因产物fukutin的功能分析。

基本信息

批准号：
13672376
负责人：
KOBAYASHI Kazuhiro
金额：
$ 1.98万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive severe muscular dystrophy accompanied by brain malformation, prevalent in Japan. This research was performed for the purpose of making the antibodies specific for the FCMD gene product fukutin, analyzing localization and function of fukutin, and creating the mouse model of this disease. Then the following things were clarified.Although some antibodies were obtained which detect overexpressed fukutin in mammalian cells, these could not detect endogenous fukutin. Since it is supposed that fukutin is a glycosyltransferase from our recent researches and that very small quantity of endogenous fukutin exists in cells like many of known glycosyltransferases, it was thought that detection of the endogenous fukutin by the antibodies cannot be made. That is, it turned out that the analysis is difficult using the fukutin antibodies. Moreover, it was shown that fukutin exists in a Golgi body by the immunohistochemical ana … More lysis of mammalian cells overexpressing fukutin, and that is not contradictory to the possibility of being a glycosyltransferase.Mutational analysis was performed in the FCMD patients and some additional mutations were newly discovered.Identification of fukutin-binding proteins is tried by affinity column chromatography using recombinant fukutin and by mass spectrometric analysis. Although some proteins were obtained, we are checking whether these are the actual fukutin-binding proteins. Moreover, another analysis is performed using 2-dimensional electrophoresis, immunoprecipitation, and two-hybrid methods in order to identify the target protein of fukutin as a possible glycosyltransferase and the partner protein of the possible fukutin complex.To create the knock-in mice which carry the retrotransposon insertion in 3'-untranslated region of the fukutin gene, the knock-in vector was constructed and introduced to embryonic stem cells.Muscle-eye-brain disease (MEB) bears a striking resemblance to FCMD, We identified the gene responsible for MEB which encodes POMGnT1 glycosyltransferase. Less

福山型先天性肌营养不良症（FCMD）是一种常染色体隐性严重肌营养不良伴脑畸形的疾病，常见于日本。本研究的目的是制备针对口蹄疫基因产物fukutin的特异性抗体，分析fukutin的定位和功能，建立口蹄疫小鼠模型。然后澄清了以下事情。虽然获得了一些检测哺乳动物细胞中过表达fukutin的抗体，但这些抗体不能检测内源性fukutin。由于我们最近的研究推测fukutin是一种糖基转移酶，并且与许多已知的糖基转移酶一样，内源性fukutin在细胞中存在的数量非常少，因此认为无法通过抗体检测内源性fukutin。也就是说，使用fukutin抗体很难进行分析。此外，通过免疫组织化学分析表明，fukutin存在于高尔基体中，对过表达fukutin的哺乳动物细胞进行了更多的裂解，这与作为糖基转移酶的可能性并不矛盾。对口蹄疫患者进行了突变分析，并发现了一些新突变。利用重组fukutin亲和柱层析和质谱分析鉴定fukutin结合蛋白。虽然获得了一些蛋白质，但我们正在检查这些是否是真正的fufutin结合蛋白。此外，利用二维电泳、免疫沉淀和双杂交方法进行了另一项分析，以确定fukutin的靶蛋白可能是糖基转移酶和可能的fukutin复合物的伴侣蛋白。为了构建携带fukutin基因3'-非翻译区反转录转座子插入的敲入小鼠，构建了敲入载体并将其引入胚胎干细胞。肌眼脑病（MEB）与手足口病（ffcmd）有着惊人的相似之处，我们发现了负责MEB的编码POMGnT1糖基转移酶的基因。少