TAY-SACHS AND RELATED GENETIC NEUROLOGICAL DISORDERS

描述：(改编自申请者摘要)GM2神经节苷脂沉积症 一组不同类型的遗传性疾病，由基因分解代谢缺陷引起 神经节苷脂GM2。这种严重的、几乎总是致命的神经系统疾病 结果当正常所需的三种组织成分中的任何一种 GM2神经节苷脂的降解是遗传异常的。人体内的异常 氨基己糖苷酶α亚单位导致一种临床上被称为 泰-萨克斯病，而亚单位的异常被认为是 桑德霍夫病。GM2激活蛋白的缺失被称为“AB” 所有三种蛋白质的cDNAs和第一个蛋白质的基因组克隆 现在有两种形式可用于分析遗传异常，网址为 核酸水平。在过去的三年里，申请人的 实验室已经确定了Tay基因13个已知突变中的7个 萨克斯病，也是桑德霍夫病中唯一已知的特定突变， 并成功克隆了该激活蛋白的编码基因。这 应用程序建议扩展他们的工作，以进一步描述自然 遗传性GM2患者中三个基因的突变 神经节苷脂。对自然发生的突变的鉴定将是 主要由标准的分子生物学技术完成，包括 基因文库构建、筛选、聚合酶链合成 反应、测序、等位基因特异性寡核苷酸筛选，以及 COS I细胞的瞬时功能表达。第二个具体目标是 获得并鉴定全长cDNAs和编码该蛋白的基因。 GM2激活蛋白，并寻找其结构要求。 甘露糖磷酸识别标记物，利用体积小的优势 并且该蛋白只有一个糖基化位点。刻画人物形象 GM2激活蛋白的cDNA和基因组结构也将被 用标准的重组DNA技术完成的，因为他们已经 全长cdna克隆，并正在获得基因组片段。 第三个具体目标是生产和提纯毫克量的 使用新的杆状病毒系统的三种蛋白质。这些蛋白质将被用于 用于随后的实验，如产生抗体。第四次 具体目标是探索 酶反应的每个成分的不同结构域， 激活蛋白和底物GM2神经节苷脂两个亚基， 以及三种蛋白质之间的相互作用。这些研究将广泛地 基于从以下方面获得的知识利用定点突变 自然发生的突变及其酶学特性耦合 利用来自计算机分子建模的信息。两者都是自然的 将对发生和突变产生的突变进行功能评估 在COS I细胞和杆状病毒系统中。即将到来的新知识 由这一系列研究产生的结果可能有助于最终 对这些遗传性神经病分子发病机制的认识 并可能对未来的基因疗法的尝试有所帮助。

DESCRIPTION: (Adapted from the applicants abstract) GM2 gangliosidosis is a heterogeneous group of genetic disorders due to defective catabolism of ganglioside GM2. This severe and nearly always fatal neurological disorder results when any one of the three tissue constituents required for normal degradation of GM2 ganglioside is genetically abnormal. Abnormality in the enzyme hexosaminidase alpha subunit leads to a disease clinically known as Tay Sachs disease, whereas abnormality in the subunit is identified as Sandhoff disease. A deficiency of the GM2 activator protein is termed "AB variant". cDNA's for all three proteins and genomic clones for the first two forms are now available for analyses of the genetic abnormalities at the nucleic acid level. During the past three years, the applicants' laboratory has identified seven of the thirteen known mutations in Tay Sachs disease, and the only known specific mutation in Sandhoff disease, and has successfully cloned cDNA's coding for the activator protein. This application proposes to extend their work to further characterize naturally occurring mutations in the three genes among patients with genetic GM2 gangliosidoses. Identification of naturally occurring mutations will be accomplished largely by standard molecular biological techniques, including cDNA and genomic library construction, screening, the polymerase chain reaction, sequencing, allele specific oligonucleotide screening, and transient functional expression in COS I cells. The second specific aim is to obtain and characterize full length cDNA's and the gene coding for the GM2 activator protein and to search for the structural requirement for the mannose phosphate recognition marker, taking advantage of the small size and only one glycosylation site of the protein. Characterization of the cDNA and the genomic structure of the GM2 activator proteins will also be done with standard recombinant DNA technologies, since they already have full length cDNA clones and are on their way to obtain genomic fragments. The third specific aim is to produce and purify milligram quantities of the three proteins using the new baculovirus system. The proteins will be used for subsequent experiments such as to produce antibodies. The fourth specific aim is to explore the structure/function relationship within the different domains of each of the constituents for the enzymatic reaction, the two subunits, the activator protein, and the substrate GM2 ganglioside, and interactions among the three proteins. These studies will extensively utilize sitedirected mutagenesis based on the knowledge gained from naturally occurring mutations and their enzymological properties coupled with information from computerized molecular modeling. Both naturally occurring and mutagenesis generated mutants will be evaluated functionally in the COS I cell and baculovirus systems. The new knowledge to be generated by this series of studies may contribute to eventual understanding of the molecular pathogenesis of these genetic neurological disorders and may be useful to future attempts at the gene therapy.