CHEMICAL PATHOLOGY OF NEUROLOGICAL DISORDERS

我计划在我的实验室继续这项工作的几个方面 在生物化学和生物化学领域得到了该项目赠款的支持 溶酶体酸性水解酶的分子遗传学。两个基因编码 溶酶体蛋白有待研究；GM1β-半乳糖苷酶和 鞘脂激活蛋白(SAPS)。这些基因的异常 导致明显的神经疾病--GM1-神经节苷脂沉着症/Morquio B病(酸性β-半乳糖苷酶缺乏症)，异染性 脑白质营养不良样和高谢样综合征-(SAP-1和SAP-2 分别为缺陷)和独特的多形性综合征(完全SAP 不足)。人类的cDNA和编码其他蛋白质的基因 以上列表已被克隆。此外，我们还克隆了该基因的c DNA 和小鼠GM1β-半乳糖苷酶的基因。研究项目 主要研究内容包括：(1)小鼠基因的克隆和鉴定； 鞘脂激活蛋白(SAP)的编码基因，(2)功能 SAP基因的翻译产物，(3)探索 SAPS和GM1β-半乳糖苷酶的结构/功能关系(4) 转基因小鼠SAP缺乏症和GM1基因缺失模型的建立 神经节苷脂缺乏症(GM1β-半乳糖苷酶缺乏症) 重组技术，以及(5)突变的持续鉴定 在因上述缺陷而患有疾病的患者中 列出的基因，并尝试通过以下方式进行基因型/表型相关性 自然发生的突变，定点突变，计算机- 辅助分子建模和表达系统。标准 方法学包括构建cDNA和基因组文库，筛选， 聚合酶链式反应、测序、等位基因特异性 寡核苷酸筛选及其在COS I中的瞬时功能表达 细胞。全长cDNA和编码基因的分离与鉴定 对于小鼠来说，SAPS将通过预期的同源性来完成 在人类和老鼠的基因之间，就像我们已经成功地为 克隆小鼠酸性β-半乳糖苷酶。的生理功能 SAP基因产品将通过代谢负荷进行评估 体内和培养系统中的潜在底物。正常的 克隆将被过度表达和纯化，这样我们就可以获得 毫克量的这些蛋白质用于随后的生化 实验。它们的结构/功能关系的研究 蛋白质将广泛利用基于基因的定点突变 从自然发生的突变中获得的知识及其 酶学特性与来自计算机的信息相结合 分子建模。转基因小鼠的品系将通过 使用适当结构的同源重组技术 小鼠β-半乳糖苷酶和SAP基因。无论是自然发生的还是 突变产生的突变体将在COS中进行功能评估 I细胞和杆状病毒系统。将产生的新知识 这一系列的研究应该有助于我们最终了解 这些严重的遗传性神经疾病的分子发病机制 以及未来对这些疾病的基因治疗的尝试。

I plan to pursue several aspects of the work in my laboratory that have been supported by this project grant in the area of the biochemistry and molecular genetics of lysosomal acid hydrolases. Two genes coding for lysosomal proteins are to be studied; GM1 beta-galactosidase and the sphingolipid activator proteins (SAPS). Abnormalities in these genes result in distinct neurological disorders, -- GM1-gangliosidosis/ Morquio B disease (acid beta-galactosidase deficiency), metachromatic leukodystrophy-like and Gaucher-like syndromes-(SAP-1 and SAP-2 deficiencies respectively), and a unique pleomorphic syndrome (total SAP deficiency). Human cDNAs and the genes coding for other proteins in the above list have been cloned. In addition, we have also cloned the cDNA and the gene for the mouse GM1 beta-galactosidase. Research projects proposed include: (1) Cloning and characterization of mouse cDNA and the gene coding for the sphingolipid activator proteins (SAPs), (2) Functions of the translation products of the SAP gene, (3) Exploration of structure/function relationship of SAPs and GM1 beta-galactosidase, (4) Transgenic production of a mouse model of total SAP deficiency and GM1- gangliosidosis (GM1 beta-galactosidase deficiency) by the homologous recombination technology, and (5) Continuing identification of mutations in patients who suffer from the disorders due to defects in the above listed genes, and attempts at genotype/phenotype correlations through naturally-occurring mutations, site-directed mutagenesis, computer- assisted molecular modeling and expression systems. Standard methodologies include cDNA and genomic library construction, screening, the polymerase chain reaction, sequencing, allele-specific oligonucleotide screening, and transient functional expression in COS I cells. Isolation and characterization of the cDNA and the gene coding for the mouse SAPS will be accomplished through the expected homology between the human and mouse genes, as we have successfully done for cloning the mouse acid beta-galactosidase. Physiological functions of the SAP-gene products will be evaluated with metabolic loading of potential substrates in vivo and also in culture systems. The normal clones will be over-expressed and purified so that we can obtain milligram quantities of these proteins for subsequent biochemical experiments. The studies of the structure/function relationship of these proteins will extensively utilize site-directed mutagenesis based on the knowledge gained from naturally-occurring mutations and their enzymological properties coupled with information from computerized molecular modeling. The transgenic lines of mice will be generated by the homologous recombination technology using appropriate constructs of the mouse beta-galactosidase and SAP genes. 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 should contribute to our eventual understanding of the molecular pathogenesis of these serious genetic neurological disorders and to future attempts at the gene therapy of these disorders.