CHEMICAL PATHOLOGY OF NEUROLOGICAL DISORDERS

我计划在我的实验室里从事几个方面的工作， 在生物化学领域得到了该项目赠款的支持， 溶酶体酸水解酶的分子遗传学。 两个基因编码 研究溶酶体蛋白; GM 1 β-半乳糖苷酶和 鞘脂激活蛋白（SAPS）。 在这些基因中缺失 导致不同的神经系统疾病，-GM 1-神经节苷脂沉积症/ 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Gaucher样综合征（SAP-1和SAP-2 缺乏分别），和一个独特的多形性综合征（总SAP 不足）。 人cDNA和编码其他蛋白质的基因在 以上列表已被克隆。 此外，我们还克隆了 和小鼠GM 1 β-半乳糖苷酶的基因。 研究项目 主要包括：（1）小鼠cDNA的克隆和鉴定， 编码鞘脂激活蛋白（SAP）的基因，（2）功能 SAP基因翻译产物的研究;（3）SAP基因翻译产物的研究; SAP和GM 1 β-半乳糖苷酶结构/功能关系，（4） 转基因生产总SAP缺陷和GM 1- 神经节苷脂沉积症（GM 1 β-半乳糖苷酶缺乏症） 重组技术，和（5）突变的持续鉴定 在由于上述缺陷而患有疾病的患者中， 列出的基因，并试图通过基因型/表型相关性， 自然发生的突变，定点诱变，计算机- 辅助分子建模和表达系统。 标准 方法包括cDNA和基因组文库构建，筛选， 聚合酶链反应，测序，等位基因特异性 寡核苷酸筛选和COS I中的瞬时功能表达 细胞 cDNA的分离和鉴定以及编码 对于小鼠SAPS，将通过预期的同源性来实现 人类和老鼠基因之间的联系，就像我们已经成功地 克隆小鼠酸性β-半乳糖苷酶。 生理功能 SAP基因产物将用代谢负荷进行评价， 在体内和培养系统中的潜在底物。 正常 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 这些结构/功能关系的研究 蛋白质将广泛利用基于 从自然发生的突变及其 酶的性质加上信息从计算机 分子模拟 小鼠的转基因品系将通过以下方法产生： 同源重组技术使用合适的构建体， 小鼠β-半乳糖苷酶和SAP基因。 天然存在的和 诱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。 新知识的产生 这一系列的研究应该有助于我们最终理解 这些严重的遗传性神经疾病的分子发病机制 本发明还涉及这些疾病的基因治疗的未来尝试。

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.