TWITCHER--MODEL OF A HUMAN GENETIC DISORDER

提出了三大类研究项目。 这些是 沿着我们现在在遗传学研究中必须采取的主要方向进行设计， 中枢神经系统疾病，其中基本遗传原因是 阐明了其发病机制和DNA异常 水平 在一系列体内研究中，将尝试确定 半乳糖神经酰胺的代谢来源， 抽搐小鼠的肾脏。 假设半乳糖神经酰胺 由于神经系统中的髓鞘变性而产生的， 运输，也许在球形细胞内的吞噬形式， 肾脏 与此相关的问题，可能存在的循环 将寻找针对抽搐中的半乳糖神经酰胺的抗体。 在 另一系列实验，单核细胞-巨噬细胞对 半乳糖神经酰胺将进行生化检查，因为半乳糖神经酰胺 具有引发球状细胞反应的独特能力。 正常 肾脏或脑组织将被移植到受影响的抽搐者中， 大脑 移植对临床、病理和免疫功能的影响 将检查受影响小鼠的酶学表现。 在 平行实验，外源性补充的影响， 半乳糖神经酰胺酶和共培养的正常组织将在 器官型CNS培养系统。 两种方法获得抗体或 提出了鼠半乳糖神经酰胺酶的部分氨基酸序列; 试图获得无活性形式的纯酶，并用它来生产 抗体，也用于氨基酸测序，并获得抗 一种贝类β-半乳糖苷酶。 该β-半乳糖苷酶具有活性 半乳糖基神经酰胺 这个项目是基于我的假设， 两种哺乳动物溶酶体β-半乳糖苷酶从一种酶进化而来 这种贝类酶可能代表了 进化分支点上的β-半乳糖苷酶。 与所述 抗体或由氨基酸制备的寡核苷酸混合物 序列，克隆半乳糖基神经酰胺酶cDNA将尝试通过 筛选在λ gt 11中构建的鼠肾cDNA文库。 如果 成功后，将对cDNA进行测序，并克隆该基因 尝试，和表征抽搐突变的水平上， 核酸将变得可行。

Three major categories of research projects are proposed. These are designed along the major directions we must take now in research on genetic diseases of the CNS in which the fundamental genetic causes have been clarified; the pathogenetic mechanisms and the abnormality on the DNA level. In a series of in vivo studies, attempts will be made to determine the metabolic origin of galactosylceramide that accumulates abnormally in the kidney of twitcher mice. The hypothesis is that galactosylceramide generated as the result of myelin degeneration in the nervous system may be transported, perhaps in the phagocytized form within the globoid cell, to the kidney. Related to this question, possible presence of circulating antibody against galactosylceramide in the twitcher will be looked for. In another series of experiments, responses of monocytes-macrophages to galactosylceramide will be examined biochemically, since galactosylceramide possesses the unique capacity to elicit the globoid cell reaction. Normal kidney or cerebral tissues will be transplanted into affected twitcher brains. The effects of the transplant on the clinical, pathological and enzymological manifestations of the affected mice will be examined. In parallel experiments, effects of exogenous supplementation of galactosylceramidase and of co-cultured normal tissues will be examined in the organotypic CNS culture system. Two approaches to obtain antibody or a partial amino acid sequence of murine galactosylceramidase are proposed; attempt to obtain the pure enzyme in an inactive form and use it to produce antibody and also for amino acid sequencing, and to obtain antibody against a shellfish Beta-galactosidase. This Beta-galactosidase has activity toward galactosylceramide. This project is based on my hypothesis that the two mammalian lysosomal Beta-galactosidases evolved from a single enzyme and that this shellfish enzyme might represent the primordial Beta-galactosidase at the branching point of evolution. With either the antibody or an oligonucleotide mixture prepared from the amino acid sequence, cloning of galactosylceramidase cDNA will be attempted by screening a murine kidney cDNA library constructed in lambda gt11. If successful, the cDNA will be sequenced, cloning of the gene will be attempted, and characterization of the twitcher mutation on the level of nucleic acids will become feasible.