TWITCHER--MODEL OF A HUMAN GENETIC DISORDER

提出了三大类研究项目。这些是 沿着我们现在在基因研究中必须走的主要方向设计 中枢神经系统疾病，其中根本的遗传原因是 阐明了其致病机制及DNA的异常 水平。在一系列体内研究中，将试图确定 体内异常蓄积的半乳糖神经酰胺的代谢来源 抽动小鼠的肾脏。假设是半乳糖基神经酰胺 可能是神经系统髓鞘变性的结果 可能在球形细胞内以吞噬的形式运输到 肾脏。与这个问题有关，可能存在循环 将在抽搐中寻找针对半乳糖神经酰胺的抗体。在……里面 另一系列实验，单核-巨噬细胞对 半乳糖基神经酰胺将进行生化检查，因为半乳糖神经酰胺 具有诱导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患者体内 大脑。移植对患者临床、病理及预后的影响 将对受影响的小鼠的酶学表现进行检查。在……里面 平行试验，外源补充剂的效果 半乳糖神经酰胺酶和共培养的正常组织将在 器官型中枢神经系统培养系统。获得抗体或抗体的两种方法 提出了小鼠半乳糖基神经酰胺酶的部分氨基酸序列； 尝试以非活性形式获得纯酶并将其用于生产 抗体和氨基酸序列测定，并获得抗体。 贝类的β-半乳糖苷酶。这种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-半乳糖苷酶由一种酶进化而来 这种贝类的酶可能代表了原始的 处于进化分支点的β-半乳糖苷酶。无论是使用 抗体或由该氨基酸制备的寡核苷酸混合物 序列，将尝试克隆半乳糖基神经酰胺酶cDNA. 构建于lambda gt11中的小鼠肾脏cdna文库的筛选。如果 成功后，将对该基因进行测序，克隆出该基因 Twitcher突变的尝试和特征 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.