TWITCHER--MODEL OF A HUMAN GENETIC DISORDER

点击翻译按钮获取中文摘要

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.