MOLECULAR STUDIES IN A MODEL OF MOTOR NEURON DISEASE

这项提案中概述的实验试图分离和表征 导致摇摆器(WR)突变的基因和基因产物 小鼠，一种脊髓性肌萎缩的模型。将使用一种新的方法， 应用差减和差异杂交技术分离腹侧 脊髓特异的cDNA。然后这些探头将被用来分离 筛选正常大鼠脊髓cDNA库的WR候选克隆 以及从WR小鼠腹侧脊髓中获得的cDNA库。 发育和成年小鼠对照和摇摆器基因的Northern杂交 将与候选的WR cDNA杂交，以确定 表达的大小和时间。这将有助于进一步缩小 潜在的WR克隆。对照、杂合子和摇摆者的Southern杂交 DNA还将与潜在的WR cDNA杂交，以确定 它们是正常动物和患病动物之间的结构差异。 DNA水平。推测的WR基因的核苷酸序列将是 确定并推导出反丁酸序列。如果蛋白质是 已知的，抗体和探针将被用来进一步表征 它在WR表型中的作用。如果它是一种未知的蛋白质，合成的 将制造多肽，并将提高抗体以分离和 确定WR蛋白的特征。还将进行染色体分析 利用作图技术，将对基因组文库进行筛选 推测的基因。这项工作将有助于更好地理解 前角细胞的生理学和病理学，希望能提供新的见解 人类运动神经元病。

The experiments outlined in this proposal seek to isolate and characterize the gene and gene product, responsible for the mutation of the wobbler (wr) mouse, a model of spinal muscular atrophy. A novel approach will be used, applying subtractive and differential hybridization, to isolate ventral spinal cord-specific cDNAs. These probes will then be used to isolate candidate wr cDNA clones by screening a spinal cord cDNA library of normal mice, and a cDNA library made from the ventral cord of the wr mouse. Northern blots of control and wobbler mRNA of developing and adult mice will be hybridized with the candidate wr cDNAs to determine differences in size and time of expression. This will help further narrow down the potential wr clones. Southern blots of control, heterozygote, and wobbler DNA will also be hybridized to the potential wr cDNAs to determine whether them are structural differences between normal and affected animals at the DNA level. The nucleotide sequence of the putative wr cDNA will be determined and antino acid sequence will be deduced. If the protein is already known, antibodies and probes will be used to further characterize its role in the wr phenotype. If it is an unknown protein, synthetic peptides will be made and antibodies will be raised to isolate and characterize the wr protein. Chromosomal analysis will also be performed using mapping techniques, and genomic libraries will be screened for the putative gene. This work will lead to an improved understanding of anterior horn cell physiology and pathology, and hopefully give new insight into human motor neuron disease.