MOLECULAR STUDIES IN A MODEL OF MOTOR NEURON DISEASE

本提案中概述的实验旨在分离和表征 基因和基因产物，负责wobbler（wr）突变 小鼠，脊髓性肌萎缩模型。 将使用一种新的方法， 应用差减杂交和差异杂交技术， 脊髓特异性cDNA。 这些探针将用于分离 通过筛选正常人脊髓cDNA文库， 小鼠，和从wr小鼠腹索制备的cDNA文库。 发育和成年小鼠对照和摆动mRNA的北方印迹 将与候选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wobbler的Southern印迹 DNA也将与潜在的wr cDNA杂交，以确定是否 它们是正常动物和患病动物之间的结构差异， DNA水平。 推定的wr cDNA的核苷酸序列将是 测定并推断其氨基酸序列。 如果蛋白质是 已知，抗体和探针将用于进一步表征 其在WR表型中的作用。 如果是未知蛋白质， 肽将被制造出来，抗体将被培养出来， 表征WR蛋白。 还将进行染色体分析 使用作图技术，基因组文库将被筛选， 推定基因 这项工作将使人们更好地了解 前角细胞生理学和病理学，并希望提供新的见解 人类运动神经元疾病

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.