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Gene analysis of the superoxide dismutase gene in amyotrophic lateral screlosis

肌萎缩侧索硬化超氧化物歧化酶基因的基因分析

基本信息

批准号：
07670725
负责人：
UENO Satoshi
金额：
$ 1.41万
依托单位：
Nara Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670725/
关键词：
antioxidant superoxide amyotrophic lateral screlosis gene SOD

项目摘要

We have characterized the Cu-Zn superoxide dismutase gene (SOD1) in six Japanese families with familial amyotrophic lateral screlosis (FALS). Sequence analysis revealed two novel mutations in this gene from two independent families. A T-to-A transversion causes a Val17Glu substitution in one family. A T-to-G mutation leads to a Leu8Val substitutions in another family. Other families had unique clinical symptoms and normal coding sequences of the SOD1 gene, suggesting that FALS is a complex of heterogeneous motor neuron diseases. We studied the gene expression in the PC 12 cell line transfected with the mutant SOD1 cDNA.The mutant cDNA was synthesized by RT-PCR of mRNA from FALS patients. The cDNA clones were ligated into the pRc/RSV expression plasmid vector, transfected into PC12 cells. After selection with neomycin, the established cell line was culture under various condition, ie. low concentration of CSF or various CO2 concentration. No morphological changes indicating the apoptosis occurred. The fibroblasts from the FALS patient were cultured under the various condition, ie. hypoxia, low concentrations of FCS,the presence of oxidant or heat stress. The mRNA was purified from the treated fibroblasts of the patient and controls. The differential display techniques detected several bands that were specifically expressed in the patients. The band was excised out from the gel, PCR amplified, cloned into the vector and sequenced. Thus obtained cDNA contained the previously unreported sequences. The human brain cDNA library was constructed and was screened using the cloned. The identified plaques contained the same sequence as that we detected in the treated fibroblasts of the patients. This results suggests that the fibroblasts from the FALS patients responds to the heat stress by expressing some specific gene.

我们研究了6个家族性肌萎缩侧索硬化症(FALS)日本家系的铜锌超氧化物歧化酶基因(SOD1)。序列分析显示，该基因有两个新的突变，来自两个独立的家族。T-to-A易位导致一个家族中的Val17Glu替换。T-to-G突变导致另一个家族中的Leu8Val替换。其他家系具有独特的临床症状和正常的SOD1基因编码序列，提示FALS是一种异质性运动神经元病的复合体。我们研究了突变型SOD1cDNA在PC-12细胞中的基因表达。克隆入pRc/RSV表达载体，转染PC12细胞。经新霉素筛选后，将所建立的细胞系在不同条件下培养。低浓度的脑脊液或不同浓度的二氧化碳。未见细胞发生凋亡的形态变化。将FALS患者的成纤维细胞在不同的培养条件下进行培养。低氧、低浓度FCS、氧化剂或热应激的存在。从处理后的患者和对照组的成纤维细胞中提纯出该基因。差异显示技术检测到了几条在患者身上特异表达的条带。从凝胶中切下该条带，经聚合酶链式反应扩增，克隆到载体上并测序。这样得到的cDNA包含了以前未报道的序列。构建人脑c DNA文库，并用克隆的文库进行筛选。已鉴定的斑块包含与我们在处理后的患者成纤维细胞中检测到的相同序列。这一结果提示FALS患者的成纤维细胞通过表达某些特定的基因来应答热应激。