Molecular analysis of the genes involved in SCA8 ataxia

SCA8 共济失调相关基因的分子分析

• 批准号: 6529952
• 负责人: MICHAEL D KOOB
• 金额: $ 33.26万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6529952
• 关键词: Escherichia coli; RNA; antisense nucleic acid; artificial chromosomes; cerebellar ataxia /dyskinesia; electroporation; gene expression; gene interaction; gene targeting; genetic recombination; genetic regulation; genetically modified animals; immunocytochemistry; in situ hybridization; laboratory mouse; molecular pathology; neural degeneration; neurons; polymerase chain reaction; regulatory gene; tissue /cell culture

DESCRIPTION (Investigator's abstract): Spinocerebellar ataxia type 8 (SCA8) is caused by a CTG expansion in an untranslated, endogenous antisense RNA that overlaps the Kelch-like 1 (KLHL1) gene. The KLHL1 promoter and open-reading frame are conserved in mouse, and a KLHL1.antisense transcript (KLHL1AS) is present in mouse as well. We have performed initial characterization of the KLHL1AS and KLHL1 genes in both man and mouse, but we are at this point left with some fundamental questions regarding these genes: 1) What is the normal function of the evolutionarily conserved KLHL1-antisense RNA?, 2) What role does the KLHL1 protein play in the neurons in which it is expressed?, and 3) How does the CTG expansion affect the KLHL1AS RNA and KLHL1 protein, and can these effects explain the neurodegeneration seen in SCA8 patients? The data we have obtained to date have allowed us to make informed hypotheses concerning these questions and to design some in vivo experimental systems to test, refine, and if necessary reformulate these hypotheses. The transcriptional organization of the KLHL1 mRNA and the KLHL1AS transcript suggests that KLHL1AS is most likely a regulator of KLHL1 expression. Based on the homology of KLHL1 to other neuron-specific kelch-like proteins and on our preliminary characterization of this protein, we speculate that KLHL1 may play a role in organizing the actin fibers that help to generate and maintain axons and dendrites. We cannot predict a priori how the SCA8 CTG expansion may affect the KLHL1/KLHL1AS gene system. Both of these genes, however, are specifically expressed in the cerebellum, and so these transcripts are likely candidates for mediating the pathogenic effect of this expansion either directly or through altered antisense interactions. We will perform multiple, iterative modifications of a BAC clone encoding the human KLHLAS gene and the first two exons of KLHL1 in E. coli using homologous recombination. We will then introduce the modified BAC clones into tissue culture cel Is to directly determine how KLHL1 and KLHL1AS interact and measure what effect these interactions have on KLHL1 expression levels. Replacing the CTG repeat in the last KLHL1AS exon with expanded repeats in these clones will also allow us to test how the SCA8 expansion affects KLHL1AS and KLHL1, and how it alters the interactions between these genes. Although we will continue to characterize the structure, protein interactions and cellular and subcellular localization of the KIHI1 protein, we will directly test the role of this protein in normal neuronal development and function by generating a KLHL1 gene knockout in mouse. We have designed our knockout strategy in a way that will allow us to both 1) study the effects of disrupting the KLHL1 gene in a tissue-specific and temporal-specific manner, and 2) determine the effect that altered levels of KLHL1AS transcription has on KLHL1 expression and function.

描述（调查员的摘要）：脊椎脑性共济失调类型8（SCA8）是 由CTG扩展在未翻译的内源性反义RNA中引起的 重叠了类似Kelch的1（KLHL1）基因。 KLHL1启动子和开放阅读 框架在鼠标中保守，klhl1.antisense转录本（KLHL1AS）为 也存在于鼠标中。我们已经对 人和老鼠的klhl1as和klhl1基因 关于这些基因的一些基本问题：1）正常是什么 进化保守的klhl1-抗抗反感RNA的功能？，2）什么作用 KLHL1蛋白在其表达的神经元中是否发挥作用？和3） CTG扩展如何影响KLHL1AS RNA和KLHL1蛋白，并且可以 这些效果解释了SCA8患者中看到的神经退行性？ 迄今为止我们获得的数据使我们能够做出明智的假设 关于这些问题，并设计一些体内实验系统 测试，完善，并在必要时重新制定这些假设。这 KLHL1 mRNA和KLHL1AS转录本的转录组织 表明KLHL1A最有可能是KLHL1表达的调节剂。基于 KLH1与其他神经元特异性kelch蛋白的同源性以及我们 该蛋白质的初步表征，我们推测KLHL1可能会发挥作用 在组织肌动蛋白纤维的作用，有助于产生和维持轴突 和树突。我们无法先验地预测SCA8 CTG扩展可能如何影响 KLHL1/KLHL1AS基因系统。但是，这两个基因都是专门的 在小脑中表达，因此这些成绩单可能是候选人 直接或通过 反义相互作用改变。 我们将对编码的BAC克隆进行多次迭代修改 人类klhlas基因和大肠杆菌中Klhl1的前两个外显子使用同源 重组。然后，我们将将改良的BAC克隆引入组织 培养CEL是直接确定KLHL1和KLHL1A的相互作用和测量 这些相互作用对KLHL1表达水平有什么影响。更换 CTG重复在最后的KLHL1AS外显子中，在这些克隆中扩展重复序列将 还允许我们测试SCA8扩展如何影响KLHL1AS和KLHL1，以及如何影响 它改变了这些基因之间的相互作用。尽管我们将继续 表征结构，蛋白质相互作用以及细胞和亚细胞 Kihi1蛋白的定位，我们将直接测试此的作用 正常神经元发育和功能中的蛋白质通过产生KLHL1基因 鼠标中的敲除。我们已经以一种方式设计了我们的淘汰赛策略 允许我们同时研究1）研究破坏A中的KLHL1基因的影响 组织特异性和时间特异性的方式，2）确定 KLHL1AS转录的水平改变了KLHL1表达和功能。