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: 6784132
• 负责人: MICHAEL D KOOB
• 金额: $ 33.25万
• 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/6784132
• 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（研究者摘要）：脊髓小脑共济失调 8 型 (SCA8) 是 由未翻译的内源性反义 RNA 中的 CTG 扩增引起 与 Kelch-like 1 (KLHL1) 基因重叠。 KLHL1启动子和开放阅读 框架在小鼠中是保守的，KLHL1.反义转录本（KLHL1AS）是 也存在于小鼠中。我们已经进行了初步表征 人类和小鼠中都存在 KLHL1AS 和 KLHL1 基因，但我们现在还剩下 关于这些基因的一些基本问题：1）什么是正常的 进化上保守的 KLHL1-反义 RNA 的功能？, 2) 有什么作用 KLHL1 蛋白在表达它的神经元中起作用吗？以及 3) CTG 扩增如何影响 KLHL1AS RNA 和 KLHL1 蛋白？ 这些效应可以解释 SCA8 患者中出现的神经退行性变吗？ 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表述这些假设。这 KLHL1 mRNA 和 KLHL1AS 转录本的转录组织 表明 KLHL1AS 很可能是 KLHL1 表达的调节因子。基于 KLHL1 与其他神经元特异性 kelch 样蛋白的同源性 通过对该蛋白的初步表征，我们推测 KLHL1 可能发挥作用 在组织肌动蛋白纤维中发挥作用，有助于生成和维持轴突 和树突。我们无法先验预测 SCA8 CTG 扩展可能会产生怎样的影响 KLHL1/KLHL1AS 基因系统。然而，这两个基因都特别 在小脑中表达，因此这些转录本可能是 直接或通过介导这种扩张的致病作用 改变反义相互作用。 我们将对编码 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。然后我们将修改后的 BAC 克隆引入组织中 培养细胞是直接确定KLHL1和KLHL1AS如何相互作用和测量 这些相互作用对 KLHL1 表达水平有何影响。更换 最后一个 KLHL1AS 外显子中的 CTG 重复以及这些克隆中的扩展重复将 还允许我们测试 SCA8 扩展如何影响 KLHL1AS 和 KLHL1，以及如何 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，我们将直接测试其作用 通过生成 KLHL1 基因来影响正常神经元发育和功能的蛋白质 小鼠中的基因敲除。我们设计的淘汰赛策略将 让我们能够 1) 研究破坏 KLHL1 基因的影响 组织特异性和时间特异性方式，以及 2) 确定效果 KLHL1AS 转录水平的改变会影响 KLHL1 的表达和功能。