ALTERNATIVE SPLICING OF A POTASSIUM CHANNEL GENE

钾通道基因的选择性剪接

• 批准号: 2609619
• 负责人: Linda E Iverson
• 金额: $ 26.66万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2609619
• 关键词: Drosophilidae; RNA splicing; animal genetic material tag; arthropod genetics; developmental genetics; developmental neurobiology; gene induction /repression; genetically modified animals; invertebrate embryology; larva; molecular cloning; myogenesis; neurogenetics; nucleic acid repetitive sequence; potassium channel; protein structure function; reporter genes; transcription factor

DESCRIPTION: Alternative splicing of precursor-RNA is a common genetic regulatory mechanism that gives rise to multiple protein isoforms with distinct functional properties. Alternative splicing is particularly common in excitable cells such as nerve and muscle where many of the genes encoding ion channels and muscle contractile proteins show extremely complex tissue-and cell-type specific splicing patterns. These gene products play critical roles in determining properties of excitability and contractility suggesting that alternative splicing is a key regulatory step in the expression of genes essential for maintenance of normal function of nerve and muscle. Most studies of alternative splicing rely on in vitro systems. As a result, little is know about the cis-elements or trans-acting factors that act in concert to determine tissue-specific splicing patterns. In the research described here, the Shaker (Sh) gene of Drosophila melanogaster is being used as a model system to examine the in vivo mechanisms of regulation of alternative splicing that result in tissue-specific splicing that result in tissue-specific expression of kinetically distinct potassium ion (K+) channels. Using lacZ as a reporter gene for regulated splicing activity in transgenic animals, we have demonstrated that a 240 nuc. intron region contains the cis-elements required for correct splice choice in the indirect flight muscles and that splicing in this tissue is positively regulated. The cis-elements will be more precisely defined by in vivo analysis of splicing patterns in transformants carrying deletions and insertions of this 240 nuc. region and by sequence comparison to the corresponding introns from Drosophila virilis. Once the cis-elements have been defined, the project will be extended to identify the trans-acting factors that recognize and bind to the cis-ehancer by UV crosslinking and Northwestern analysis of nuclear proteins to synthetic RNA containing the cis-elements. Isolation of cDNA clones encoding the trans-acting splicing factors that recognize and bind to the cis-will be accomplished either by direct screening of an embryonic expression library using the cis-elements as probes, screening an expression library using a yeast three-hybrid system, or biochemical purification followed by peptide sequence determination and screening the library by conventional hybridization methods. The results of these experiments will provide a crucial link between in vitro and in vivo splicing studies and are expected to be applicable to mechanisms of alternative splicing in all organisms. The importance of examining the regulation of alternative splicing of a K+ channel gene is underscored by recent evidence indicating that many of the mammalian K+ channels genes are alternatively spliced and a number of human hereditary diseases of muscle including Long QT syndrome.

描述：前体-RNA的替代剪接是一种常见的遗传 引起多种蛋白质同工型的调节机制 不同的功能特性。 替代剪接特别普遍 在神经和肌肉等可激发细胞中，许多基因编码 离子通道和肌肉收缩蛋白表现出极其复杂的 组织和细胞型特异性剪接模式。 这些基因产品播放 在确定兴奋性和收缩性的特性中的关键作用 表明替代剪接是关键的调节步骤 表达对维持正常功能神经功能必不可少的基因的表达 和肌肉。 大多数替代剪接的研究都依赖于体外系统。 结果，几乎不了解顺式元素或反式因素 该行动共同确定组织特异性的剪接模式。 在 在这里描述的研究，果蝇的动摇基因是Melanogaster 被用作模型系统检查体内调节机制 导致组织特异性剪接的替代剪接 在动力学不同的钾离子（K+）的组织特异性表达中 频道。 使用LACZ作为报告基因进行调节的剪接活性 转基因动物，我们已经证明了240 nuc。 内含子区域 包含间接中正确剪接选择所需的顺式元素 飞行肌肉和该组织中的剪接受到积极调节。 顺式元素将通过体内分析更精确地定义 带有删除和插入的转化子中的剪接模式 240 nuc。 区域和按顺序比较相应的内含子 来自果蝇病毒。 一旦定义了顺式元素， 项目将扩展以确定识别的跨性别因素 并通过紫外线交联和西北分析结合到顺式欧洲 核蛋白与包含顺式元素的合成RNA。 隔离 cDNA克隆编码识别和 通过直接筛选来完成与顺式绑定 胚胎表达式库使用顺式元素作为探针，筛选 使用酵母三杂交系统或生化的表达式库 纯化，然后进行肽序列测定和筛选 通过常规杂交方法库。 这些结果 实验将在体外和体内提供至关重要的联系 剪接研究，预计将适用于 在所有生物体中的替代剪接。 检查 K+通道基因的替代剪接的调节被强调 最近的证据表明许多哺乳动物K+通道基因是 或者剪接的肌肉和许多人类遗传疾病 包括长QT综合征。