ALTERNATIVE SPLICING OF A POTASSIUM CHANNEL GENE

描述：选择性剪接是一种常见的遗传学现象， 产生多种蛋白质同种型的调节机制， 不同的功能特性。 选择性剪接特别常见 在神经和肌肉等易兴奋的细胞中， 离子通道和肌肉收缩蛋白显示出极其复杂的 组织和细胞类型特异性剪接模式。 这些基因产物 在决定兴奋性和收缩性的特性中的关键作用 这表明选择性剪接是基因表达的关键调控步骤， 维持神经正常功能所必需的基因的表达 和肌肉 大多数选择性剪接的研究依赖于体外系统。 因此，对顺式元件或反式作用因子知之甚少 共同决定组织特异性剪接模式。 在 本文所述的研究表明，黑腹果蝇的Shaker（Sh）基因是 被用作模型系统来检查体内调节机制 选择性剪接导致组织特异性剪接， 在动力学不同钾离子（K+）的组织特异性表达中， 渠道 使用lacZ作为报告基因用于调节剪接活性， 在转基因动物中，我们已经证明了240 nuc. 内含子区 包含在间接剪接中正确选择所需的顺式元件 飞行肌肉，并且该组织中的剪接是正调控的。 顺式元件将通过体内分析更精确地定义， 在携带这种缺失和插入的转化体中的剪接模式 240 nuc. 区域，并通过与相应内含子的序列比较 来自果蝇Drosophila virilis 一旦定义了顺式元件， 该项目将被扩展到确定的trans-acting因素，认识到 并通过UV交联和Northwestern分析与顺式增强剂结合， 将核蛋白转化为含有顺式元件的合成RNA。 分离 编码反式作用剪接因子的cDNA克隆， 与顺式结合将通过直接筛选 以顺式元件为探针构建胚胎表达文库， 使用酵母三杂交系统的表达文库，或生物化学 纯化，然后进行肽序列测定和筛选， 通过常规杂交方法构建文库。 的结果予以 实验将提供体外和体内之间的关键联系， 剪接研究，并预计将适用于机制， 所有生物体的可变剪接。 审查的重要性 K+通道基因可变剪接的调节被强调为 最近的证据表明，许多哺乳动物的K+通道基因， 选择性剪接和一些人类遗传性肌肉疾病 包括长QT综合征

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.