ANALYSIS OF THE CALMODULIN CA++-BINDING SITES BY GENE

真核生物的许多细胞内过程都受钙离子的调控。 钙调蛋白是一种普遍存在的钙结合蛋白，它介导了许多 这些钙离子引起的调节变化。对其功能的阐释 钙调蛋白的四个独立的钙结合位点是 对这种蛋白质调节如此广泛的 细胞内突起的阵列。建议的研究旨在 两位强人对四个结合位点功能的研究 利用最近开发的三种技术的方法：i) 体外定点诱变，II)真核蛋白的表达 在细菌细胞中，以及iii)重新引入克隆的独特能力 基因回到黑腹果蝇的生物体并获得野生型 表情。 果蝇钙调蛋白基因的修改版本将是 制备，其中每个都呈现一个钙结合部位 通过对配体结合氨基的密码子进行突变而导致的无功能 酸。将对两个基因组的钙调蛋白进行相同的修改 基因和该基因的一个cDNA版。C DNA的修饰版本 基因将用于制备修饰的钙调素蛋白 细菌细胞。这将使体外研究的影响成为可能。 I)总的钙离子结合，ii)上每个结合位点的失能 Ca~(++)诱导构象变化，以及III)Ca~(++)诱导的相互作用 靶蛋白。基因组基因的修改版本将是 重新引入生物体并在钙调蛋白为零的胚胎中进行检测 它们支持钙调蛋白正常细胞功能的能力 在胚胎发育和随后的发育过程中。这些平行的研究 这四个结合位点在体内和体外的功能应产生 对他们的个人贡献的全新见解 钙调蛋白的调节功能。

Many intracellular processes in eukaryotes are regulated by Ca++ ions. Calmodulin is a ubiquitous Ca++-binding protein which mediates many of these Ca++-induced regulatory changes. An elucidation of the function of the four individual Ca++-binding sites of calmodulin is central to an understanding of the mechanism whereby this protein regulates such a wide array of intracellular processes. The studies proposed are aimed at an investigation of the function of the four binding sites by two powerful approaches which take advantage of three recently developed techniques: i) site-directed, in vitro mutagenesis, ii) expression of eukaryotic proteins in bacterial cells, and iii) the unique capability to re-introduce cloned genes back into the organism Drosophila melanogaster and obtain wild-type expression. Modified versions of the calmodulin gene of Drosophila melanogaster will be prepared, in each of which one Ca++-binding site has been rendered non-functional by mutagenesis of the codons for the ligand-binding amino acids. Identical modifications will be made to both the genomic calmodulin gene and a cDNA version of the gene. The modified versions of the cDNA gene will be used to prepare modified calmodulin proteins via expression in bacterial cells. This will permit in vitro studies of the effects of incapacitation of each of the binding sites on i) overall Ca++-binding, ii) Ca++-induced conformational changes, and iii) Ca++-induced interaction with target proteins. The modified versions of the genomic gene will be reintroduced into the organism and assayed in calmodulin null embryos for their capacity to support the normal cellular functions of calmodulin during embryogenesis and subsequent development. These parallel studies of the function of the four binding sites in vivo and in vitro should produce completely novel insights into their individual contributions to the regulatory functions of calmodulin.