CONTROL OF CALCIUM AND PHOSPHORYLATION-REGULATED SIGNALLING PATHWAYS

对哺乳动物cDNA和基因组克隆的催化亚基的分析 钙调蛋白依赖性蛋白磷酸酶，钙调神经磷酸酶 表明由选择性剪接产生多种分子同种型 不同的基因。这些基因之间的进化关系 已被表征，因为它们在人类染色体上的定位， mRNA表达的差异。为了研究这种基因的起源 复杂性及其监管控制，我们正在比较相应的 低等真核生物的基因。在这些简单的生物体中， 在催化和调节结构域中高度保守，而 羧基末端附近的不相关序列表明可能赋予 独特的底物特异性。在链孢菌内含子/外显子边界中 与我们在哺乳动物基因中发现的基因相对应，这表明 逐步进化的DNA控制功能。在脉孢菌和 在网骨藻中，mRNA表达似乎受发育控制， 并且正在进行检查启动子调节的研究。一 具有高酶活性的细菌表达的真菌催化亚基 并产生了一组哺乳动物蛋白的缺失突变体 描绘了CaM结合所需的最小结构域; 基于该区域的合成肽表现出抑制常数为 四分之一5 nM。对磷酸二酯酶（PDE）的发育研究表明， 在大多数大脑中与突触发生一致的表达模式 区域; E.M.现在正在研究PDE在神经元中的定位， 提出其可能的职能作用。一种新的脑特异性钙调素结合蛋白 140 kDa（天然MW）的蛋白质已被表征为定位于 主要是在边缘结构中。

Analysis of mammalian cDNA and genomic clones for the catalytic subunit of the calmodulin (CaM)-dependent protein phosphatase, calcineurin (CN) indicate multiple molecular isoforms that result from alternative splicing of distinct genes. The evolutionary relationship between these genes has been characterized, as have their localizations on human chromosomes and differences in mRNA expression. To study the origins of this genetic complexity and its regulatory controls, we are comparing the corresponding genes in lower eukaryotes. In these simpler organisms primary structure is highly conserved in catalytic and regulatory domains, while regions of unrelated sequence near the carboxyl terminus suggest areas that may impart unique substrate specificity. In Neurospora intron/exon boundaries corresponded to those we have found in the mammalian gene, suggesting a step-wise evolution of DNA control features. In both Neurospora and Dictyostelium, mRNA expression appears to be developmentally controlled, and studies to examine promoter regulation are being pursued. A bacterially-expressed fungal catalytic subunit having high enzyme activity was produced and a set of deletion mutants of the mammalian protein delineated the minimum structural domain needed for CaM binding; a synthetic peptide based on this region exhibited an inhibitory constant of one-fourth 5 nM. Developmental studies on phosphodiesterase (PDE) showed a pattern of expression that coincided with synaptogenesis in most brain areas; the e.m. localization of PDE in neurons is now being studied to suggest its possible functional roles. A novel brain-specific CaM binding protein of 140 kDa (native MW) has been characterized that is localized primarily in limbic structures.