Regulation Of Temporal And Spatial Gene Expression In Th

我们的单位感兴趣的是在时间和空间的调控模式的基因表达的分子机制。在2000财年，我们扩展了对昼夜节律电子盒的上下文特性的研究。这项工作很重要，因为E-box是一个极其混杂的DNA共识元素；因此，为了了解不同的信号通路如何区分不同的e -box，需要对原型启动子进行系统分析。通过对细胞周期蛋白B1 （CYC）、抗利尿激素（AVP）和芳基烷基胺n -乙酰转移酶（NAT） E-box-containing启动子进行这样的分析，我们获得了关于这些调节盒的模块化性质以及它们如何组合在一起以实现特异性的重要新信息。在一项相关的合作研究中，我们建立了两个新的转基因大鼠系，它们携带由野生型或e -box突变的NAT启动子驱动的荧光素酶报告基因。我们相信这些转基因细胞系将帮助我们验证我们的假设，即NAT E-box在视网膜主振荡细胞的NAT调控中起重要作用。相反，我们预测松果体对NAT的控制，围绕cAMP响应元件，将被证明对E-box失活是难以抑制的。第二个项目源于我们在1999财年发现的一个新基因，该基因使用酵母双杂交系统，似乎与周期（Per） 1特异性相互作用，周期（Per） 1是细胞机制的核心组成部分之一，在转录输出中产生自我维持的24小时周期。我们现在已经开发出一种针对这种新基因产物的优秀抗血清，这使我们能够确定这种蛋白质只在神经元中表达。确定该基因在体内功能的敲除方法仍在进行中。

Our Unit is interested in the molecular mechanisms underlying regulated patterns of gene expression, both in time and space. During FY 00 we have extended our studies of the contextual properties of circadian E-boxes. This work is important because the E-box is an extremely promiscuous DNA consensus element; thus, in order to understand how different signaling pathways can differentiate among different E-boxes, a systematic analysis of prototypic promoters is needed. By subjecting the cyclin B1 (CYC), vasopressin (AVP) and arylalkylamine N-acetyltransferase (NAT) E-box-containing promoters to such an analysis, we have obtained significant new information regarding the modular nature of these regulatory cassettes and how they are put together in order to achieve specificity. In a related collaborative study we have established two new lines of transgenic rats carrying a luciferase reporter gene driven by either the wild type or an E-box-mutated NAT promoter. We believe that these transgenic lines will help us test our hypothesis that the NAT E-box plays an important role in the regulation of NAT in the master oscillator cells of the retina. In contrast, we predict that pineal control of NAT, which revolves around cAMP responsive elements, will prove refractory to E-box inactivation. A second project is derived from our discovery during FY 99 of a novel gene, using a yeast two hybrid system, that appears to interact specifically with Period (Per) 1, one of the core components of the cellular machinery that generates the self-sustained 24 hr cycle in transcriptional output. We have now developed an excellent antiserum directed against this novel gene product, which allowed us to establish that the protein is expressed exclusively in neurons. The knockout approach to determine the function of this gene in vivo is still underway.