Bioluminescence: Molecular Mechanisms and Biochemical Control in Microorganisms

9631935黑斯廷斯这个项目涉及细菌和甲藻中发光系统的基本生化机制，这是两个主要的、进化上独立的生物发光有机体。对模型化学系统的研究将并行进行。在费氏弧菌Y-L菌株中，实验旨在确定辅助黄色荧光蛋白(YFP)不仅导致蓝色发光向黄色发光转变和发光反应速度增加的机制，而且还赋予反应负的温度系数，使得在4至20℃的较低温度下发光强度更大，YFP通过与反应中间体相互作用而作用，重组蛋白将被用于确定YFP是否翻转，即催化作用。一个目标是精确定位YFP与之反应的过氧化荧光素酶中间体(S)；反应速度非常慢的Dim V.fecheri荧光素酶突变体将通过定点突变构建，以测试YFP对改变动力学突变体的速度的影响。在甲藻系统中，荧光素酶基因最近被发现具有三个相似序列的区域。我们将表征由这些重复序列表达的单个活性多肽的催化特性，并确定活性所需的结构特征，以及串联催化位点的机制作用。我们将完成全长荧光素酶基因组和cDNA的序列和结构的测定，并寻找启动子和内含子。我们将寻找一种参与荧光素酶翻译控制的调节mRNA结合蛋白。在草酸酯的高效化学发光中，计划进行实验以确定反应活跃、能量丰富的中间体(S)以及电子或电荷转移在化学激发中的作用。黄昏时萤火虫的闪光和夜晚海洋中明亮的“磷光”是生物发光的两个例子，也存在于许多不同生物群的几个成员中。生物发光的功能也非常不同；一些动物用它来躲避或躲避捕食者，而另一些动物用它来吸引猎物。还有其他的，如萤火虫，使用闪光灯进行交流，例如在求爱时。对生物和化学发光的基本机制的理解有望促进我们在几个基本领域的知识，例如能量代谢和由活性氧物种(ROS)引起的细胞损伤，包括ROS作为细胞信使的可能作用。发光系统在医学上也有许多应用，特别是在药物和代谢物测量的临床分析中，以及在基因研究中敏感和明确的基因表达可视化。本实验室要研究的系统在很大程度上是未被探索的，所解决的问题在许多方面都是新的；因此，预计结果将对基础知识及其应用都有很大贡献。***

9631935 Hastings This project is concerned with the fundamental biochemical mechanisms of light emitting systems in bacteria and dinoflagellates, two major and evolutionarily independent groups of bioluminescent organisms. Studies of model chemical systems will be carried out in parallel. In the bacterium Vibrio fischeri strain Y-l, experiments are designed to determine the mechanism by which an accessory yellow fluorescent protein (YFP) causes not only a shift from blue to yellow luminescence and an increase in the rate of the light-emitting reaction, but also confers a negative temperature coefficient to the reaction, such that the intensity is greater at lower temperatures over the range of 4 to 20 C. YFP acts by interacting with a reaction intermediate, and recombinant proteins will be used to determine if YFP turns over, i.e., acts catalytically. A goal will be to pinpoint the peroxidic luciferase intermediate(s) with which YFP reacts; dim V. fischeri luciferase mutants in which the reaction rate is very slow will be constructed by site directed mutagenesis in order to test the effect of YFP on the rate of altered kinetic mutants. In the dinoflagellate system, the luciferase gene has recently been found to possess three regions with similar sequences. We will characterize the catalytic properties of the individual active peptides expressed by these repeat sequences, and determine the structural features required for activity, as well as a mechanistic role for the tandem catalytic sites. We will complete the determination of the sequence and structure of full length luciferase genomic and cDNAs and look for the promoter and for introns. We will search for a regulatory mRNA binding protein involved in the translational control of luciferase. In the efficient chemiluminescence of oxalate ester, experiments are planned to identify the reactive, energy-rich intermediate(s) and the role of electron or charge transfer in chemiexcitation. %%% The flash of fireflies at dusk and the brillia nt "phosphorescence" of the ocean at night are two examples of bioluminescence, present also in a few members of each of many different groups of organisms. The functions of bioluminescence are also very different; some animals use it to avoid or escape predators while others use it to attract prey. Still others, like fireflies, use flashing for communication, in courtship for example. An understanding of the basic mechanisms in bio- and chemiluminescence can be expected to advance our knowledge in several fundamental areas, for example energy metabolism and cellular damage caused by reactive oxygen species (ROS), including the possible roles of ROS as cellular messengers. There are also many applied uses of luminescent systems in medicine, particularly in clinical analysis for measurements of drugs and metabolites, and in genetic research for the sensitive and unambiguous visualization of gene expression. The systems to be studied in this laboratory are largely unexplored, and the problems addressed are novel in many respects; it is thus expected that the results will contribute significantly to both basic knowledge and its applications. ***