Bioluminescence:Molecular Mechanisms and Biochemical Control

这个项目试图定义和理解生物发光的基本方面，从生物功能到遗传学和生物化学。生物发光是一种非常有用的标记(通过光发射)来检测基因的表达和定位，这已经导致了在生物研究和生物技术应用中的许多应用。该项目侧重于两种不同的生物发光系统：海洋甲藻和双翅目(蝇)幼虫。在刺激下，鞭毛虫的闪光由细胞质中的小细胞器发出，由类似神经冲动的膜信号激活。独特的荧光素酶和荧光素位于细胞器中，该反应由pH变化触发。荧光素酶有三个分子内同源和连续的重复单元，它们各自具有活性，这是一种不寻常的结构。一个主要的目标是获得它的完整的晶体结构，然后是它与荧光素的络合物及其相关的结合蛋白。与甲藻不同的是，来自新西兰和美国的两种双翅目有不同的发光系统，但它们占据着相似的生态位，构建网来捕捉猎物，进化成长得像成虫的苍蝇，并发出所有昆虫中最蓝的光(460-484 nm)。Arachnocampa反应需要ATP，就像在甲虫中一样；获得了其荧光素酶的序列，并发现其与甲虫的荧光素酶同源。其表达和鉴定工作正在计划中。荧光素的结构将与Y.Kishi教授合作确定。在奥菲利亚，光发射的生物化学似乎不符合已知的模型。它涉及三个组分：一个荧光素酶(~140 kDa)，一个小分子荧光素，以及一个大分子量组分，如果不是聚集体，它可能是一个蛋白质，当有DDT或抗坏血酸存在时，它会释放或激活荧光素与荧光素酶反应。这些结果表明了一种新的系统。广泛的影响：生物发光的基础研究通常是为了揭示它是如何以及为什么发生的，可以归功于在无关领域的显著进步和具有重大生物技术意义的应用。一个重要的例子来自对细菌生物发光的研究，在这个研究中，自我诱导的发现导致了“群体感应”的概念，从而导致了细菌之间的化学交流。另一个例子是绿色荧光蛋白(GFP)，它是腔肠动物生物发光的辅助发射体；GFP及其颜色突变体现在被广泛用作跟踪体内生化过程的报告。该项目还将涉及本科生培训和教育活动。为本科生开设了一个关于生物发光的新生研讨会。

This project seeks to define and understand basic aspects of bioluminescence, from biological functions to genetics and biochemistry. Bioluminescence is remarkably useful as a marker (by light emission) for the detection of gene expression and localization, which has already led to many applications in biological research and biotechnology applications. This project focuses on two different bioluminescent systems: marine dinoflagellates and dipteran (fly) larvae. Upon stimulation, dinoflagellates flashes are emitted from small organelles in the cytoplasm, activated by membrane signals similar to nerve impulses. The unique luciferase and luciferin are located in the organelle, the reaction being triggered by a pH change. Luciferase has three intramolecularly homologous and contiguous repeat units, which are individually active, an unusual structure. A major goal is to acquire its complete crystal structure, then that of its complex with luciferin and its associated binding protein. In contrast to the dinoflagellates, the two diptera, from New Zealand (Arachnocampa luminosa) and the US (Orfelia fultoni), have different light-emitting systems, yet they occupy similar niches, build webs to catch prey, evolve into look-alike adult flies and emit the bluest light of all insects (460-484 nm). The Arachnocampa reaction requires ATP, as in beetles; the sequence of its luciferase was obtained and found to be homologous to beetle's. Work on its expression and characterization is planned. The structure of luciferin will be determined in collaboration with Prof. Y. Kishi. In Orfelia, the biochemistry of light emission appears to fit no known model. Three components are involved: a luciferase (~140 kDa), a small molecular weight luciferin, and a component of large molecular weight, presumably a protein if not an aggregate, which releases or activates luciferin to react with luciferase when either DDT or ascorbate are present. The results suggest a novel system.Broader Impacts: Basic research on bioluminescence, often carried out for the pleasure of uncovering how and why it occurs, can be credited with remarkable advances in unrelated fields and with applications of great biotechnological significance. One important example comes from the study of bacterial bioluminescence, where the discovery of autoinduction led to the notion of "quorum sensing", and thus of chemical communication between bacteria. Another example is the green fluorescent protein (GFP), an accessory emitter in coelenterate bioluminescence; GFP and its color mutants are now widely used as reporters for tracking biochemical processes in vivo. The project will also involve undergraduate training and educational activities. A freshman seminar on bioluminescence is offered for undergraduate students.