Bioluminescence:Molecular Mechanisms and Biochemical Control

该项目旨在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。生物发光作为基因表达和定位检测的标记物（通过光发射）是非常有用的，这已经导致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（苍蝇）幼虫。在刺激下，甲藻闪光从细胞质中的小细胞器发出，被类似于神经冲动的膜信号激活。独特的荧光素酶和荧光素酶蛋白位于细胞器中，反应由pH变化触发。荧光素酶具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其完整的晶体结构，然后是其复合物与Escherin及其相关的结合蛋白。与腰鞭毛虫相反，来自新西兰（Arachnocampa luminosa）和美国（Orfelia fultoni）的两种双翅目昆虫具有不同的发光系统，但它们占据相似的生态位，建立网络捕获猎物，进化成看起来相似的成年苍蝇，并发出所有昆虫中最蓝的光（460-484 nm）。蜘蛛网的反应需要ATP，就像甲虫一样;它的荧光素酶序列被获得，并发现与甲虫的同源。关于其表达和定性的工作已列入计划。将与Y.岸在Orfelia中，发光的生物化学似乎不符合任何已知的模型。涉及三个组成部分：荧光素酶（~140 kDa）、小分子量的荧光素酶和大分子量的组分，如果不是聚集体，则可能是蛋白质，当存在DDT或抗坏血酸盐时，其释放或激活荧光素酶与荧光素酶反应。更广泛的影响：对生物发光的基础研究通常是为了揭示它是如何发生的以及为什么会发生，可以在不相关的领域取得显着进展，并具有重大的生物技术意义。一个重要的例子来自细菌生物发光的研究，其中自诱导的发现导致了“群体感应”的概念，从而导致了细菌之间的化学通讯。另一个例子是绿色荧光蛋白（GFP），腔肠动物生物发光中的辅助发射体; 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基因。该项目还将涉及本科生培训和教育活动。为本科生提供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.