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.
