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. ***
