PROJECT SUMMARY Robert Dores Gene duplication is a recurring theme in the evolution of vertebrate polypeptide hormones and neuropeptides. These duplication events lead to the formation of gene families in which divergence of function is the usual outcome. In the case of the opioid-coding genes, duplication events have proceeded along two paths: a) an apparent duplication of function (both Proenkephalin and Prodynorphin circuits function as inhibitory networks in the central nervous system); or b) divergence of function as seen in analgesic activity of Proenkephalin and Prodynorphin products, as compared to the heightened pain responsiveness (nociceptic) activity of Pronociceptin products, or the melanocortin (color change and chronic stress regulation) activity of Proopiomelanocortin products. Are these duplication events entirely random, or do they correspond to discrete points of radiation of the vertebrates? This proposal develops the hypothesis that the duplication-driven expansion of the opioid-coding gene family (Proenkephalin, Prodynorphin, Pronociceptin, and Proopiomelanocortin) corresponds to periods when polyploidization (replication of the entire genome) altered the course of vertebrate evolution. To identify these "burst" periods, Dr Dores and colleagues have combined a comparative approach with a molecular approach. The species selected for this analysis (lungfish, urodele amphibians, and a ancient lineage of anuran amphibians) represent lineages that bracketed one of the predicted genome duplication events (the rise of the lobed finned fish and tetrapods in the Devonian). Thus, by taking a comparative approach it is possible to select taxa that fit into a logical phylogenetic hypothesis based on the fossil record. By employing the molecular approach, it is possible to test that hypothesis. In this study, the opioid-coding gene family will be used as a model to analyze how natural selection acts on duplicated genes to alter sequence, and potenti ally alter function. The key to the molecular approach is to focus on an ancestral character common to all members of this gene family - the opioid core sequence YGGF(M/L). By taking this approach, we have already detected genes in ray-finned fish and lobe finned fish (previous period of support) which were previously unsuspected. In addition, novel opioid peptides have been revealed whose opiate agonist potential has not been evaluated. While it is appreciated that none of the taxa used in this study are a "living fossil," by taking a comparative/molecular approach it is possible to reconstruct, through cladistic paradigms (maximum parsimony), the most likely pathways which have lead to the extant opioid-coding genes, and the potential ramifications of these genes with respect to evolution of neuronal circuits in the vertebrate central nervous system.
