Vision has profound effects on the evolution of organisms by affecting survivorship through such basic behaviors as mate choice and foraging strategies. Its importance is strongly indicated by the presence of visual pigments in a whole array of organisms ranging from bacteria to human. Here, we propose to continue our molecular evolutionary analyses by using a diverse range of species: marine lamprey (Petromyzon marinus), coelacanth (Latemeria chalumnae), American chameleon (Anolis carolinensis), and pigeon (Columba livia). Using these species, we plan to investigate molecular genetic mechanisms that underlie the processes of dim and color visions in vertebrates. By constructing genomic DNA libraries, cloning and nucleotide sequencing, we plan to characterize all opsin genes from these species. These sequence and other available opsin gene sequences will be subjected to extensive phylogenetic analyses. From statistical analyses, we intend to learn the processes of the opsin gene evolution, including gene duplications and functional differentiations of the orthologous and paralogous opsin genes in vertebrates. Using information collected from sequence comparisons and knowledge of naturally-occurring species-specific wavelength of maximal absorptions (lambda-max), we will also identify the potentially important amino acid (and nucleotide) substitutions which may cause changes in the lambda-max values. The opsin genes found will be expressed in cultured COS cells, reconstituted with 11-cis-retinal, and measured for the absorption spectra of the opsins encoded. The effects of the unique nucleotide (and amino acid) substitutions will be investigated by generating mutants using site- directed mutagenesis and conducting the COS cell expression experiments. In this way, we will rigorously evaluate the effects of specific nucleotide substitutions on the functional changes of any opsin.
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