9600772 Ma Rhodopsin is a light-absorbing protein residing in the outer segments of rods and mediates dim light vision. Amphibian photoreceptors are widely used models in vision research as they provide several advantages over mammalian cells. Salamanders have two types of rod cells in the retina: red rods and green rods, which have distinct absorbance spectra. The proposed research focuses on the molecular basis underlying the distinct absorbance peaks in the red and green rods, with the hypothesis that there exist two distinct rhodopsins (red and green) which are responsible for the distinct absorbance peaks. The first objective of this project is to clone and sequence the red and green rod rhodopsins in order to elucidate the primary structures and divergent residues of these two rhodopsin molecules. The second objective is to express the red and green rod rhodopsins in COS cells, regenerate pigments with a given chromophore, and measure the absorbance of the resulting pigments. The measurements using pure forms of the opsins and chromophores will provide precise determination of the absorbance spectra. The third objective is to identify amino acids responsible for the absorbance difference of the red and green rods using site-directed mutagenesis techniques. The long term goal of these studies is to understand the visual transduction system at a molecular level, which is of broad biological significance in that the visual receptor rhodopsin is the prototype of the large family of G protein-coupled receptors. %%% A career development program is proposed for integrating research that focuses on the molecular mechanisms of visual transduction with educational activities that bring molecular biology techniques into pre-college classroom settings. Dim light vision is mediated by a class of photoreceptor cells called rods. Salamanders provide a good source of rods for vision research because they have two types of rod cells, green rods and red rods, which mediate vision at different wavelength s. The molecular basis accounting for the difference has not been discovered. The goal of the research project is to clone the proteins responsible for the distinct color sensitivities in the two types of rods, and determine the structural difference of these two molecules. By changing the structure of the proteins at selected locations by site-directed mutagenesis, the part which is critical in controlling the function and color sensitivity of the molecules will be determined. An innovative education plan is also proposed using the MUSC Summer Institute for precollege teachers as a platform for bringing molecular biology and molecular genetics into South Carolina's high school classrooms. The Principal Investigator will serve as an instructor in the laboratory activities of molecular biology which include DNA sequencing, PCR, restriction enzymatic digestion, Southern blot analysis and site-directed mutagenesis. ***
