The aim of the research proposed in this grant is to gain insight into the cellular and molecular basis of phototransduction in the neuronal photoreceptor cell. It is expected that the results obtained from these studies will help our understanding of the basis of sensory reception and information processing in biological systems. Over the past 22 months we have concentrated our efforts in the isolation and characterization of genes important for photoreceptor cell function. We have isolated the genes encoding four distinct Drosophila opsins. One is expressed in the six outer photoreceptor cells (R1-R6), the second in the central R8 photoreceptor cell, and the other two in the UV sensitive R7 photoreceptor cells. We have determined the structure and nucleotide sequence of each of these genes. We have used P element-mediated gene transfer to introduce the cloned structural gene for the R1-R6 opsin into the Drosophila germline and restored the ninaE mutant phenotype to wild-type. In an attempt to study the contribution of the various opsins to the specific functional properties of the different photoreceptor cell types, we have genetically engineered Drosophila lines that express R8 opsin in the R1-R6 photoreceptor cells. The experiments proposed in this grant are a continuation, and an extension, of these studies. Focusing on the Drosophila visual system, we will: (1) Attempt to isolate the additional genes encoding the different opsins expressed in each photoreceptor cell type of Drosophila. (2) Determine the primary sequence and gene structure of each of these opsin genes. (3) Determine the anatomical sites of expression of the different rhodopsins, and (4) attempt to determine the basis for the photoreceptor cell specificity of the different genes. We will use oligonucleotide site-directed mutagenesis to mutate selected amino acids and regions of the rhodopsin molecule and reintroduce the mutated genes into the germline of Drosophila melanogaster by P element-mediated gene transfer to: (5) Study the basis of the different spectral sensitivities of the various opsin proteins, and (6) analyze structure-function relationships in the rhodopsin molecule. The latter two aims will also be approached by constructing hybrid genes between the different opsins. These constructs will also allow the expression of the different opsins in photoreceptor cells in which they are not normally expressed so that we can study the physiological activities and specific properties of the different rhodopsins in relation to the various photoreceptor cell types. (7) Finally, we will isolate genes encoding other polypeptides involved in the phototransduction process.
| Cook, Boaz; Hardy, Robert W; McConnaughey, William B et al. (2008) Preserving cell shape under environmental stress. Nature 452:361-4 |
| Zelhof, Andrew C; Hardy, Robert W; Becker, Ann et al. (2006) Transforming the architecture of compound eyes. Nature 443:696-9 |
