The goal of the proposed work is to gain insight into the function of ras in eukaryotic cells. For this, we will use the relatively simple developmental system Dictyostelium discoideum. We have constructed a missense mutation (Gly12-Thr12) in the Dictyostelium ras gene. When this is transformed into Dictyostelium, the transformed cells develop abnormally and do not form mature fruiting bodies. The abnormalities suggest that the observed phenotypes result from an alteration of the extracellular-cAMP-receptor signal transduction pathway. We propose experiments to examine the function of the ras protein in this pathway during Dictyostelium development. This will involve expressing wild-type ras and the ras-Thr12 missense mutation using promoters from Dictyostelium genes which are expressed at specific developmental stages. This will be combined with a biochemical analysis of the different steps within the signal transduction pathway in normal and transformed cells. We will also select and analyze second site suppressor mutations of the ras-Thr12 gene. It is hoped that the proposed study of ras in Dictyostelium will result in a better understanding of its function both in this organism and in other organisms including mammals. Because ras overexpression or missense mutations have been associated with a large number of human cancers and can transform cells in tissue culture, we hope that the work described in this proposal will also lead to a better understanding of these processes.
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