Genetic, cellular, and biochemical basis of functional senescence: The proposed research will attempt to characterize translational fidelity in human cells and transgenic mice in relation to age. The experimental approach will focus on two kinds of mistranslation: frameshifting (both 'programmed' and fortuitous) and readthrough of termination codons, at particular defined sites on transgenic constructs. Specifically, we will relate the frequency and extent of these phenomena to age of cell line, starvation, and immortalization status. Human cells in culture will be transfected with constructs designed to detect mistranslation events by enzyme activity. A defined site for frameshifting or nonsense readthrough will be flanked by genes for beta-galactosidase and luciferase. Successful transits of the mistranslation site will result in a minority population of fusion protein bearing both enzyme activities, and the ratio of these activities will describe the fraction of mistranslation events. This approach gives us the most sensitive method to date to investigate the impact of aging on protein synthesis.
