A fundamental question in aging research concerns the molecular mechanisms of gene regulation in cells undergoing programmed differentiation. Dictyostelium provides a model system of programmed aging in which developmentally-controlled and cell-type- specific gene expression are highly responsive to external experimental conditions. One of the pivotal steps in the aging process in Dictyostelium is the conversion of glycogen to the vital structural end-products of terminal differentiation. The enzyme that initiates this biochemical pathway, glycogen phosphorylase, is developmentally- and cAMP-regulated, and is known to exist in two forms, gp1 and gp2. In this proposal we will test the hypothesis: (1) that one or both of the enzymes is critical to the differentiation process, (2) that cAMP mediates the expression of both gp1 and gp2 at the level of the gene, and (3) that elements upstream of the genes encoding glycogen phosphorylase 1 and 2 contain sequences that are involved in stage and cell-type specific gene expression. cDNA clones coding for both gp1 and gp2 will be used in a series of experiments that will examine: the expression of the phosphorylase genes during the normal time course of differentiation, the factors (initial signals and secondary messengers) that influence the levels of gp1 and gp2 transcription, and the cell-type enrichment for the gp1 and gp2 transcript. To determine the developmental significance of the enzymes, gp-mutants will be prepared by traditional chemical mutagenesis or by the recently developed DNA-mediated transfection techniques for Dictyostelium, including the isolation of homologous recombinants and the introduction of antisense constructs. We will attempt to: (1) rescue gp- mutants by transfection with a complete gp1 and gp2 cDNA coding region, and (2) study the effect of increased amounts of glycogen phosphorylase or displaced developmental expression of gp1 and gp2 by placing the coding regions under alternate Dictyostelium promoters. The physiological significance of post- translational modifications of the protein will be evaluated by site-directed mutagenesis of regions of DNA coding for suspected regulatory sites. Mutagenized sequences will be transfected into gp- cells, and the transformants, will then be assayed for the levels of phosphorylase activity and the response to perturbations in liquid culture. For the study of gp1 and gp2 gene regulation, genomic fragments containing the phosphorylase genes will be isolated, mapped, and sequenced, and areas 5' to the transcription initiation site will be compared to known Dictyostelium promoters and upstream elements for cAMP regulated genes in other systems. Regions critical for efficient cell-type or stage-specific transcription will be determined by deletion analysis or by in vitro mutagenesis of certain sequences in conjunction with the transformation of Dictyostelium cells. Future experiments, if applicable, will include footprinting or gel retention techniques for the study of trans-acting regulatory factors.
