The promoters of the Dictyostelium cyclic nucleotide phosphodiesterase (PDE) regulate its expression during two critical periods in development. The first period occurs as the cells begin to starve and have to chose between growth and development. The second period occurs about eight hours later when a second promoter causes the PDE to be secreted by prestalk cells. Amoebae beginning development secrete a unique glycoprotein that inhibits the phosphodiesterase. Building on work that has examined the time and place of expression of the PDE and the PDI, and using new techniques that have become applicable to the organism, we will ask the following three questions: l. What genes and signal transduction pathways regulate the PDE and the PDI during the transition from growth to development? Using Restriction Enzyme Mediated Integration (REMI) and existing signal transduction mutants we will isolate and study genes that control the earliest events of development. Several variants of REMI will be described. 2. What are the important genes in cell-type specification? We have recovered the genes that are affected in two mutants created by REMI and others are in earlier stages of study. Methods for systematic analysis of these mutants and their genes will be described, as will a rationale for choosing the tight aggregate stage as a point of blockade. 3. Can fusogenic membranes produced during an alternative life cycle of this organism be used for a DNA delivery system that would increase the efficiency of transformation enough to carry out genetic complementation? Although REMI is a powerful technique, a full exploitation of the genetic potential of the organism requires genetic complementation with genomic or cDNA libraries.
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