The cellular slime mold, Dictyostelium discoideum, provides an ideal model system to study eukaryotic development, cell differentiation, and aging. A crucial developmental event in Dictyostelium is glycogen degradation. The degradation of glycogen provides glucose monomers that are used to synthesize structural components necessary for cellular differentiation. Glycogen degradation is catalyzed by glycogen phosphorylase, and two developmentally regulated glycogen phosphorylase activities have been discovered in Dictyostelium. Glycogen phosphorylase 1 (gp-1) activity is predominant early in development, and is dependent upon 5’ AMP as a positive allosteric modifier; glycogen phosphorylase 2 (gp-2) activity peaks late in development and is independent of 5° AMP. I showed that these two glycogen phosphorylase activities are associated with unique proteins that are the products of two distinct, but related, genes. Both genes were observed to be typical Dictyostelium genes in a number of respects. The gp-1 and gp-2 enzymes were also found to be similar to glycogen phosphorylases from other organisms. I also examined the developmental expression of these genes and found that both mRNAs are developmentally regulated; gp-1 mRNA levels fluctuate during development, while gp-2 mRNA levels increase late in development. The expression of the gp-1 and gp-2 genes is regulated by exogenous cAMP. Exogenous cAMP enhances the level of gp-1 mRNA, apparently through a mechanism that requires intracellular cAMP signaling. Specific DNA sequence elements appear to be required for maximal vegetative and late developmental expression of gp-1. Exogenous cAMP induces the appearance of gp-2 mRNA via a mechanism that appears to be independent of intracellular cAMP signaling. Repeated TA-rich sequences located between nucleotides 193 and 305 upstream of the transcriptional start site are necessary for maximal cAMP induction of gp-2. I also examined the cell type specific expression of gp-1 and gp-2. gp-1 is expressed predominantly in pre-stalk cells. gp-2 1s expressed in both cell types in a temporally regulated fashion; this type of expression has not been reported for other Dictyostelium genes, but, given the importance of glycogen degradation in both stalk and spore cells, it is not inconceivable that such regulation 1s necessary. / Ph. D.

Development of multicellular organisms is one of the most fundamental but least understood biological processes. Due to its simple life cycle, the lower eukaryote Dictyostelium has been used as a model system to study several basic biological problems, such as cell differentiation, cell motility, cell adhesion, signal transduction, and especially gene regulation. Glycogen phosphorylase is the enzyme that initiates one of the key biochemical pathways, glycogen degradation, during Dictyostelium discoideum development. Two forms of glycogen phosphorylase, gpl and gp2, exist in D. discoideum with gp1 being active in vegetative cells and gp2 in differentiating cells. Study of glycogen phosphorylase gene regulation clearly will provide insight into the molecular mechanism of D. discoideum development and facilitate understanding of development in general. Two distinct genes that encode the two forms of glycogen phosphorylase were cloned. The nucleotide sequence analysis of the gp2 gene revealed an open reading frame of 2976 bp, that consists of three exons separated by two introns. An interesting feature in the gene is a 45 bp sequence in the second exon that contains 11 CAA trinucleotide repeats. The entire 5' and 3' non-coding regions of the gp2 gene and the whole 5' noncoding region of the gp1 gene have also been cloned. The regulation of the gp2 gene by Dictyostelium developmental signals was studied. Both cyclic AMP (cAMP) and Differentiation Inducing Factor (DIF) were discovered to induce gp2 gene expression during differentiation. DIF was also found to inhibit the cAMP responsiveness of the gene. Both cAMP and DIF induction of the gene were repressed by NH₃. Another developmental signalling molecule, adenosine, was involved in gp2 gene regulation through the inhibition of the DIF-mediated expression. The cell-type-specificity of the gp2 gene were also investigated. The gene was found to be expressed in both prestalk/stalk and prespore/spore cells. This is in agreement with the cAMP and DIF inducibility of the gene since the former molecule is a spore-cell morphogen, while the latter is a stalk-cell morphogen. A model of gp2 gene regulation during development is proposed, based on these findings. The two gp? introns and the 45 bp CAA repeat were studied by deletion of these elements. However, there were no alterations of gp2 gene expression observed after these deletions. Also investigated was genomic structural alteration in gp1- mutants that were obtained through homologous recombination and antisense RNA. Southern analysis revealed that the normal gp1 gene was disrupted in all homologous recombination transformants and in half of the antisense RNA transformants. Finally, for the first time, an extrachromosomal luciferase reporter vector has been established for the study of cis-acting regulatory elements in D. discoideum. / Ph. D.