| The life cycle of Dictyostelium discoideum is characterized by a multicellular differentiation process culminating in the formation of a fruiting body primarily composed of two cell-types; stalk cells and spores. Precursor cells, prestalk cells and prespore cells, are induced upon formation of the multicellular aggregate and are distinguishable by the expression of cell-type specific genes. Induction of these aggregation-specific genes, and other essential events in Dictyostelium development, are regulated by cAMP interaction with cell-surface receptors.; In this thesis, a previously identified regulatory element of the cyclic AMP inducible aggregate-stage gene, pst-cath/CP2, and regulatory regions for three other coordinately regulated genes are examined for function in vivo and for the ability to interact with putative trans-acting factors.; A DNA-binding activity, denoted AT factor, was identified in nuclear extracts of developmentally induced cells. The factor binds to the regulatory region of the pst-cath/CP2 gene, and interacts with poly (dA-dT) sequences. Activity of the AT factor is induced in a cyclic AMP-independent manner, a few hours into development.; A second trans-acting factor, termed GBF (G-box binding factor) was identified in nuclear extracts of aggregate-stage cells. This factor recognizes specifically a G-rich regulatory element of the pst-cath/CP2 promoter. The specific activity of GBF correlates with the transcription of the pst-cath/CP2 gene during development, and increases dramatically following addition of cyclic AMP to developmentally competent Dictyostelium cells. Single point mutations, that affect the pst-cath/CP2 G-rich regulatory element and significantly reduce the ability of this element to activate pst-cath/CP2 expression in vivo, similarly affect the affinity of GBF for these sequences, suggesting a direct involvement of GBF in the transcriptional induction of this gene in vivo.; The putative involvement of GBF in the regulation of other cyclic AMP-inducible aggregate-stage genes was also examined. The factor interacts specifically with G/C-rich elements located in the upstream regions of some these genes. The affinity of GBF for each of these dissimilar G/C-rich elements correlates with the ability of the elements to rescue activity of a pst-cath/CP2 promoter deletion mutant, suggesting a similar GBF-dependent function for the G/C-rich elements in vivo. Mechanistic models for cyclic AMP-induction of aggregate-stage gene expression, that incorporate GBF as an essential element, are presented. |
