Enhancer regions responsible for temporal and cell-type-specific expression of a spore coat gene in Dictyostelium

The extracellular spore coat of Dictyostelium discoideum is composed of three major proteins, SP96, SP70, and SP60, encoded by the cotA, cotB, and cotC genes, respectively. The spore coat proteins are coordinately synthesized in prespore cells shortly after aggregation, stored in prespore vesicles during the slug stage, and secreted during encapsulation of spores. We have ligated various portions of the upstream region of cotB to lacZ such that a protein consisting of the first nine amino acids of SP70 fused to beta-galactosidase is synthesized in prespore cells. Individual cells that accumulate the enzyme can be observed in situ during early aggregation due to the sensitivity of the assay. We have found that prespore cells first appear in a random distribution throughout the aggregates with no indication of spatial localization. They subsequently sort out from prestalk cells that form a tip on the aggregates. The cotB regulatory region was subdivided into a proximal and a distal region, each of which could independently direct proper temporal and cell-type control. Transcriptional activity directed by these two regions appears to be additive in the full-length regulatory region. The proximal region was shown to be complex in that removal of certain portions partially reduced transcriptional activity while removal of other portions abolished all activity. Nevertheless, cells transformed with constructs showing attenuated activity expressed the fusion gene at the proper time in development and the activity was localized to prespore cells. The cis-acting regions responsible for all aspects of cotB regulation appear to be closely opposed within the minimal essential sequence of the proximal region.