CSM News Electronic Edition Volume 1, number 7 June 26, 1993 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. ---------------------------------------------------------------------- cAMP-Dependent Protein Kinase Differentially Regulates Prestalk and Prespore differentiation during Dictyostelium development Sandra K.O. Mann and Richard A. Firtel University of California, San Diego Accepted by Development ABSTRACT We and others have previously shown that cAMP-dependent protein kinase (PKA) activity is essential for aggregation, induction of prespore gene expression, and multicellular development in Dictyostelium. In this manuscript, we further examine this regulatory role. We have overexpressed the Dictyostelium PKA catalytic subunit (PKAcat) in specific cell types during the multicellular stages, using prestalk and prespore cell-type-specific promoters to make PKA activity constitutive in these cells independent of cAMP concentration). To examine the effects on cell-type differentiation, we co-transformed the PKAcat-expressing vectors with reporter constructs expressing lacZ from four cell-type-specific promoters: ecmA (specific for prestalk A cells); ecmB (specific for prestalk B and anterior-like cells in the slug; ecmB-delta-89 (specific for stalk cells); and SP60 (prespore-cell-specific). By staining for beta-galactosidase expression histologically at various stages of development in individual strains, we were able to dissect the morphological changes in these strains, examine the spatial localization of the individual cell types, and understand the possible roles of PKA during multicellular development. Expression of PKAcat from either the ecmA or ecmB prestalk promoters resulted in abnormal development that arrested shortly after the mound stage, producing a mound with a round apical protrusion at the time of tip formation. Prestalk A and prestalk B cells were localized in the central region and the apical mound in the terminal differentiated aggregate, while prespore cells showed an aberrant spatial localization. Consistent with a developmental arrest, these mounds did not form either mature spores or stalk cells and very few cells expressed a stalk-cell-specific marker. Expression of PKAcat from the prespore promoter resulted in abnormal morphogenesis and accelerated spore cell differentiation. When cells were plated on agar, a fruiting body was formed with a very large basal region, containing predominantly spores, and a small, abnormal sorocarp. Mature spore cells were first detected by 14 hrs, with maximal levels reached by 18-20 hrs, in contrast to 24-26 hrs in wild-type strains. When cells were plated on filters, they produced an elongated tip from a large basal region, which continued to elongate as a tubular structure and produce a "slug-like" structure at the end. The slug was comprised predominantly of prestalk cells with a few prespore cells restricted to the junction between the "slug" and tube. As the slug migrated, these prespore cells were found in the tube, while new prespore cells appeared at the slug/tube junction, suggesting a continual differentiation of new prespore cells at the slug's posterior. The slug eventually produced a fruiting body-like structure that in many cases was abnormal and showed only a low level of SP60 expression. Models are presented of the roles cAMP dependent kinase plays in regulating various aspects of Dictyostelium differentiation. ================================================================= Characterization of the Signal Transduction Pathways and cis-Acting DNA Sequence Responsible for the Transcriptional Induction during Growth and Development of the Lysosomal a-Mannosidase Gene in Dictyostelium discoideum John Schatzle(1,2), John Bush(1), Su Dharmawardhane(3) Richard Firtel(3), Richard Gomer(4) and James Cardelli(1) (1)Department of Microbiology and Immunology, LSU Medical Center, Shreveport, LA 71130, Tel. 318 674 5756/FAX 318 674 5764, (2, Present address) Department of Microbiology, University of Texas, Austin, TX 78712-1095, (3)Department of Biology, University of California, La Jolla, CA 92093-0634, (4)Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892 J. Biol. Chem, in press ABSTRACT The lysosomal a-mannosidase gene in D. discoideum is representative of a small group of genes that are expressed under two different conditions: 1) immediately upon removal of the bacterial food source from exponentially growing cells at <5x10^5 cells/ml (which also initiates the developmental cycle), and 2) when the concentration of a secreted glycoprotein termed the prestarvation response factor (PSF) reaches a critical threshold in cultures growing at densities >5x105 cells/ml. In this report, we show that transcription of the a-mannosidase gene induced by starvation did not require protein synthesis in axenic wild-type strains whereas protein synthesis was required for the transcriptional induction observed in response to PSF. Northern blot analysis was also done using mRNA from Ga1 and Ga2 gene disruption mutants. These genes encode subunits of heterotrimeric G proteins found at the cell surface in growing cells and cells early in development. The pattern of a-mannosidase gene expression was normal in these mutants as well as in mutants unable to produce the secreted glycoprotein conditioned medium factor (CMF) or the cAMP receptor-1 (cAR1) protein. These genes have been shown to regulate the expression of many genes during early development. Promoter analysis studies identified a 145 bp sequence element containing a TTG box that was required for a-mannosidase transcriptional induction under both starvation conditions and in response to PSF. The TTG box identified is an important regulatory element in the promoter of another PSR response gene, the discoidin Ig gene. A ts mutant was found to misregulate the expression of both discoidin *I and a-mannosidase expression at restrictive temperatures. Taken together these results suggest that the PSR genes may be coordinately regulated possibly perhaps through the TTG box. ====================================================================== [[END CSM-NEWS. Vol. 1, number 7]]