Dicty News Electronic Edition Volume 11, number 7 October 10, 1998 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ============= Abstracts ============= The Glycogen Phosphorylase-2 Promoter Binding Protein in Dictyostelium is Replication Protein A Xiao Wen, Pawjai Khampang, and Charles L. Rutherford Biology Department, Molecular and Cellular Biology Section, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 J. Mol. Biol., in press SUMMARY During Dictyostelium development, glycogen degradation is a crucial event that provides glucose monomers used in the synthesis of the essential structural components for cellular differentiation. The product of the developmentally- regulated glycogen phosphorylase-2 gene (gp2) catalyzes the degradation. DNA binding proteins were found to bind to a regulatory site of the gp2 gene in a stage-dependent pattern. Gel-shift analysis of undifferentiated amoebae cell extract revealed a protein migrating at 0.40 Rf, while 17 h differentiated cell extract produced a species migrating at 0.32 Rf. Both the 0.32 and 0.40 Rf proteins were purified and found to consist of three subunits of 18, 35 and 62 kDa (for 0.40 Rf) or 81 kDa (for 0.32 Rf). Database searches identified the protein as the Dictyostelium homologue of replication protein A (DdRPA). Amino acid sequence analysis showed identity between the 62 and 81 kDa subunits. Incubation of cell-free extracts under appropriate conditions at low pH, resulted in conversion of the 81 kDa to the 62 kDa subunit. Northern blot analysis revealed that the levels of expression of the large subunit of DdRPA were constant throughout differentiation and the size of the mRNA was the same at all stages of development. The results raise the possibility that pH induced post-translational modifications of DdRPA are involved in events that halt cell proliferation and induce differentiation in Dictyostelium . ---------------------------------------------------------------------------- Two-component Signal Transduction Systems in Eukaryotic Microorganisms William F. Loomis, Adam Kuspa* and Gad Shaulsky# Department of Biology, University of California San Diego, La Jolla CA 92093 *Department of Biochemistry, and #Department of Molecular Genetics, Baylor College of Medicine, Houston, TX 77030 Curr. Opinions in Microbiology (in press) SUMMARY Conserved signal transduction pathways using phosphorelay from histidine kinases through an intermediate transfer protein (H2) to response regulators have been found in a variety of eukaryotic microorganisms. Several of these pathways are linked to MAP kinase cascades. These networks control different physiological responses including osmoregulation, cAMP levels and cellular morphogenesis. ---------------------------------------------------------------------------- A NOVEL ADENYLYL CYCLASE DETECTED IN RAPIDLY DEVELOPING MUTANTS OF DICTYOSTELIUM Hyun-Ji Kim, Wen-Tsan Chang, Marcel Meima, Julian D. Gross and Pauline Schaap Department of Biochemistry, University of Oxford, Oxford OX13QU, UK Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, 2333 AL Leiden, The Netherlands. J. Biol. Chem., in press SUMMARY Disruption of either the RDEA or REGA genes leads to rapid development in Dictyostelium. The RDEA gene product displays homology to certain H2 type phosphotransferases, while REGA encodes a cAMP phosphodiesterase with an associated response regulator. It has been proposed that RDEA activates REGA in a multi step phosphorelay. To test this proposal, we examined cAMP accumulation in rdeA and regA null mutants and found that these mutants show a pronounced accumulation of cAMP at the vegetative stage, that is not observed in wild-type cells. This accumulation was due to a novel adenylyl cyclase and not to the known Dictyostelium adenylyl cyclases, ACA or ACG, since it occurred in an acaA/rdeA double mutant and, unlike ACG, was inhibited by high osmolarity. The novel adenylyl cyclase was not regulated by G-proteins and was relatively insensitive to stimulation by Mn2+ ions. Addition of the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) permitted detection of the novel AC activity in lysates of an acaA/acgA double mutant. The fact that disruption of the RDEA gene as well as inhibition of the REGA-phoshodiesterase by IBMX permitted detection of the novel AC activity supports the hypothesis that RDEA activates REGA. ---------------------------------------------------------------------------- TWO DISTINCT SIGNALING PATHWAYS MEDIATE DIF INDUCTION OF PRESTALK GENE EXPRESSION IN DICTYOSTELIUM Irene Verkerke-van Wijk, Raymond Brandt, Laurens Bosman and Pauline Schaap Cell Biology Section, Institute for Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands Experimental Cell Research, in press ABSTRACT During Dictyostelium development, the Differentiation Inducing Factor (DIF) triggers expression of the prestalk gene ecmB and induces stalk cell differentiation, a form of programmed cell death. The effects of DIF are mediated by a sustained increase in cytosolic Ca2+ levels. The Ca2+-ATPase inhibitor, BHQ, causes a similar rise in Ca2+ levels and also induces prestalk gene expression. We show here that Ca2+ is a specific intermediate for prestalk gene induction, since BHQ represses transcription of the cAMP-inducible aggregative gene PDE, the post-aggregative gene CP2, and the prespore gene D19. The prestalk gene ecmA is also induced by DIF, but induction appears to occur in two steps, which occur within 1 hour and after 2 hours respectively. The slow step shows the same kinetics as ecmB induction and similar to ecmB induction, this step is BHQ-inducible and requires an initial round of protein synthesis. The fast step does not require protein synthesis and cannot be induced by BHQ. This indicates that in addition to the slow Ca2+-mediated pathway, there is probably a second fast Ca2+-independent signal transduction pathway for DIF. ---------------------------------------------------------------------------- [End Dicty News, volume 11, number 7]