Dicty News Electronic Edition Volume 18, number 6 May 4, 2002 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============= Abstracts ============= Rapid patterning and zonal-differentiation in a two-dimensional Dictyostelium cell mass: a role of pH and ammonia. Satoshi Sawai, Takashi Hirano, Yasuo Maeda and Yasuji Sawada Graduate School of Information Sciences, Biological Institute, Graduate School of Science and Research Institute of Electrical Communication, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan J. Exp. Biol., in press SUMMARY Recently it was demonstrated that a rapidly forming self-organizing pattern which emerges within two-dimensional Dictyostelium discoideum cell cultures could later give rise to stripes of distinct zones, each comprised of different cell-types. Here we report physiological aspects of the initial rapid patterning and its relation to cell differentiation. We found that as temperature is lowered the characteristic length of the pattern increases. From this the activation energy of the patterning kinetics was estimated. Fluorescence of fluorescein conjugated dextran revealed that the cytosolic pH of cells in the zone inside becomes lower than that in the other zone facing the air. The patterning could be inhibited by addition of the plasma-membrane proton pump inhibitors diethystilbestrol (DES) or miconazole. Preincubation of cells with weak acid delayed the timing of the patterning, whereas weak base hastened it. A pH indicating dye revealed localized accmulation of ammonia in the extracelluar space. These results suggest that gradients of secreted metabolites may be directly responsible for the rapid patterning and its consequence on cell differentiation in confined geometry. Possible diffusible candidate molecules and a reaction scheme coupled to the imposed oxygen gradient is discussed. ----------------------------------------------------------------------------- Sequence and Analysis of Chromosome 2 of Dictyostelium discoideum Gernot Glckner*, Ludwig Eichinger , Karol Szafranski*, Justin A. Pachebat! , Alan T. Bankier! , Paul H. Dear! , Rdiger Lehmann*, Cornelia Baumgart*, Gens Parra, Josep F. Abril, Roderic Guig, Kai Kumpf*, Budi Tunggal , the Dictyostelium Genome Sequencing Consortium, Edward Cox, Michael A. Quail, Matthias Platzer*, Andr Rosenthal*# and Angelika A. Noegel *IMB Jena, Dept of Genome Analysis, Beutenbergstr. 11, 07745 Jena, Germany Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Kln, Germany ! PNAC Biotech, Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH, UK Grup de Recerca en Informatica Biomedica, Institut Municipal d'Investigaci Mdica, Universitat Pompeu Fabra, Centre de Regulaci Genmica, 08003 Barcelona, Spain Princeton University, Princeton, NJ 08544, USA The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK #Friedrich Schiller Universitt Jena, 07743 Jena, Germany Nature, accepted Abstract The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8Mb represents about 25% of the genome. Despite an A+T content of nearly 80% the chromosome encodes 2,799 predicted protein coding genes and 73 tRNA genes (http://genome.imb-jena.de/dictyostelium/chr2/Chr2map.html and http://www.uni-koeln.de/dictyostelium/chr2/index.shtml). This gene density, about 1 gene per 2.6kb, is surpassed only by Saccharomyces cerevisiae (one per 2kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5kb). If we assume that the other chromosomes have a similar gene density, we expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located prior to the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution. ----------------------------------------------------------------------------- Establishing direction during chemotaxis in eukaryotic cells Wouter -Jan Rappel, Peter Thomas, Herbert Levine, and William F. Loomis Departments of Physics and Biology, UCSD, La Jolla, CA 92093 and Salk Institute for Biological Studies, La Jolla, CA 92186 Biophys. J (in press). ABSTRACT Several recent studies have demonstrated that eukaryotic cells including amoeboid cells of Dictyostelium discoideum and neutrophils, respond to chemoattractants by translocation of PH-domain proteins to the cell membrane where these proteins participate in the modulation of the cytoskeleton and relay of the signal. When the chemoattractant is released from a pipette, the localization is found predominantly on the proximal side of the cell. The recruitment of PH-domain proteins, particularly for Dictyostelium cells, occurs very rapidly (<2 seconds). Thus, the mechanism responsible for the first step in the directional sensing process of a cell must be able to establish an asymmetry on the same time scale. Here, we propose a simple mechanism in which a second messenger, generated by local activation of the membrane, diffuses through the interior of the cell, suppresses the activation of the back of the cell and converts the temporal gradient into an initial cellular asymmetry. Numerical simulations show that such a mechanism is plausible. Available evidence suggests that the internal inhibitor may be cGMP which accumulates within less than a second following treatment of cells with external cAMP. ----------------------------------------------------------------------------- mRNA processing in Dictyostelium: sequence requirements for termination and splicing Francisco Rivero Institut fr Biochemie I, Medizinische Fakultt, University of Cologne. Joseph-Stelzmann-Strasse 52, D-50931 Kln, Germany Protist, in press Criteria for the identification of termination regions in Dictyostelium discoideum genes have been established and the sequence requirements for termination in 33 genes have been analyzed. A canonical hexamer signal AATAAA was present 15-30 nucleotides upstream of the cleavage site, usually a TA, and was embedded in a particularly A-rich environment. T- or GT-rich downstream elements characteristic of animal cells could not be identified. In a sample of 102 introns we have established the consensus AG/GTAAGT and ATAG/ for the 5' and 3' splice sites, respectively. Most introns are 75-150 nucleotides long and the A+T content is high (90%). A putative branch point was identified in half of the introns 20-60 nucleotides upstream of the 3' splice site and the consensus TACTAAY was derived. A polypyrimidine tract required for branching in vertebrates was not identified, but weak preference for pyrimidine was found 10-45 nucleotides upstream of the 3' splice site. ----------------------------------------------------------------------------- Brabek, J., Mojzita, D., Puta, F. Folia Biologica(Prague)vol.48 p.73-76 (2002). Abstract. We achieved the production of the v-Src of the low-oncogenic strains Prague C (PRC) and H19 in Dictyostelium discoideum, an emerging host system suitable for the synthesis of heterologous proteins. To accomplish the expression, the first 6 codons of the N-terminus of v-src had to be changed according to the D. discoideum codon preference. Alternatively, N-terminal fusions, of 6xHis-tag or GFP, were sufficient to overcome the incompatibility in codon usage. D. discoideum expressed v-Src kinases of expected molecular weight were recognized by Src-specific antibodies; GFP-PRC was distributed uniformly in the cytosol. In contrast to other lower eukaryotes, where the accumulation of v-Src leads to growth inhibition, D. discoideum cells silenced the kinase activity of PRC derived v-Src and showed no developmental or growth defects. ----------------------------------------------------------------------------- [End Dicty News, volume 18, number 6]