Dicty News Electronic Edition Volume 15, number 9 October 28, 2000 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" ============== Abstracts ============== Cell cycle phase, cellular Ca2+and development in Dictyostelium discoideum M. Azhar1, P. K. Kennady2, Gopal Pande2, Michael Espiritu3, Wesley Holloman, Derrick Brazill, Richard H. Gomer3 and Vidyanand Nanjundiah1* 1 Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore-560012, India. 2 Centre for Cellular and Molecular Biology, Hyderabad-500007, India. 3 Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251-1892, USA. International Journal of Developmental Biology, in press. In Dictyostelium discoideum the initial differentiation of cells is regulated by the phase of the cell cycle at starvation.Cells that are between S and early G2 at starvation (or with a low DNA content) have relatively high cellular Ca2+ and display a prestalk tendency, while cells in mid to late G2 (or with a high DNA content) have relatively low levels of Ca2+ and display a prespore tendency. As expected on the basis of this correlation, cell cycle inhibitors influence the proportions of amoebae containing high or low Ca2+. However, in the mutant rtoA, which upon differentiation gives rise to many more stalk cells than spores (compared to the wild type), initial cell-type choice is independent of cell cycle phase at starvation (Wood et al., 1996). In contrast to the wild type, a disproportionately large fraction of rtoA amoebae fall in the high Ca2+ class, possibly due to an altered ability of this mutant to transport Ca2+. These results show that Ca2+ is yet another aspect of early functional heterogeneity between cells of D. discoideum. The list of such aspects keeps growing and, as of today, includes cell size (Bonner et al; 1971), nutritional status (Leach et al; 1973), cell cycle phase (McDonald and Durston, 1984; Weijer et al; 1984), sequestered Ca 2+ (Azhar et al; 1996) and the sensitivity of freshly starved amoebae to DIF (Azhar et al; 1997). From an evolutionary point of view, these can be thought of as so many correlates of the phenotype with relative cellular fitness (what we have previously termed 'qualities' ; Atzmony et al; 1995). Phenotypic selection at the level of the individual cell ensures that following aggregation, high-quality amoebae display traits of prespore cells on the whole and low-quality amoebae display prestalk traits (again, on the whole). Seen thus, it appears that while the notion of morphogens and positional information is a useful way of describing collective behaviour during the later stages of differentiation and pattern formation in D. discoideum, the essence of that behaviour must be sought in the myriad and interlocking ways in which cells differ from each other at all stages of the life cycle. ---------------------------------------------------------------------------- Phg1p: A Nine-Transmembrane Protein Superfamily Member Involved in Dictyostelium Adhesion and Phagocytosis Sophie Cornillon, Emmanuel Pech, Mohammed Benghezal, Kissia Ravanel, Erin Gaynor, Fran�ois Letourneur, Franz Br�ckert and Pierre Cosson Universit� de Gen�ve, Centre M�dical Universitaire, D�partement de Morphologie, 1 rue Michel Servet, CH-1211 Gen�ve 4, Switzerland J. Biol. Chem., In press To identify the molecular mechanisms involved in phagocytosis, we generated random insertion mutants of Dictyostelium discoideum and selected two mutants defective for phagocytosis. Both represented insertions in the same gene, named PHG1. This gene encodes a polytopic membrane protein with an N-terminal lumenal domain and nine potential transmembrane segments. Homologous genes can be identified in many species, however their function is yet to be elucidated. Disruption of PHG1 caused a selective defect in phagocytosis of latex beads and E. coli, but not K. aerogenes bacteria. This defect in phagocytosis was caused by a decrease in the adhesion of mutant cells to phagocytosed particles. These results indicate that the Phg1 protein is involved in the adhesion of Dictyostelium to various substrates, a crucial event of phagocytosis and demonstrate the usefulness of a genetic approach to dissect the molecular events involved in the phagocytic process. ---------------------------------------------------------------------------- Novel Aromatic Substances, Dictyomedin A and B, from Dictyostelium Cellular Slime Molds and Their Inhibitory Effects on Dictyostelium Development Yoshiaki Takaya1, Haruhisa Kikuchi1, Yuichi Terui1, Jun Komiya, Yasuo Maeda2, Akira Ito3 and Yoshiteru Oshima1* 1Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Sendai 980-8578, 2Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai 980-8578 and 3Kyorin Pharmaceutical Co. Ltd., Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan Tetrahedron Lett. (in press) Abstract To elucidate the diversity of secondary metabolites of Dictyostelium cellular slime molds and search for physiologically active substances affecting their development, we investigated the constituents of D. medium. >From the methanol extract of its fruit body, we obtained two novel aromatic compounds named dictyomedin A (1) and B (2), and their structures were elucidated by means of physicochemical data. Biological evaluation of the compounds showed that they delayed the differentiation of D. discoideum cells. ---------------------------------------------------------------------------- Efficient transformation of Dictyostelium discoideum with a particle inflow gun Birgit Wetterauer1*, Klaus Salger1, Petra Demel2 and Hans-Ulrich Koop2 1 Zoologisches Institut der Ludwig Maximilians-Universitaet, Luisenstra�e 14, 80333 Muenchen, Germany 2 Botanisches Institut der Ludwig Maximilians-Universitaet, Menzingerstra�e 67, 80683 Muenchen, Germany BBA, Section: Molecular Cell Research We report experiments to transform Dictyostelium discoideum using a simple home made particle gun. Stable transformants were obtained at frequencies of up to 2500 clones/�g DNA. This is five times more than we achieve with the same vector using electroporation protocols. We also show that the particle inflow gun can be used for analysis of developmentally regulated gene expression in a transient assay. ---------------------------------------------------------------------------- Identification and characterisation of DdPDE3, a cGMP-selective phosphodiesterase from Dictyostelium Hidekazu Kuwayama, Helena Snippe, Mari Derks, Jeroen Roelofs and Peter J.M. Van Haastert GBB, Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands Biochemical Journal, in press Abstract In Dictyostelium cAMP and cGMP play important functions as first and second messengers in chemotaxis and developments. Two cyclic nucleotide phosphodiesterases (DdPDE 1 and 2) have been identified previously, an extracellular dual-specificity enzyme and an intracellular cAMP-specific enzyme (encoded by the psdA and regA gene, respectively). Biochemical data suggest the presence of at least one cGMP-specific PDE that is activated by cGMP. Using bioinformatics we identified a partial sequence in the Dictyostelium EST database that shows a high degree of amino acid sequence identity with mammalian PDE catalytic domains (DdPDE3). The deduced amino acid sequence of a full-length DdPDE3 cDNA isolated in this study predicts a 60 kDa protein with a 300 amino acid C-terminal PDE catalytic domain, which is preceded by about 200 amino acids that are rich in asparagine and glutamine residues. Expression of the DdPDE3 catalytic domain in E. coli shows that the enzyme has Michaelis-Menten kinetics and a higher affinity for cGMP (Km = 0.22 microM) than for cAMP (Km = 145 microM); cGMP does not stimulate enzyme activity. The enzyme requires divalent cations for activity; Mn2+ is preferred to Mg2+ , whereas Ca2+ yields no activity. DdPDE3 is inhibited by 3- isobutyl-1-methyl-xanthine (IBMX) with an IC50 of about 60 mM. Overexpression of DdPDE3 catalytic domain in Dictyostelium confirms these kinetic properties without indications for its activation by cGMP. The properties of DdPDE3 resemble those of mammalian PDE9 that also shows the highest sequence similarity within the catalytic domains. DdPDE3 is the first cGMP-selective PDE identified in lower eukaryotes. ---------------------------------------------------------------------------- EXPRESSION AND ONE STEP PURIFICATION OF PLASMODIUM PROTEINS IN DICTYOSTELIUM Miguel X. van Bemmelen1, Carole Beghdadi-Rais 2, Chantal Desponds+, Esmeralda Vargas3, S�crates Herrera3, Christophe D. Reymond1 and Nicolas Fasel2 1 Institut de Biologie Cellulaire et de Morphologie, Universit� de Lausanne, Rue du Bugnon 9, CH-1005 Lausanne, Switzerland 2 Institut de Biochimie, Universit� de Lausanne, ch. des Boveresses 155, CH-1066 Epalinges, Switzerland 3 Secci�n del Immunolog�a, Facultad de Salud, Universidad del Valle, Cali, Colombia Molecular and Biochemical Parasitology, In Press Abstract Nearly full length CSP from Plasmodium falciparum, the C-terminal fragments from both P. falciparum and P. yoelii CSP and a fragment comprising 351 amino acids of P.vivax MSP1 were expressed in the slime mold Dictyostelium discoideum. Discoidin-tag expression vectors allowed both high yields of these proteins and their purification by a nearly single-step procedure. We exploited the galactose binding activity of Discoidin Ia to separate the fusion proteins by affinity chromatography on Sepharose-4B columns. Inclusion of a thrombin recognition site allowed cleavage of the Discoidin-tag from the fusion protein. Partial secretion of the protein was obtained via an ER independent pathway, whereas routing the recombinant proteins to the ER resulted in glycosylation and retention. Yields of proteins were ranging from 0.08 to 3 mg per Litre depending on the protein sequence and the purification conditions. The recognition of purified MSP1 by sera from P. vivax malaria patients was used to confirm the native conformation of the protein expressed in Dictyostelium. The simple purification procedure described here, based on Sepharose-4B, should facilitate the expression and the large-scale purification of various Plasmodium polypeptides. ---------------------------------------------------------------------------- [End Dicty News, volume 15, number 9]