Dicty News Electronic Edition Volume 12, number 8 April 17, 1999 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 ============== Folic acid stimulation of the G(alpha)4 G protein-mediated signal transduction pathway inhibits anterior prestalk cell development in Dictyostelium Jeffrey A. Hadwiger and Jaishree Srinivasan Department of Microbiology and Molecular Genetics, Oklahoma State University Stillwater, OK 74078-3020 Differentiation (in press) ABSTRACT In Dictyostelium discoideum, several G proteins are known to mediate the transduction of signals that direct chemotactic movement and regulate developmental morphogenesis. The G protein alpha subunit encoded by the G(alpha)4 gene has been previously shown to be required for chemotactic responses to folic acid, proper developmental morphogenesis, and spore production. In this study, cells overexpressing the wild type G(alpha)4 gene, due to high copy gene dosage (G(alpha)4HC), were found to be defective in the ability to form the anterior prestalk cell region, express prespore- and prestalk-cell specific genes, and undergo spore formation. In chimeric organisms, G(alpha)4HC prespore cell-specific gene expression and spore production were rescued by the presence of wild type cells, indicating that prespore cell development in G(alpha)4HC cells is limited by the absence of an intercellular signal. Transplanted wild type tips were sufficient to rescue the G(alpha)4HC prespore cell development suggesting that the rescuing signal originates from the anterior prestalk cells. However, the deficiencies in prestalk-specific gene expression were not rescued in the chimeric organisms. Furthermore, G(alpha)4HC cells were localized to the prespore region of these chimeric organisms and completely excluded from the anterior prestalk region, suggesting that the G(alpha)4 subunit functions cell autonomously to prevent anterior prestalk cell development. The presence of exogenous folic acid during vegetative growth and development delayed anterior prestalk cell development in wild type but not g(alpha)4 null mutant aggregates, indicating that folic acid can inhibit cell type-specific differentiation by stimulating of the G(alpha)4- mediated signal transduction pathway. The results of this study suggest that G(alpha)4-mediated signals can regulate cell type-specific differentiation by promoting prespore cell development and inhibiting anterior prestalk cell development. ---------------------------------------------------------------------------- Polycistronic transcription and editing of the mitochondrial small subunit (SSU) ribosomal RNA in Dictyostelium discoideum. C. Barth, U. Greferath, M. Kotsifas and P.R. Fisher. Microbiology Dept., La Trobe University, Melbourne, Australia. Current Genetics. In Press. Summary: Northern analyses and reverse transcription-polymerase chain-reaction (RT-PCR) experiments were performed on total RNA of Dictyostelium discoideum. The mitochondrial genes encoding the small subunit ribosomal RNA (SSU), cytochrome b (CYTB) and subunit 3 of the NADH dehydrogenase (ND3) were found to be cotranscribed. Further posttranscriptional processing resulted in a dicistronic transcript for CYTB and ND3, and a monocistronic SSU transcript. Markedly higher steady state transcript levels were detected for the mature SSU ribosomal RNA. A comparison of the SSU cDNA sequence with the mitochondrial DNA sequence of the SSU gene revealed C-to-U substitutional editing of the SSU ribosomal RNA at a single site, as a consequence of which the cDNA contained a PvuII site not present in the genomic DNA. The editing was shown to be highly efficient and to occur in the primary transcript before the release of the mature mRNA, rRNA and tRNAs. It is suggested that the editing may be required for normal pseudoknot formation in the 530 loop of the RNA and thus important for efficient, accurate translation in the mitochondria. ---------------------------------------------------------------------------- Molecular Basis of Localized Responses During Chemotaxis in Amoebae and Leukocytes Saskia van Es and Peter N. Devreotes Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore Maryland 21205 Cellular and Molecular Life Sciences, in press Abstract Chemotaxis and phagocytosis are fundamental responses shared by many eukaryotic cells. In chemotaxis, motile cells sense and respond directionally to chemical gradients; in phagocytosis, they bind and engulf foreign organisms and debris. Both processes are central to the inflammatory responses mounted by leukocytes and are displayed by cells of the primitive immune systems of sponges, nematodes and flies. Moreover, free-living amoebae display both chemotaxis and phagocytosis. One such amoeba, Dictyostelium discoideum, provides a powerful system for molecular genetic analysis of these processes. In this review, we will describe the general features of chemotaxis shared by amoebae and leukocytes. We will not focus on motility per se, since this subject has been extensively reviewed and, although motility and chemotaxis are related, these phenomena can be separated. We will outline the genetic and biochemical tools available in D. discoideum to study chemotaxis and update our current understanding. Finally, we will discuss the relationship between phagocytosis and chemotaxis. ---------------------------------------------------------------------------- The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialised substrate binding domain Stefan Steinbacher1, Peter Hof, Ludwig Eichinger2, Michael Schleicher2, Jan Gettemans3, Joël Vandekerckhove3, Robert Huber and Jörg Benz4 Abteilung Strukturforschung, Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany; 2) A.-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität, 80336 München, Germany; 3) Flanders Interuniversity Institute of Biotechnology, University of Gent, 9000 Gent, Belgium; 4) Present address: Department für Chemie und Biochemie, Universität Bern, 3012 Bern, Switzerland EMBO J., in press ABSTRACT Coordinated temporal and spatial regulation of the actin cytoskeleton is essential for diverse cellular processes such as cell division, cell motility and the formation and maintenance of specialised structures in differentiated cells. In plasmodia of Physarum polycephalum the F-actin capping activity of the actin-fragmin complex is regulated by phosphorylation of actin. This is mediated by a novel type of protein kinase with no sequence homology to eukaryotic-type protein kinases. Here we present the crystal structure of the catalytic domain of the first cloned actin kinase in complex with adenosine-monophosphate at 2.9 Å resolution. The three-dimensional fold reveals a catalytic module of approximately 160 residues, in common with the eukaryotic protein kinase super-family, which harbours the nucleotide binding site and the catalytic apparatus in an inter-lobe cleft. Several kinases that share this catalytic module differ in the overall architecture of their substrate recognition domain. The actin-fragmin kinase has acquired a unique flat substrate recognition domain which is supposed to confer stringent substrate specificity. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 8]