Dicty News Electronic Edition Volume 16, number 4 February 10, 2001 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 the Dictyostelium Web Page "http://dicty.cmb.northwestern.edu/dicty" ============== Abstracts ============== WD-repeat domains target Dictyostelium myosin heavy chain kinases by binding directly to myosin filaments Paul A. Steimle*, Teresa Naismith†, Lucila Licate*; and Thomas T. Egelhoff* *Department of Physiology and Biophysics Case Western Reserve University School of Medicine Cleveland, Ohio 44106-4970 †Current address: Department of Cell Biology and Physiology Washington University St. Louis, MO 63130 Journal of Biological Chemistry (In Press) Abstract: Myosin heavy chain kinase A (MHCK A) phosphorylates mapped sites at the carboxyl-terminal tail of Dictyostelium myosin II heavy chain (MHC), driving disassembly of myosin filaments both in vitro and in vivo. MHCK A is organized into three functional domains that include an amino-terminal coiled-coil region, a central kinase catalytic domain unrelated to conventional protein kinases, and a WD-repeat domain at the carboxy-terminus. MHCK B is a homologue of MHCK A that possesses structurally related catalytic and WD-repeat domains. In the current study, we explored the role of the WD-repeat domains in defining the activities of both MHCK A and MHCK B using recombinant bacterially-expressed truncations of these kinases either with or without their WD-repeat domains. We demonstrate that substrate targeting is a conserved function of the WD-repeat domains of both MHCK A and MHCK B and that this targeting is specific for Dictyostelium myosin II filaments. We also show that the mechanism of targeting involves direct binding of the WD-repeat domains to the myosin substrate. To our knowledge, this is the first report of WD-repeat domains physically targeting attached kinase domains to their substrates. The examples presented here may serve as a paradigm for enzyme targeting in other systems. ----------------------------------------------------------------------------- The complex repeats of Dictyostelium discoideum Gernot Glöckner1*+, Karol Szafranski1+, Thomas Winckler2, Theodor Dingermann2, Michael A. Quail3, Edward Cox4, Ludwig Eichinger5, Angelika Anna Noegel5, André Rosenthal1,6 1 IMB Jena, Dept of Genome Analysis, Beutenbergstr. 11, D-07745 Jena, Germany 2Institut für Pharmazeutische Biologie, Biozentrum, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany 3The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK 4 Dept. of Molecular Biology, Princeton University, Princeton, NJ 08544, USA 5Institut für Biochemie I, Medizinische Einrichtungen der Universität zu Köln, Joseph-Stelzmann-Str. 52, D-50931 Köln, Germany 6 Friedrich Schiller Universität Jena, D-07743 Jena, Germany * corresponding author Genome Research, in press Abstract In the course of determining the sequence of the Dictyostelium discoideum genome we have characterised in detail the quantity and nature of interspersed repetitive elements present in this species. Several of the most abundant small complex repeats and transposons (DIRS-1; TRE3-A,B; TRE5-A; skipper; Tdd-4; H3R) have been described previously. In our analysis we have identified additional elements. Thus we can now present a complete list of complex repetitive elements in D. discoideum. All elements add up to 10 % of the genome. Some of the newly described elements belong to established classes (TRE3-C, D; TRE5-B,C; DGLT-A,P; Tdd-5). However, we have also defined two new classes of DNA transposable elements (DDT; thug) that have not been described so far. Based on the nucleotide amount we calculated the least copy number in each family. These vary between less than 10 up to over 200 copies. Unique sequences adjacent to the element ends and truncation points in elements gave a measure for the fragmentation of the elements. Furthermore, we describe the diversity of single elements with regard to polymorphisms and conserved structures. All elements show insertion preference into loci in which other elements of the same family reside. The analysis of the complex repeats is a valuable data resource for the ongoing assembly of whole D. discoideum chromosomes. ----------------------------------------------------------------------------- Large Scale Sequencing and Analysis of AT rich Eukaryote Genomes Gernot Glöckner, Institut für Molekulare Biotechnologie E.V., Department of Genome Analysis, D-07745 Jena, Germany Current Genomics (2000), 1, 289-299 Abstract Environmental pressures can direct genomes from a normal to a more or less pronounced imbalance in the base composition. These pressures seem to occur relatively often since genomes with a deviation from a normal base composition are widespread throughout lower eukaryotes. These genomes show altered codon usage and enrichment for the preferred bases in intron and intergenic regions. Techniques designed for large scale sequencing and assembly of genomes with normal base composition will fail with these unusual genomes. Additionally, the currently available analysis tools are mainly suitable for gene finding in genomes with normal base composition. In recent years some large scale genome analysis projects involving species with a skew directed to a very high AT content were initiated. These projects are encountered with sequencing, assembly, and gap closure problems due to the high AT content. These problems can only be overcome with additional techniques, which partly were developed and used in the ongoing projects. In this review some characteristic aspects of AT rich genomes, the progress of the Dictyostelium discoideum and the Plasmodium falciparum projects, as well as techniques specifically used for the sequencing of these genomes are highlighted. ----------------------------------------------------------------------------- A role for myosin VII in dynamic cell adhesion Richard I. Tuxworth, Igor Weber, Deborah Wessels, Gregory C. Addicks, David R. Soll, Günther Gerisch, and Margaret A. Titus Current Biology, in press. Abstract Background The initial stages of phagocytosis and cell motility resemble each other. The extension of a pseudopod at the leading edge of a migratory cell and the formation of a phagocytic cup are actin-dependent and each rely on the plasma membrane adhering to a surface during dynamic extension. Results A myosin VII null mutant exhibited a drastic loss of adhesion to particles, consistent with the extent of observed decrease in particle uptake. Additionally, cell-cell adhesion and the adhesion of the leading edge to the substratum during cell migration were defective in the myosin VII null cells. GFP-myosin VII rescues the phagocytosis defect of the null mutant and was distributed in the cytosol and recruited to the cortical cytoskeleton, where it appeared to be enriched at the tips of filopods. It was also localized to phagocytic cups, but only during the initial stages of particle engulfment. During migration, GFP-myosin VII is found at the leading edge of the cell. Conclusions Myosin VII plays an important role in mediating the initial binding of cells to substrata, a novel role for an unconventional myosin. ----------------------------------------------------------------------------- Genetic analysis of phototaxis in Dictyostelium. P.R. Fisher Comprehensive Series in Photosciences Vol. 1. "Photomovement". Chapter 19. pp. 517-558. Ed. D-P. Häder and M. Lebert. Publisher: Elsevier Science Ltd. In Press. Preprint version available from http://www.latrobe.edu.au/www/mcbg/my.html. Abstract. Dictyostelium discoideum slugs are doubtless the simplest multicellular eukaryotes in which classical and molecular genetic approaches to the study of the signal transduction pathways controlling photomovement are possible. The slugs are formed by chemotactic aggregation of starving amoebae, so that it is a simple matter to generate, from clonally-derived mutant cell lines, hundreds of genetically identical multicellular individuals for study. Adding to the already extensive knowledge of the unicellular stages of the D. discoideum life cycle, classical and molecular genetics have begun to unravel transduction of signals controlling phototaxis (and thermotaxis) in the slugs. Distributed over all seven genetic linkage groups are at least 20, but possibly as many as 55 genes of importance for slug behaviour. The encoded proteins appear from pharmacological studies and mutant phenotypes to govern transduction pathways involving extracellular cAMP signals and corresponding receptors, heterotrimeric and small GTP-binding proteins, the intracellular second messengers cyclic AMP, cyclic GMP, Ca2+ and IP3 as well as cytoskeletal proteins such as Actin Binding Protein-120 and myosin II. Pathways from the photo- and thermoreceptors converge first with each other and thence with those from extracellular tip activation (cyclic AMP) and inhibition (Slug Turning Factor and/or ammonia and/or adenosine) signals that control slug movement and morphogenesis. ----------------------------------------------------------------------------- The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation Celia Briscoe, John Moniakis, Ji Yun Kim, Jason M. Brown, Dale Hereld, Peter N. Devreotes, and Richard A. Firtel Developmental Biology, in press. ABSTRACT cAMP receptors mediate some signalling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis, induce postaggregative and cell-type-specific gene expression, and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development. ----------------------------------------------------------------------------- [End Dicty News, volume 16, number 4]