Dicty News Electronic Edition Volume 9, number 9 11 October 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== Gene trapping with GFP: the isolation of developmental mutants in the slime mold Polysphondylium. Petra Fey and Edward C. Cox Department of Molecular Biology, Princeton University Princeton NJ 08544 Current Biology, in press. Abstract In order to study how a cell mass undergoes a transition from one symmetry to another in the slime mold Polysphondylium, we developed a genetic screen in which mutant phenotype and gene expression can easily be visualized in the living organism. The screen combines restriction enzyme mediated integration (REMI), and green fluorescent protein (GFP) expression. In REMI, a restriction enzyme is electroporated along with linearized vector into cells, thus determining the site of plasmid insertion and often increasing the integration frequency. A set of transforming plasmids carrying the GFP coding sequence in three reading frames was used for transformation. The plasmids were constructed so that GFP could be expressed only under control of a host promoter. Living transformants expressing GFP spatially and temporally could be rapidly identified in a very large background of non-expressing cells and fruiting bodies. The phenotypes of representative mutants range from cells that cannot aggregate and initiate cell-cell interactions, through mutant fruiting bodies, to apparently wild-type fruiting bodies expressing GFP in all or a sub-population of cells. The ability to screen mutant living cells and tissues for GFP expression is rapid and effective, and likely to have application in many transformable systems where screening by gene and promoter trapping is essential for understanding temporal and spatial gene regulation. ------------------------------------------------------------------------- A novel, negative selectable marker for gene disrution in Dictyostelium Morrison, A. Marschalek, R., Dingermann, T. & Harwood, A.J. Gene, in press Abstract Expression of an ochre suppressor mutant of the GluII(UUA) tRNA appears to be lethal to Dictyostelium, and offers a novel positive-negative strategy to select for targeted gene disruption by homologous recombination. Inclusion of the suppressor tRNA gene decreases the overall transformation frequency by approximately twenty fold. This increases the proportion of targeted gene disruptions to over ninety percent. ------------------------------------------------------------------------- [Correction: The following abstract appeared in Dicty News v9, number 7, but contained several errors. The corrected abstract appears in its entirety below:] Phosphorylation of Chemoattractant Receptors is Not Essential for Chemotaxis or Termination of G-Protein-Mediated Responses* Ji-Yun Kim#, Ron D.M. Soede%, Pauline Schaap%, Romi Valkema&, Jane A. Borleis#, Peter J.M. Van Haastert&, Peter N. Devreotes#, and Dale Hereld#,@ # Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; % Institute for Molecular Plant Sciences, University of Leiden, Leiden, The Netherlands; & Department of Biochemistry, University of Groningen, Groningen, The Netherlands. @ Present address: Microbiology and Molecular Genetics, The University of Texas Health Science Center, Houston, Texas. J. Biol. Chem., in press SUMMARY: In several G-protein-coupled signaling systems, ligand-induced receptor phosphorylation by specific kinases is suggested to lead to desensitization via mechanisms including receptor/G-protein uncoupling, receptor internalization, and receptor down-regulation. We report here that elimination of phosphorylation of a chemo- attractant receptor of Dictyostelium, either by site-directed substitution of the serines or by truncation of the C-terminal cytoplasmic domain, completely prevented agonist-induced loss-of-ligand binding but did not impair the adaptation of several receptor-mediated responses including the activation of adenylyl and guanylyl cyclases and actin polymerization. In addition, the phosphorylation-deficient receptors were capable of mediating chemotaxis, aggregation, and differentiation. We propose that, for chemoattractant receptors, agonist-induced phosphorylation regulates surface binding activity but other phosphorylation-independent mechanisms mediate response adaptation. ------------------------------------------------------------------------- [End Dicty News, volume 9, number 9]